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PMC4965202

today s world is a world of organizations , and their main operators are the people who invigorate them and prepare the grounds for the realization of their objectives . without people , the concepts of organization and management would become meaningless ( 1 ) . therefore , professional satisfaction on the part of manpower is one of the most important factors in the efficacy of organizations . the issue of professional satisfaction has been studied repeatedly with respect to various organizations since the 1920s , and , according to many experts , it is one of the most challenging organizational concepts and the basis behind the many management policies adopted to improve organizational effectiveness ( 2 ) . health care organizations and treatment centers are responsible for maintaining the health of all citizens in a society , and nurses have a key role . their success in fulfilling this role depends on the measures taken by competent managers who recognize the prominent factors involved in the activities of nurses and seek to create favorable work conditions in order to enhance their professional satisfaction ( 1 ) . previous studies have shown that nurses should demonstrate a passion for their work and have the necessary proficiency and expertise to achieve the highest level of efficiency ( 2 ) . it could be argued that any person would provide better services if he or she were motivated and enthusiastic about the work . despite the great significance of the professional satisfaction of nurses due to its considerable impact on them , their patients , their organizations , and the very profession of nursing ( 3 ) , most previous studies have reported the satisfaction level to be low among nurses ( 4 ) . the results from the study conducted by lou et al . indicated that only half of the nurses in the study were moderately or highly satisfied with their work ( 5 ) . another study was conducted to compare the level of professional satisfaction among different groups of people in korea , and it was found that only 41.5% of nurses were satisfied with their work , whereas the percentages for other experts working at hospitals , social workers , and middle school teachers were 50.1 , 58.2 , and 89% , respectively . these statistics indicate that there is a lower level of satisfaction among nurses than other professionals ( 6 ) . various studies have been conducted regarding the extent of professional satisfaction among the nurses , and , sadly , all have indicated low degrees of satisfaction ( 7 ) . most nurses who work in internal medicine and surgical departments ( 54.4% ) demonstrated low levels of professional satisfaction ( 8) . overall , these studies suggested that the situation concerning nurses professional satisfaction is unacceptable . it should be noted that a lack of sufficient concern towards professional satisfaction will result in serious consequences in the long run . it could give rise to rebellion , diminished feelings of responsibility , and eventually resignation from the job ( 9 ) . professional dissatisfaction among nurses could result in emotional detachment , depression , anger , evasion from work , and ineffectiveness ( 10 ) . according to a qualitative study done in 2010 in india , the reasons nurses quit their jobs were determined to be professional exhaustion , stress at work , and lack of a friendly environment , all of which contributed to their diminished professional satisfaction . in addition , it was indicated that a strong relationship exists between professional dissatisfaction and quitting work ( 11 ) . according to released statistics , the insufficient number of available nurses around the world is a tangible phenomenon . in australia , for instance furthermore , according to estimations , by 2010 , canada will need an additional 113,000 nurses , and , by 2020 , the united states would incur a 20% shortage in the number of nurses ( approximately 581,500 nurses ) ( 12 , 13 ) . in iran , one of the most problematic issues in nursing is the shortage of nursing staff . the shortage of nursing staff has different causes , including job dissatisfaction and organizational and sociocultural factors ( 14 ) . in iran , many studies have reported that nurses are not happy because of the shortage of nursing staff , high numbers of patients , the lack of independence and authority , the lack of appreciation and acknowledgment , low income , heavy workloads , low level of involvement in management , disagreements with physicians , managerial issues , and a lack of appropriate work conditions in hospitals ( 15 ) . iran regret having become nurses , whereas 43.9% of them feel disappointed and dissatisfied that they became nurses . the most challenging problem in nursing in iran is the shortage of nurses working in hospitals because of their dissatisfaction and lack of motivation , and , unfortunately , most nurses in iran would like to change their work ( 14 ) . because satisfaction with nursing is related to improving the health system and patient satisfaction , this study was conducted to assess how the satisfaction of nurses with their work can be improved , their situation in hospitals can be improved , and also how patients satisfaction could be increased ( 15 ) . the objective of this research was to determine iranian nurses experiences of the concept of professional satisfaction . today s world is a world of organizations , and their main operators are the people who invigorate them and prepare the grounds for the realization of their objectives . without people , the concepts of organization and management would become meaningless ( 1 ) . therefore , professional satisfaction on the part of manpower is one of the most important factors in the efficacy of organizations . the issue of professional satisfaction has been studied repeatedly with respect to various organizations since the 1920s , and , according to many experts , it is one of the most challenging organizational concepts and the basis behind the many management policies adopted to improve organizational effectiveness ( 2 ) . health care organizations and treatment centers are responsible for maintaining the health of all citizens in a society , and nurses have a key role . their success in fulfilling this role depends on the measures taken by competent managers who recognize the prominent factors involved in the activities of nurses and seek to create favorable work conditions in order to enhance their professional satisfaction ( 1 ) . previous studies have shown that nurses should demonstrate a passion for their work and have the necessary proficiency and expertise to achieve the highest level of efficiency ( 2 ) . it could be argued that any person would provide better services if he or she were motivated and enthusiastic about the work . despite the great significance of the professional satisfaction of nurses due to its considerable impact on them , their patients , their organizations , and the very profession of nursing ( 3 ) , most previous studies have reported the satisfaction level to be low among nurses ( 4 ) . the results from the study conducted by lou et al . indicated that only half of the nurses in the study were moderately or highly satisfied with their work ( 5 ) . another study was conducted to compare the level of professional satisfaction among different groups of people in korea , and it was found that only 41.5% of nurses were satisfied with their work , whereas the percentages for other experts working at hospitals , social workers , and middle school teachers were 50.1 , 58.2 , and 89% , respectively . these statistics indicate that there is a lower level of satisfaction among nurses than other professionals ( 6 ) . various studies have been conducted regarding the extent of professional satisfaction among the nurses , and , sadly , all have indicated low degrees of satisfaction ( 7 ) . most nurses who work in internal medicine and surgical departments ( 54.4% ) demonstrated low levels of professional satisfaction ( 8) . overall , these studies suggested that the situation concerning nurses professional satisfaction is unacceptable . it should be noted that a lack of sufficient concern towards professional satisfaction will result in serious consequences in the long run . it could give rise to rebellion , diminished feelings of responsibility , and eventually resignation from the job ( 9 ) . professional dissatisfaction among nurses could result in emotional detachment , depression , anger , evasion from work , and ineffectiveness ( 10 ) . according to a qualitative study done in 2010 in india , the reasons nurses quit their jobs were determined to be professional exhaustion , stress at work , and lack of a friendly environment , all of which contributed to their diminished professional satisfaction . in addition , it was indicated that a strong relationship exists between professional dissatisfaction and quitting work ( 11 ) . according to released statistics , the insufficient number of available nurses around the world is a tangible phenomenon . in australia , for instance furthermore , according to estimations , by 2010 , canada will need an additional 113,000 nurses , and , by 2020 , the united states would incur a 20% shortage in the number of nurses ( approximately 581,500 nurses ) ( 12 , 13 ) . in iran , one of the most problematic issues in nursing is the shortage of nursing staff . the shortage of nursing staff has different causes , including job dissatisfaction and organizational and sociocultural factors ( 14 ) . in iran , many studies have reported that nurses are not happy because of the shortage of nursing staff , high numbers of patients , the lack of independence and authority , the lack of appreciation and acknowledgment , low income , heavy workloads , low level of involvement in management , disagreements with physicians , managerial issues , and a lack of appropriate work conditions in hospitals ( 15 ) . approximately 45% of nurses in iran regret having become nurses , whereas 43.9% of them feel disappointed and dissatisfied that they became nurses . the most challenging problem in nursing in iran is the shortage of nurses working in hospitals because of their dissatisfaction and lack of motivation , and , unfortunately , most nurses in iran would like to change their work ( 14 ) . because satisfaction with nursing is related to improving the health system and patient satisfaction , this study was conducted to assess how the satisfaction of nurses with their work can be improved , their situation in hospitals can be improved , and also how patients satisfaction could be increased ( 15 ) . the objective of this research was to determine iranian nurses experiences of the concept of professional satisfaction . this was a qualitative study that was conducted with a targeted sampling of 10 nurses ( 4 men and 6 women ) , with a minimum of one year and a maximum of 25 years of work experience . all participants had earned a bachelor s degree and cared for patients directly as staff members in general or intensive care units at public or private hospitals in tehran . in this study , data were collected using semi - structured interviews . before conducting the interviews , the objective of the study was explained to the participants , and their consent to participate in the study was obtained in writing . all interviewees were ensured that their identities would not be disclosed in the reports and that they could have access to the results . the interviews were conducted in a place where the participants felt comfortable , such as in the break room in the hospital where they worked . in addition to covering the general questions of the study , attempts were made to personalize each interview depending on its flow . the qca is an analytical procedure that is utilized to provide a subjective interpretation of textual data contents ( 16 ) . in this method , codes and categories are extracted from raw data directly and inductively through a systematic categorization process ( 17 ) . content analysis is much more than the extraction of objective contents out of textual data , i.e. , key concepts and hidden patterns may be revealed from the content of the participant - generated data through this method ( 16 ) . in the qca , data collection and analysis are done simultaneously ( 17 ) . in this method , the researcher collects and analyzes data back and forth in order to collect new data to be able to provide answers to the research questions ( 16 ) . an analytical unit is a part of text that can be analyzed to help achieve the research objectives . initial codes were extracted from meaning units , which are important and reliable parts of the analytical units . initial codes could contain either the exact content of the participant s interview or the abstract of the content . based on their similarities and differences , the initial codes were reduced to sub - categories , which , in turn , were used to abstract categories and key concepts ( 17 ) . in the first step of this study , the researchers listened to the interviews , and then they wrote the results of the interviews word for word . in the next step , then , based on similarities and differences , the initial codes were reduced to sub - categories , abstracts were prepared , and the key codes were identified . in order to achieve reliability in this research , the data were controlled by the participants and investigated by an investigation team . in addition to restoring the speech and experiences of the participants during the interviews by replaying them , the researcher gave them full typed texts of the first four interviews together with their initial codes to those with whom he or she had conducted the initial interviews for verified or revision . in addition , coding and initial categories with respect to content analysis were submitted to the researcher s supervisor , and verification was received on her / his part regarding the implementation , coding , and initial categories . in order to increase the reliability of the data , the researcher attempted to establish a deeper relationship with the participants and also allocated sufficient time for the interviews . the researcher also attempted to provide a friendly atmosphere in order to gain the participants trust , so that more accurate data and information could be elicited . the participants were willing to participate in the interviews , and they express their feelings , experiences , and thoughts with no fear of censorship or pressure . also , to increase the degree of reliability , the researcher attempted to avoid including his personal preferences in the study . from the qualitative analysis of the data collected from the interviewees , fair conduct was extracted as the turning point and basis upon which professional satisfaction of nurses could be achieved , which , in turn , consisted of three sub - categories , i.e. , 1- expectation of fairness in social - professional settings , 2- expectation of fairness in receiving professional benefits , and 3- expectation of fairness in the area of professional interactions . many of the participants maintained that there was a gross distinction between what the society said and how people reacted , in that , even though nurses were academically educated , cared for patients , and dealt with their problems in a never - ending manner , they were not regarded by patients , their families , or even society as having an equal status to those engaged in other professions , including medicine . one of the nurses expressed her feelings by saying , nursing is a difficult job . you study for four years at university just like any other discipline , you get a bachelor degree , yet at the end , people regard nurses as a bunch of illiterates with no independence or literacy whatsoever , and who merely obey what doctors command them to do . society regards nurses as mere observers who tend their patients , but view doctors as even those engaged in other fields such as accounting , engineering , etc . with equivalent bachelor degrees enjoy a better salary and are held in greater esteem than the people employed in nursing . yet another participant stressed the significance of professional satisfaction in making nurses continue to do their jobs ; i become very upset when i see the respectful way by which people of other professions who have bachelor degrees just like we do , are referred to ( e.g. the engineer man / lady ) , or when i compare the level of respect shown to doctors with that shown to nurses . people do not have high regards for the community of nurses ; they view them as ill - humored or bad people . , nurses are shown wearing dressed with all kinds of colors running after doctors . yet , in those same movies , doctors are displayed as distinguished people helping patients . most of the participants stated that unfair practices at work with respect to financial matters had left them disappointed and lowered their professional satisfaction , in such a way that their attempts were directed towards quitting their job or finding a new one with better salary . concerning this matter , one of the nurses stated , in today s world my basic needs have to be fulfilled , and currently a nurse s salary is not in any sense proportionate to that earned by those working in other medical sectors . on lack of a financial fairness , another nurse maintained , it might be true that a doctor s education is higher than that of a nurse , but a nurse devotes more of his / her time to patients , in return . the thing that bothers the iranian nurses the most is their awareness of all professional benefits that nurses working in other countries enjoy . in advanced countries , nursing has a high status , while this is not the case for iran , and that is exactly the reason behind many dissatisfactions . a male nurse expressed his discontent of nursing as follows , with 35 years of age , i suffer from diabetes due to the stress at work , which i try to handle with strict regiments and such . i am trying to change my job , since no matter how hard i work i end up having nothing . i have to hold two or three jobs to be able to buy a house . yet , this is not the case for those engaging in other professions , with little or no difference than nursing from an academic point of view ? another male nurse expressed his discontent of nursing as follows , i know for a fact that , nurses in the great britain earn an equal amount of salary compared to surgeons , which is something to reflect upon . that how the monthly income of a nurse is equal to that of a surgeon is something that requires serious contemplation . furthermore , they merely work one shift and tend two patients all night long , unlike in iran , where two nurses tend 45 patients simultaneously . another nurse with two years experience said , with rising inflation , doctors salaries and fees increase day by day , whether officially or unofficially , and the authorities regard this as their inalienable right . however , nurses live off of a petty salary and then they complain about the few number of working nurses , and that why it is that they continually quit their job ! most of the participants viewed a number of factors as influencing the satisfaction they felt in doing their job , the most important of which was the viewpoint and attitude of hospital managers towards nurses . one of the participants in this study voiced her / his opinion on this matter as such , the treatment received by nurses from hospital management is unfair . they do not show them the level of respect they deserve , yet always support doctors rights . one of the nurses pointed to decision making by doctors or nurses as one of the factors causing professional dissatisfaction , during all these years that i have worked as a nurse , i have seen with my own eyes that many decisions are made for us by those in the medical department . they are just not fair ! another participant stated , justice is not observed at iranian hospitals . one of the nurses working in the department of psychiatry pointed to the following as one of the reasons behind his / her professional satisfaction , since , as you know , working with mentally - challenged patients drives one to exhaustion . yet , despite all these hardships , our relationship with our managers is at a very high level . they act very reasonably , and this brings about satisfaction and makes us care for patients with greater motivation . the belief in establishing a kind of justice on the part of some of the participants was so strong that they viewed the existing difficulties in the nursing profession to be more bearable if some sense of justice was in place . the following statement could be brought to attention in this regard , despite the high level of physical and mental exhaustion that our work brings about , a fair treatment by managers and equality with respect to observing the rules would make the hardships of this job , including lack of sleep , intensity of work , low salary , etc . many of the participants maintained that there was a gross distinction between what the society said and how people reacted , in that , even though nurses were academically educated , cared for patients , and dealt with their problems in a never - ending manner , they were not regarded by patients , their families , or even society as having an equal status to those engaged in other professions , including medicine . one of the nurses expressed her feelings by saying , nursing is a difficult job . you study for four years at university just like any other discipline , you get a bachelor degree , yet at the end , people regard nurses as a bunch of illiterates with no independence or literacy whatsoever , and who merely obey what doctors command them to do . society regards nurses as mere observers who tend their patients , but view doctors as even those engaged in other fields such as accounting , engineering , etc . with equivalent bachelor degrees enjoy a better salary and are held in greater esteem than the people employed in nursing . yet another participant stressed the significance of professional satisfaction in making nurses continue to do their jobs ; i become very upset when i see the respectful way by which people of other professions who have bachelor degrees just like we do , are referred to ( e.g. the engineer man / lady ) , or when i compare the level of respect shown to doctors with that shown to nurses . people do not have high regards for the community of nurses ; they view them as ill - humored or bad people . , nurses are shown wearing dressed with all kinds of colors running after doctors . yet , in those same movies , doctors are displayed as distinguished people helping patients . most of the participants stated that unfair practices at work with respect to financial matters had left them disappointed and lowered their professional satisfaction , in such a way that their attempts were directed towards quitting their job or finding a new one with better salary . concerning this matter , one of the nurses stated , in today s world my basic needs have to be fulfilled , and currently a nurse s salary is not in any sense proportionate to that earned by those working in other medical sectors . on lack of a financial fairness , another nurse maintained , it might be true that a doctor s education is higher than that of a nurse , but a nurse devotes more of his / her time to patients , in return . the thing that bothers the iranian nurses the most is their awareness of all professional benefits that nurses working in other countries enjoy . in advanced countries , nursing has a high status , while this is not the case for iran , and that is exactly the reason behind many dissatisfactions . a male nurse expressed his discontent of nursing as follows , with 35 years of age , i suffer from diabetes due to the stress at work , which i try to handle with strict regiments and such . i am trying to change my job , since no matter how hard i work i end up having nothing . i have to hold two or three jobs to be able to buy a house . yet , this is not the case for those engaging in other professions , with little or no difference than nursing from an academic point of view ? another male nurse expressed his discontent of nursing as follows , i know for a fact that , nurses in the great britain earn an equal amount of salary compared to surgeons , which is something to reflect upon . that how the monthly income of a nurse is equal to that of a surgeon is something that requires serious contemplation . furthermore , they merely work one shift and tend two patients all night long , unlike in iran , where two nurses tend 45 patients simultaneously . another nurse with two years experience said , with rising inflation , doctors salaries and fees increase day by day , whether officially or unofficially , and the authorities regard this as their inalienable right . however , nurses live off of a petty salary and then they complain about the few number of working nurses , and that why it is that they continually quit their job ! most of the participants viewed a number of factors as influencing the satisfaction they felt in doing their job , the most important of which was the viewpoint and attitude of hospital managers towards nurses . one of the participants in this study voiced her / his opinion on this matter as such , the treatment received by nurses from hospital management is unfair . they do not show them the level of respect they deserve , yet always support doctors rights . one of the nurses pointed to decision making by doctors or nurses as one of the factors causing professional dissatisfaction , during all these years that i have worked as a nurse , i have seen with my own eyes that many decisions are made for us by those in the medical department . they are just not fair ! another participant stated , justice is not observed at iranian hospitals . one of the nurses working in the department of psychiatry pointed to the following as one of the reasons behind his / her professional satisfaction , since , as you know , working with mentally - challenged patients drives one to exhaustion . yet , despite all these hardships , our relationship with our managers is at a very high level . they act very reasonably , and this brings about satisfaction and makes us care for patients with greater motivation . the belief in establishing a kind of justice on the part of some of the participants was so strong that they viewed the existing difficulties in the nursing profession to be more bearable if some sense of justice was in place . the following statement could be brought to attention in this regard , despite the high level of physical and mental exhaustion that our work brings about , a fair treatment by managers and equality with respect to observing the rules would make the hardships of this job , including lack of sleep , intensity of work , low salary , etc . more bearable . as was discussed , one of the main components of professional satisfaction is fair conduct , which consists of three sub - categories , i.e. , expectation of fairness in social - professional settings , expectation of fairness in receiving professional benefits , and expectation of fairness in the area of professional interactions . the subject of fairness is one of the few fundamental topics relevant to various spheres of science and philosophy , and it can be applied to any situation in terms of people s needs and expectations . the concept of fairness is integrated with moral and social values and within the sphere of morality , in particular social morality , supporting fairness and equity is of the most fundamental moral values . researchers regard the establishment of justice as one of the most important factors affecting professional satisfaction ( 18 ) . one of the long - term challenges of the nursing profession is its public image ( 19 ) . there are various ups and downs to nursing due to the challenging nature of this profession from the initial education at university until retirement . dropping out of university , although low salaries and benefits and professional exhaustion are among the factors causing discontent among nurses , according to conducted studies , lack of consideration towards the status and public image of the nursing profession is responsible for 70% of professional dissatisfaction ( 20 ) . generally , people form opinions or mental images of other individuals based on their look , way of clothing , reaction , communication , behavior and attitude ( 21 ) . the image is the perception or assumption that the general public makes about an individual , profession , or organization . the people within those organizations use this image as a tool to communicate with people , and they , in turn , interact with those organizations based on the projected image , whether positive or negative ( 20 ) . the social status of a profession and the society s perspective towards it are of effective motivational factors influencing the extent of professional satisfaction , which , in turn , leaves an impact on the attitude shown by individuals in fulfilling their professional functions . these statuses include placing a high value on a profession and its required proficiencies and the value and status of a profession in a particular society . whenever the status is damaged on a societal scale , the status of the profession is also diminished ( 22 ) . in nursing , one of the most important concerns with regards to professional status is lack of a high value and social status within society . with regards to the importance of this issue , stevens stated that public opinion acts very strongly in determining the social structure and norms of a society ( 23 ) . a positive public image will leave a considerable effect on gaining social and situational support . as was understood from the findings of this study , most nurses in this research regarded professional satisfaction as manifested through expectation of fairness in social - professional settings . regarding the second important finding of this study , i.e. , the expectation of fairness in receiving professional benefits , the participants stated that unequal salaries among the personnel of the medical department and a gross distinction between the payments to the medical staff have been sources of disappointment and dissatisfaction . markovsky was among the first theoreticians to recognize the significance of the issue of salary satisfaction in observing fairness ( 24 ) . therefore , the findings of this study , that is , feeling a sense of fairness in receiving professional benefits has an intermediary role in bringing professional satisfaction , is in line with markovsky s theory . furthermore , expectation of fairness in receiving professional benefits gives the indication that , if individuals deem a condition to be unfair , they will have an emotional response to it ( 25 ) . yet another important finding of this study was the expectation of fairness in professional interactions . the participants of this study stated that the treatment they receive from managers is unfair and disrespectful compared to other health teams . defined interactional justice as the feeling experienced by employees when interacting with their supervisor and state ; cooperation with supervisors and other members of the work team and respectful behavior , and providing the required information would result in a high satisfaction level among employees ( 25 ) . kim et al . maintained that interactional fairness , fair treatment by managers towards employees , and instituting behavioral justice significantly affect professional satisfaction ( 26 ) . regarding the issue of fairness , the results of the present study were in line with those obtained by lou et al . ( 21 ) , in which it was demonstrated that british nurses were more displeased compared to australian nurses . the british nurses felt that their professional statuses were lower , their relationships with hospital managers were weaker , and their work conditions were less favorable than those of australian nurses . the results of this study confirm the point that the existence of a relative social status to profession , and of justice within the work environment , and also respectful treatment towards nurses by management . generally speaking , fair conduct is an integral part of professional satisfaction in the nursing profession , which could encourage nurses to employ the related measures and interventions with a higher degree of motivation ( 18 ) . furthermore , a review of the previous studies indicated that management and administrative experiences , which refer to organizational structures , such as ( de)centralization and fair and transparent organizational policies and procedures are among the factors affecting professional satisfaction . researchers have found out that decentralization and all kinds of cooperative management models are more to nurses liking , and that they resent being looked at with indifference , and this leads to a lower professional satisfaction level in their eyes ( 12 ) . the study conducted by farsi et al . indicated that the low public image of nursing in the iranian society has caused nurses to feel unappreciated and disrespected ( 16 ) . as was demonstrated in other studies conducted in iran , the motivation level of iranian nurses in fulfilling their professional tasks , which to a large degree is dependent upon the public image , is not acceptable ( 20 ) . in general , since professional satisfaction is a single concept that can not be deconstructed , it is possible to be defined by individuals in vague manners . therefore , professional satisfaction can be viewed as a general feeling , or as the attitude taken by an individual with respect to part of her or his job . overall , it is possible to improve the professional satisfaction of nurses through emphasizing the ( in)visible aspects of professional satisfaction , such as fair conduct , removing tension - generating factors , improving the attitude of society towards the nursing profession , establishing justice among all hospital staff , improving communication , organizing the nursing profession , and enhancing the payment system and making it fair .

introductionthe professional satisfaction of staff is one of the most challenging organizational concepts that can enhance the efficiency level of organizations . in a similar vein , the professional satisfaction of nurses is of considerable importance , in that , professional dissatisfaction among nurses could result in emotional detachment , depression , anger , evasion from work , and inefficacy and would negatively impact the organization s work rate . the aim of this study was to understand iranian nurses experiences of the concept of professional satisfaction.methodsthis was a qualitative study conducted with a targeted sampling of 10 nurses ( 4 men and 6 women ) in 2015 . the data were collected through conducting in - depth interviews , and textual data were analyzed subsequently using the qualitative content analysis ( qca ) method.resultsthe findings of this study pointed to fair conduct , which was comprised of three sub - categories , i.e. , expectation of fairness in social - professional settings , expectation of fairness in receiving professional benefits , and expectation of fairness in the area of professional interactions.conclusionsthere are various ups and downs in nursing due to the challenging nature of the profession , from the initial education at the university until retirement . according to the findings of this study , a lack of fairness in social - professional settings , a lack of fairness in receiving professional benefits , and a lack of fairness in the area of professional interactions were among the factors that have great impacts on the degree of professional dissatisfaction among nurses .

1. Introduction 1.1. Background 1.2. Statement of the problem and study logic 1.3. Objective 2. Material and Methods 3. Results 3.1. Expectation of fairness in social-professional settings 3.2. Expectation of fairness in receiving professional benefits 3.3. Expectation of fairness in the area of professional interactions 4. Discussion 5. Conclusions

PMC5026097

the success of restorative procedures depend on effective removal of infected dentin prior to the placement of the restorative material . failure to mechanically remove any carious portion and not being able to achieve complete disinfection may lead to microleakage , increased pulp sensitivity , pulpal inflammation , and eventually may cause secondary caries , thereby making it necessary to replace the restoration . therefore , application of cavity disinfectant before tooth restoration is gaining acceptance . the main objective of adhesive dentistry is to create an effective , durable union between tooth structure and the restorative material . the resin to dentin adhesion occurs through the infiltration into and polymerization of hydrophilic resins within the collagen matrix exposed through acid decalcification of dentin , thus forming a hybrid layer . the durability of the dentine adhesive interface is directly related to the quality of hybrid layer and the hydrophilic nature of the adhesive . in case of etch and rinse adhesives , it is pertinent that the adhesive resin monomer penetrates the acid - etched exposed dentine collagen fibrils . certain mechanisms have been proposed to improve dentin adhesion i.e. , adjunctive collagen pretreatment , matrix metalloproteinases ( mmp ) inhibitors , etc . different types of mmps have been identified from human dentin , including mmps 2 , 3 , 8 , 9 , 20 . both mmps and cysteine cathepsins contribute to the degradation of denuded collagen within the hybrid layer . chlorhexidine ( chx ) has been widely used as a cavity disinfectant because of its antimicrobial property . it also has an inhibitory effect on the mmps ( against mmps 2 , 8 , 9 ) in the dentin . this effect can be useful in preventing collagen degradation and disintegration of the bonding interface over time . in recent years , the potential for the inhibition of mmps by substances derived from natural products has gained increasing attention . a. barbadensis miller ( aloe vera ) is a short succulent herb resembling a cactus , with green fleshy , spiny , and well marginated leaves filled with a clear viscous gel . a. vera has been used to relieve thermal burn , sunburn , and promote wound healing and has antimicrobial activity and can help stimulate the body 's immune system . recent study has revealed a. vera exhibits mmp inhibitory effect against mmp 2 and 9 . conducted a randomized clinical trial in 2015 and advocated the use of a. vera as cavity disinfectant . there have been many researches to prove antimicrobial efficacy a. vera against caries causing microorganisms , i.e. , streptococcus mutans . therefore , we intended to use a. vera as a cavity disinfectant in our study . in order to use these agents for extending the longevity of resin - dentin bonds , it is necessary to first evaluate whether these agents interfere with the dentin bond strength following their use to pretreat the acid - etched dentin . the aim of our study was to evaluate a. vera for resin - dentin bond stabilization . this was the first study of its kind to test a. vera for resin - dentin bond stabilization . sixty freshly extracted non carious human mandibular molars were thoroughly cleaned and stored in 0.1% thymol until use . they were horizontally cut using diamond disk ( markus ink . , michigan , usa ) in a high - speed hand piece under air and water spray ; the long axes of the teeth were perpendicular to the surfaces cut . after the removal of enamel , the mid coronal dentin was exposed . the dentine surface was examined for the lack of enamel or pulp tissue under stereomicroscope ( olympus ; zoom type , japan ) . the sections of the teeth including the roots were embedded in auto polymerizing acrylic resin to form cylinders 2.5 cm in diameter and 5 cm high . dentin surfaces were flattened with 1200 grit silicon carbide paper under running water , so that a very smooth surface and to obtain a standardized smear layer . acid etching of the exposed dentine was performed for 15 s with 37% phosphoric acid gel ( scotchbond etchant , 3 m espe , st . the teeth in experimental groups were treated with one of the following cavity disinfectants i.e. group 2 : 2% chx solution was prepared from dilution of 20% chx solution using distilled water ( basic pharma , gujrat , india ) . group 3 : a. barbadensis miller ( a. vera ) solution was prepared using a. vera powder of 99% purity and dissolving 20 mg of a. vera powder in 10 ml of distilled water ) . the acid etched dentin was pretreated with 2% chx in group 2 and a. barbadensis miller solution in group 3 for 30 s , active application with a brush applicator ( microbrush international , wi , usa ) and the excess removed with cotton pellet prior to the application of bonding agent ( adper single bond 2 , 3 m espe , st . adhesive tape with a 3 mm diameter hole in it was used to define the bonding agent . the dentin surfaces of the teeth were then dried with air for 10 s , resin composite was applied in 56 increments ( filtek z 350 , 3 m espe , st . paul , mn , usa ) with the aid of polyethylene tubes ( 3 mm diameter , 2 mm height and 0.5 mm thickness ) and individually light cured for 40 s using light curing unit spectrum 800 ( dentsply , caulk , milford , usa ) with an output of 600 mw / cm . the teeth were then stored in distilled water at room temperature for 24 h. for shear bond strength testing , each tooth was secured in a specially designed attachment jig to hold the specimens to the universal testing machine ( instron , admet , enkay enterprises , new delhi ) . load was applied by the testing machine through a wire loop adjusted to the bonded interface at a cross head speed of 0.5 mm / min . shear bond strength in mpa was calculated from the peak load at failure divided by the specimen surface area . after testing , the fracture modes were evaluated under a stereomicroscope ( olympus , zoom type ) and classified according to the predominant mode of fracture as adhesive fracture at the resin - cement dentin interface , cohesive fracture in the resin cement , cohesive fracture in dentin , or mixed adhesive and cohesive fracture in the resin cement . data were analyzed using one - way anova and post hoc tukey test for multiple comparison between the groups . the test was done at a 0.05 level statistical significance [ tables 1 and 2 ] . shear bond strength to dentin ( meanstandard deviation ) comparison of immediate mean shear bond strength values ( mpa ) of all groups from the results analyzed , it was noted that there was statistically significant difference between the groups control and chx and control and a. vera ( p < 0.05 ) , specifically values of control < chx and control < a. vera ( p < 0.05 ) . however , there was no statistically significant difference between chx and a. vera ( p > 0.05 ) . hence , the following result for the shear bond strengths to dentin was obtained : control < chx a. vera . fracture analysis indicated that most specimens showed adhesive fracture after 24 h [ table 3 ] . acid etching creates a low ph which activates the dentin mmps in the presence of zinc and calcium ions . it has been advocated that to improve the durability of restorations , pretreatment of acid - etched dentin with mmp inhibitors should be carried out . chx increased the immediate shear bond strength in this study , which was in support of the study by brackett et al . , the use of chx after etching prevents collagen fiber degradation and preserves the hybrid layer due to its inhibitory effect on mmps , thereby increasing the bond strength . yet few studies have showed that applying chx before acid etching did not significantly affect the bond strength . this increase in the immediate bond strength can be attributed to the mmp inhibitory action of chx . the present study was conducted from agents derived from natural products that has been recently reported to possess anti - mmp potential , especially against mmp-2 and mmp-9 . the results of this study revealed that the application of a. barbadensis miller to acid - etched dentine improves the longevity of resin dentin bonds by inhibiting mmps . the purpose of this study was to introduce new herbal product with possible potential for the inhibition of mmps in order to maintain the dentin adhesive interface . the shear bond testing was performed only after 24 h of storage as this was the first study of its kind and it aimed to determine whether the tested concentration of the herbal product would be really effective and thereafter conduct longitudinal studies . as if immediate bond strength was negatively affected by the use of a. vera , there would have been no point for conducting long duration studies . although the use of a. barbadensis miller on acid - etched dentin prevents the degradation of collagen and improves the longevity of composite restorations , it is imperative that they do not adversely affect adhesive bonding to dentin . as pretreatment with the herbal used was able to maintain immediate dentin bond strength , there is not enough evidence to reject the null hypothesis . the ability of this agent to improve the durability of resin - dentin should be evaluated in future studies . within the limitations of this in vitro study , it may be stated that the use of chx or a. barbadensis miller , as pretreatment agents of acid demineralized dentin collagen , has no adverse effect on the shear immediate bond strength of a two - step etch and rinse adhesive to dentin . further , in vitro and in vivo studies are still warranted to evaluate the effect of a. barbadensis miller for cavity disinfection .

introduction : the main objective of adhesive dentistry is to create an effective , durable union between the tooth structure and restorative material . however , degradation of adhesive dentine interface remains largely responsible for the relatively short lifetime of tooth colored resin restoration.aim:the aim of the study is to compare the dentin collagen stabilization property of chlorhexidine ( chx ) and aloe barbadensis miller using shear bond strength testing.materials and methods : occlusal reduction was done in sixty extracted human mandibular molars to expose the mid coronal dentin and divided into three groups n = 20 . following the surface pretreatment ( group 1 = control , group 2 = chx , group 3 = aloevera ) , dentine bonding agent and composite resin were applied and cured . the specimens were then subjected to shear bond strength testing.results:from the results analyzed , it was noted that there was statistically significant difference between the groups control and chx and control and a. barbadensis miller ( p < 0.05 ) , specifically the values of control < chx and control < a. barbadensis miller ( p < 0.05 ) . however , there was no statistically significant difference between chx and a. barbadensis miller ( p > 0.05 ) . hence , the following result for the shear bond strengths to dentin was obtained : control < chx a. barbadensis miller.conclusion:chx and a. barbadensis miller , as pretreatment agents of acid demineralized dentin collagen , has no adverse effect on the immediate shear bond strength of a two - step etch and rinse adhesive to dentin .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest

PMC4013981

five thoroughbred horses ( two females and three geldings ) , aged 4 to 7 years old with body weight of 493.8 26.6 ( mean sd ) kg , belonging to the equine research institute , japan racing association , were assigned to the experiment . these horses had been routinely grazed for 2 months and specially trained for treadmill exercise prior to this study . hydrogen - rich water ( hw ) was made by special equipment ( high density hydrogen water , melodian co. , tokyo , japan ) for the present study . the hw consisted of filtrated water ( ph=6.82 ) with a concentration of molecular hydrogen ( h2 ) higher than 1 ppm . fresh hw that was made about 3 hr before the treadmill exercise was stored in an aluminum bag ( 10 l in volume ) and tightly sealed in order to prevent hydrogen gas escape . two liters of hw were administered into the esophagus via a nasogastric catheter 30 min before the treadmill exercise . for the placebo trial , the same volume of normal water without hydrogen was administered by the same method . horses were exercised with increment of load every 2 min at speeds of 1.6 , 3.6 , 7 , 10 , 12 and 13 m / s until they were exhausted . the first treadmill exercise was performed after placebo administration and the second treadmill exercise was performed after hydrogen - rich water ( hw ) administration , after one - week interval . two litters of placebo water or hw were administered to the esophagus by a nasogastric catheter in each horse at 30 min before the treadmill exercise . blood samples ( serum ) were collected 4 times : 30 min before the exercise , immediately before the exercise , post - exercise , and at 30 min after the exercise .. treadmill exercise on a 6% incline was performed by each horse . each horse was exercised on the treadmill with stepwise increases of exercise intensity every 2 min , at treadmill running speed of 1.6 , 3.6 , 7 , 10 , 12 , 13 and 14 m / sec , until the horses became thoroughly exhausted . by thoroughly exhausted , we mean that the horses were too exhausted to maintain their position at the front of the treadmill with humane encouragement . horses were exercised with increment of load every 2 min at speeds of 1.6 , 3.6 , 7 , 10 , 12 and 13 m / s until they were exhausted . the first treadmill exercise was performed after placebo administration and the second treadmill exercise was performed after hydrogen - rich water ( hw ) administration , after one - week interval . two litters of placebo water or hw were administered to the esophagus by a nasogastric catheter in each horse at 30 min before the treadmill exercise . blood samples ( serum ) were collected 4 times : 30 min before the exercise , immediately before the exercise , post - exercise , and at 30 min after the exercise . the first treadmill exercise was performed after the placebo administration , and the second treadmill exercise was performed after the hw administration , after one week interval . in order to measure serum d - roms and bap , venous blood samples of 10 ml the blood samples were collected from the jugular vein immediately before the administration of placebo water ( distilled water ) or hw , 30 min before the treadmill exercise . blood samples were also collected at pre - exercise , immediately before the onset of treadmill exercise , at post - exercise , immediately after treadmill exercise , and at 30 min after the end of treadmill exercise . in addition to the samples , blood was collected at 10:00 and 13:00 on a day without treadmill exercise from all five horses to determine their background levels of d - roms and bap . the measurement of d - roms was performed using a colorimetric method of final derivatives , i.e. , hydroperoxide produced by free radicals , in which hydroperoxide in the serum reacts with n , n - diethyl - p - phenylenediamine to form [ a - nh2 ] using a free radical analyzer ( free carpe diem , wismerll , tokyo , japan ) . this d - roms test was invented and developed by carratelli , m. and the validity of this method has been demonstrated by comparisons with the results of the electron spin resonance ( esr ) method , which serves as a direct measurement of unpaired electrons [ 2 , 39 , 40 ] . bap was determined by color reaction of thiocyanate which reflects reduction potency from fe to fe due to electrons ( e ) in the blood using the same free radical analyzer . the d - roms , bap and bap / d - roms values were statistically evaluated using two - way repeated - measures analysis of variance ( two - way repeated - measures anova ) . in addition , the statistical significance of differences was tested by two - factor anova with only one observation in each cell at pre - exercise , post - exercise and 30 min after exercise for each of the placebo and hw groups , and the dunnet test was used to compare post - exercise and 30 min after exercise data with that at pre - exercise . wilcoxon s signed - rank test was also used to examine the data of the blood samples at 30 min before treadmill exercise , before the placebo or hw administration , in order to examine if the basal conditions were the same . for all the data , differences were considered significant if the p value was less than 0.05 . five thoroughbred horses ( two females and three geldings ) , aged 4 to 7 years old with body weight of 493.8 26.6 ( mean sd ) kg , belonging to the equine research institute , japan racing association , were assigned to the experiment . these horses had been routinely grazed for 2 months and specially trained for treadmill exercise prior to this study . hydrogen - rich water ( hw ) was made by special equipment ( high density hydrogen water , melodian co. , tokyo , japan ) for the present study . the hw consisted of filtrated water ( ph=6.82 ) with a concentration of molecular hydrogen ( h2 ) higher than 1 ppm . fresh hw that was made about 3 hr before the treadmill exercise was stored in an aluminum bag ( 10 l in volume ) and tightly sealed in order to prevent hydrogen gas escape . two liters of hw were administered into the esophagus via a nasogastric catheter 30 min before the treadmill exercise . for the placebo trial , the same volume of normal water without hydrogen was administered by the same method . horses were exercised with increment of load every 2 min at speeds of 1.6 , 3.6 , 7 , 10 , 12 and 13 m / s until they were exhausted . the first treadmill exercise was performed after placebo administration and the second treadmill exercise was performed after hydrogen - rich water ( hw ) administration , after one - week interval . two litters of placebo water or hw were administered to the esophagus by a nasogastric catheter in each horse at 30 min before the treadmill exercise . blood samples ( serum ) were collected 4 times : 30 min before the exercise , immediately before the exercise , post - exercise , and at 30 min after the exercise .. treadmill exercise on a 6% incline was performed by each horse . each horse was exercised on the treadmill with stepwise increases of exercise intensity every 2 min , at treadmill running speed of 1.6 , 3.6 , 7 , 10 , 12 , 13 and 14 m / sec , until the horses became thoroughly exhausted . by thoroughly exhausted , we mean that the horses were too exhausted to maintain their position at the front of the treadmill with humane encouragement . horses were exercised with increment of load every 2 min at speeds of 1.6 , 3.6 , 7 , 10 , 12 and 13 m / s until they were exhausted . the first treadmill exercise was performed after placebo administration and the second treadmill exercise was performed after hydrogen - rich water ( hw ) administration , after one - week interval . two litters of placebo water or hw were administered to the esophagus by a nasogastric catheter in each horse at 30 min before the treadmill exercise . blood samples ( serum ) were collected 4 times : 30 min before the exercise , immediately before the exercise , post - exercise , and at 30 min after the exercise . the first treadmill exercise was performed after the placebo administration , and the second treadmill exercise was performed after the hw administration , after one week interval . in order to measure serum d - roms and bap , venous blood samples of 10 ml the blood samples were collected from the jugular vein immediately before the administration of placebo water ( distilled water ) or hw , 30 min before the treadmill exercise . blood samples were also collected at pre - exercise , immediately before the onset of treadmill exercise , at post - exercise , immediately after treadmill exercise , and at 30 min after the end of treadmill exercise . in addition to the samples , blood was collected at 10:00 and 13:00 on a day without treadmill exercise from all five horses to determine their background levels of d - roms and bap . the measurement of d - roms was performed using a colorimetric method of final derivatives , i.e. , hydroperoxide produced by free radicals , in which hydroperoxide in the serum reacts with n , n - diethyl - p - phenylenediamine to form [ a - nh2 ] using a free radical analyzer ( free carpe diem , wismerll , tokyo , japan ) . this d - roms test was invented and developed by carratelli , m. and the validity of this method has been demonstrated by comparisons with the results of the electron spin resonance ( esr ) method , which serves as a direct measurement of unpaired electrons [ 2 , 39 , 40 ] . bap was determined by color reaction of thiocyanate which reflects reduction potency from fe to fe due to electrons ( e ) in the blood using the same free radical analyzer . the d - roms , bap and bap / d - roms values were statistically evaluated using two - way repeated - measures analysis of variance ( two - way repeated - measures anova ) . in addition , the statistical significance of differences was tested by two - factor anova with only one observation in each cell at pre - exercise , post - exercise and 30 min after exercise for each of the placebo and hw groups , and the dunnet test was used to compare post - exercise and 30 min after exercise data with that at pre - exercise . wilcoxon s signed - rank test was also used to examine the data of the blood samples at 30 min before treadmill exercise , before the placebo or hw administration , in order to examine if the basal conditions were the same . for all the data , differences were considered significant if the p value was less than 0.05 . on the days of placebo and hw administration , the mean values of d - roms immediately before administration of placebo or hw at 30 min preceding the treadmill exercise were 148.3 15.3 ( placebo ) and 152.8 9.4 ( hw ) , respectively . likewise , the mean values of bap at 30 min before treadmill exercise were 2,555.9 92.5 ( placebo ) and 2,774.1 32.7 ( hw ) , respectively . there were no significant d - roms or bap between the placebo and hw treatment trials . d - roms values of each horse at pre - exercise , post - exercise and 30 min after the treadmill exercise are shown in fig . 2fig . 2.d - roms values at pre - exercise , post - exercise and 30 min after exercise of each horse after administration of placebo and hw .. in all horses , d - roms tended to elevate at post - exercise , i.e. , immediately after the treadmill exercise . the average changes in d - roms induced by the treadmill exercise in the placebo and hw trials are shown in fig . 3fig . 3.average changes of d - roms of all horses after administration of placebo and hw.30 min : at 30 min after the treadmill exercise . data are shown as means se .. a significant difference in d - roms was found among the three measurement times of pre - exercise , post - exercise and 30 min after exercise in the placebo ( p<0.005 ) and hw ( p<0.005 ) trials . the d - roms value significantly increased at post - exercise , compared to pre - exercise , in the placebo ( p<0.005 ) and hw ( p<0.001 ) trials . no significant changes in d - roms were observed at 30 min after exercise in both the placebo and hw trials . the d - roms value at pre - exercise was lower for the hw trial ( 148 14.3 u.carr ; mean se ) than for the placebo trial ( 179 12.1 u.carr ; mean se ) , but the difference was not significant . d - roms values at pre - exercise , post - exercise and 30 min after exercise of each horse after administration of placebo and hw . average changes of d - roms of all horses after administration of placebo and hw . the bap values of each horse at pre - exercise , post - exercise and 30 min after the treadmill exercise are shown in fig . 4.bap values at pre - exercise , post - exercise and 30 min after exercise of each horse after administration of placebo and hw .. the bap value increased in all horses at post - exercise . the average change in bap values of the placebo and hw trials are shown in fig . 5.average changes of bap of all horses after administration of placebo and hw.30 min : at 30 min after the treadmill exercise . data are shown as means se .. there were significant differences among the three measurement times in the placebo ( p<0.0001 ) and hw ( p<0.0001 ) trials ; the bap values at post - exercise increased to 150% and 153% of the pre - exercise value in the placebo and hw trials , respectively . significant differences from pre - exercise were found at post - exercise ( p<0.0001 ) and 30 min after exercise ( p<0.005 ) in the placebo trial , whereas a significant difference was present only at post - exercise ( p<0.0001 ) in the hw trial . bap values at pre - exercise , post - exercise and 30 min after exercise of each horse after administration of placebo and hw . the value of the bap / d - roms ratio is shown in fig . 6.average changes of bap / d - roms of all horses after administration of placebo and hw.30 min : at 30 min after the treadmill exercise . data are shown as means se .. significant differences from pre - exercise were found in the placebo ( p<0.005 ) and hw ( p<0.05 ) trials ; the differences were greatest at post - exercise in the placebo ( p<0.001 ) and hw ( p<0.01 ) trials , and they were also significant at 30 min after the exercise in the placebo ( p<0.001 ) and hw ( p<0.05 ) trials . as for comparisons between the placebo and hw trials on d - roms , bap and bap / d - roms ratio , a significant difference ( p=0.04 ) was observed only for bap / d - roms ( two - way repeated - measures anova ) without interaction between substance ( placebo or hw ) and measurement time points ( p=0.87 ) . average changes of bap / d - roms of all horses after administration of placebo and hw . the excessive production of intramuscular or extra - muscular ros induced by intensive exercise such as supra - maximum exercise might play an important role in enhancing inflammation of the muscle . however , concurrent action of protective factors , represented by various antioxidative substances such as super oxide dismutase ( sod ) , catalase , peroxidase , glutathione ( gsh ) , homocysteine , ascorbic acid ( vitamin c ) , -tocopherol ( vitamin e ) and some minerals , might be enhanced or recruited against the oxidative stress . therefore , the balance of oxidative and antioxidative functions in tissues and blood are thought to be more important than the production of oxidative substance alone . in the present study , significant increases in d - roms and bap were induced by the intensive treadmill exercise ( figs . 3 and 5 ) , and were accompanied by elevation of the bap / d - roms ratio ( fig . 6 ) . there are several reports on the association of exercise with oxidative stress in equine . it has been suggested that biochemical parameters such as lipid hydroperoxides , which are indicative of oxidative stress , are changed by exercise , and that the changes are exacerbated during exercise at high temperature and humidity . also , the exercise - induced increase in plasma lipid peroxide was reduced by allopurinol - induced inhibition of xanthine oxidase , which resulted in the formation of ros during exercise . changes in blood contents of malondialdehyde ( mda ) and gsh were evident in race horses ( ten 3 year - old stallions ) subjected to physical exercise with a progressive strength of training , where the plasma mda and gsh significantly increased after the ride and the increase in mda was still present at 18 hr after the exercise . the present study demonstrated that the oxidative parameter ( d - roms ) and antioxidative parameter ( bap ) in the blood were clearly and simultaneously elevated by intensive treadmill exercise of thoroughbred horses . the finding of increase in d - roms may be supportive of the finding of increased lipid hydroperoxidase in horses after exercise [ 25 , 26 ] . moreover , in the present study the marked increase in bap observed at post - exercise and rapid return to the pre - exercise level at 30 min after the exercise is worthy of note . this evidence implies that antagonistic action to strong oxidative stress is designed to onset in rapid time - course and bap recovery to the pre - exercise level occurs within a short period after the exercise . this antioxidative function may be reinforced by exercise training of thoroughbred horses because the antioxidative capacity has been suggested to be improved by exercise training . some evidence of the efficacy of dietary supplementation with antioxidants has previously been presented . the antioxidative influence of vitamin e and selenium supplementation in 3-year - old stallions has been reported . the antioxidant capacity , total antioxidant activities and thiobarbiturate reactive substances in thoroughbred race horses were elevated after a race , and the intravenous administration of ascorbate reduced the oxidative stress , although the creatine kinase activities were not influenced by the administration . in recent years , possible therapeutic effects of hw , which contains molecular hydrogen ( h2 ) , have been noticed in experimental studies with animals . in rats with periodontitis , the intake of hw for 4 weeks lowered serum levels of ros and oxidised low - density lipoprotein - cholesterol . in mice subjected to physical restraint stress for 10 hr per day for 6 days each week , in addition , the inhalation of h2 gas protected the brain from ischemia and reperfusion - induced damage in the rat . brain slices derived from mice administered hydrogen - rich pure water for 33 days showed significantly less superoxide formation than the control . in a rat model of sepsis , the intraperitoneal administration of hydrogen - rich saline inhibited the increase of oxidative responses , such as ros and malondialdehyde , in the hippocampus in a dose - dependent manner . also , drinking hw significantly suppressed intimal hyperplasia of the inferior vena cava in the rat . it is of interest to understand the biochemical mechanism of the antioxidative action of hw . the antioxidative action of molecular hydrogen ( h2 ) has been described in detail . h2 dissolved in culture medium of pc12 cells significantly decreased the level of hydroxyl radicals ( oh ) without decreasing the level of in super oxide anion radicals ( o2 ) or hydrogen peroxide ( h2o2 ) derived from mitochondria . the absorption of hw from the digestive organs and its time - course change in the body was described in a rat study . hydrogen in the blood was detected at 3 min after the direct instillation of saturated hw into the stomach , and the half - life of hydrogen in the muscle was estimated to be approximately 20 min after the instillation . therefore , it is assumed that the molecular hydrogen is rapidly absorbed by the digestive organs and most of it can be metabolized within one hour in the living body . in the present study , the d - roms value showed a tendency to decrease already at pre - exercise after the intake of hw , compared to the placebo intake . in addition , there were significant increases in the bap / d - roms ration in the hw trial , compared with the placebo trial , at pre - exercise , post - exercises and 30 min after exercise ( two - way repeated - measures anova ) , while no significant difference was found in the same horses at 30 min before the treadmill exercise . this finding may indicate that the hw administered into the digestive tract of the horse is rapidly distributed throughout the body after administration and acts to reduce a part of free radicals in the blood . in addition , the earlier recovery of the bap / d - roms ratio in the hw trial , compared to the placebo trial , may reflect lesser amounts of free radicals ( d - roms ) during and immediately after the exercise , as compared to the placebo trial . in conclusion , the present study demonstrated that oxidative and antioxidative changes in the blood are significantly induced by treadmill exercise of thoroughbred horses , and that the recent developed measurements , i.e. d - roms and bap tests , are useful and convenient methods for determining exercise - related physiological changes in horses . furthermore , we suggested a possibility that the supplementation of hydrogen - rich water has efficacy in lowering oxidative stress in horses .

the present study aimed to clarify changes of oxidative stress and antioxidative functions in treadmill - exercised thoroughbred horses ( n=5 , 3 to 7 years old ) , using recently developed techniques for measurement of serum d - roms for oxidative stress , and bap for antioxidative markers . also , the effect of nasogastric administration of hydrogen - rich water ( hw ) or placebo water preceding the treadmill exercise on these parameters was examined . each horse was subjected to a maximum level of treadmill exercise in which the horses were exhausted at an average speed of 13.2 0.84 m / sec . blood samples were taken 4 times , immediately before the intake of hw or placebo water at 30 min preceding the treadmill exercise , immediately before the exercise ( pre - exercise ) , immediately after the exercise ( post - exercise ) and at 30 min following the exercise . in all horses , both d - roms and bap values significantly increased at post - exercise . the increase in d - roms tended to be lower in the hw trial , as compared to the placebo trial at pre - exercise . the increase in bap was considerable at approximately 150% of the pre - exercise values in both the hw and placebo treatment trials . the bap / d - roms ratio was significantly elevated at post - exercise in both treatment trials , while a significant elevation was also observed at pre - exercise in the hw trial . bap , d - rom , and the bap / d - rom ratio tended to decline at 30 min after the exercise , except bap and bap / d - roms in the placebo trial . these results demonstrate that the marked elevation of oxidative stress and anitioxidative functions occurred simultaneously in the intensively exercised horses , and suggest a possibility that hw has some antioxidative efficacy .

Materials and Methods Horses Supplementation of hydrogen water Experimental protocol Measurements of d-ROMs and BAP Statistical analysis Results Discussion

PMC3381533

in 1902 , albert moll published his comprehensive , 650-page book rztliche ethik : die pflichten des arztes in allen beziehungen seiner thtigkeit [ medical ethics : the doctor s duties in all relations of his work ] . at this time , moll was already well known as a medical author : his 1889 book on hypnotism and his 1891 monograph on homosexuality had gone through several editions and translations , and his studies of the libido sexualis had come out in 1897/8 . with his work on medical ethics he contributed to a literary genre that had been flourishing at the end of the nineteenth century . books on medical deontology that is , on doctors professional duties had been authored in the 1890s by the berlin physician and historian of medicine julius pagel ( 18511912 ) , the berlin medical practitioner jacob wolff , the bremen psychiatrist friedrich scholz ( 18311907 ) and others . the common context for all of them was the so - called overcrowding of the medical profession in those years . doctors not only competed for lucrative private patients , but also for contracts with the health insurance organisations [ krankenkassen ] , which gave them access to the insured working class and lower - middle - class patients . books on the doctors duties were seen as one means to counteract overly competitive behaviour among medical practitioners , and to emphasise a need for collegiality and solidarity . other means to safeguard professional conduct were the disciplinary tribunals of the medical societies and doctors chambers [ rztekammern ] . in prussia , such medical courts of honour [ rztliche ehrengerichte ] , attached to the regional doctors chambers , had been established by law in 1899 . moreover , many medical societies had adopted professional codes of conduct that served as guidance for their disciplinary committees . accordingly , writing about medical ethics predominantly meant discussing the requirements of fair conduct among practitioners and how to display behaviour that would enhance the reputation of the profession in the eyes of the public . moll s medical ethics contained much of this type of professional ethics , yet he took a distinctive approach to medical ethics an approach that was fuelled by his outrage about abuses in clinical experimentation on hospital patients . one of the most notorious cases , which had recently been discussed in the daily press , was that of dr alexander strubell , who had locked up a patient with diabetes insipidus in an attic of the jena university clinics without any access to water in order to try to break the patient s allegedly , the patient became so desperately thirsty that he ended up drinking his own urine . another widely debated case was that of the breslau professor of dermatology albert neisser ( 18551916 ) , who had injected , without consent , eight female hospital patients , some of them minors and some of them prostitutes , with blood serum from syphilis patients in the hope of developing a vaccination for the disease . none of these patients suffered from syphilis at the time of the experiment ( 1892 ) ; they had been hospitalised because of other skin or venereal diseases . four of the subjects , all of them prostitutes , developed syphilis some years later , which raised the question of whether the infection had been caused by the experimental injections or through their occupation . moll was particularly upset about the many bacteriological experiments that were carried out on hospital patients around this time , including inoculation of dying patients with the germs of gonorrhoea and other infectious diseases . against this background , moll advocated a medical ethics that focused on the doctor s relationship and duties to his patients , not just his professional obligations to medical colleagues . in this article , i will first outline moll s patient - centred type of ethics and illustrate it with some moral issues that he regarded as more important than professional etiquette : truth - telling and the justifiability of deceiving patients ; the question of euthanasia ; and the so - called perforation of the fetus ie . late abortion when a natural birth turned out to be impossible . i will complement these in the second part of the paper with two cases of moll s personal involvement in publicly debated ethical issues : the above - mentioned issue of human experimentation in hospitals ; and the so - called patient trade scandal of 1908/9 , in which four professors of the berlin university clinics were accused of paying middlemen to bring them lucrative private patients . in the final sections , i will assess the impact of moll s ethics and his contemporary image as a guardian of medical ethics. in writing medical ethics , moll had received advice from two philosophers : his friend max dessoir ( 18671947 ) , with whom he also collaborated in the berlin society for experimental psychology on questions of hypnotism and psychical research ; and georg simmel ( 18581918 ) , who had been appointed to an extraordinary professorship at berlin university in 1901 . their input may , to some extent , explain why moll started his discussion of medical ethics by delineating its relationship to moral philosophy , an aspect that one does not usually find in other works of this genre at that time . moll made it clear that none of the current systems of moral philosophy could provide a basis for medical ethics because one system of thought could be used to argue for entirely contrary positions , thus failing to give reliable guidance for the doctor . average morality [ durchschnittsmoral ] of the people , a kind of intuitive common - sense ethics in contemporary society , that the doctor shared with the layperson . this did not mean , however , as moll cautioned , that the doctor s ethical decisions were determined by public opinion . with his critical stance towards moral theories , moll echoed positions of simmel , who in his introduction to ethics ( 1892 ) had argued that ethics had to be developed into an inductive science in order to progress , and that this task was separate from the normative role of ethics . moreover , as moll stressed , moral systems such as evolutionary ethics and utilitarianism could lead to conclusions that negated the role of the doctor as a healer . for instance , from an evolutionary standpoint , it might be argued that patients with hereditary illnesses or disabilities should not be treated in order not to be helped to pass on their condition to the next generation , that is , a social darwinist and eugenic position might be supported . or from a utilitarian perspective , experimentation on a dying patient might be justified in the interest of developing treatment for future patients . none of these conclusions appeared acceptable to moll , who asserted that the essential characteristic of the doctor was that of the healer who is committed to the well - being of the individual patient . while this principle of salus aegroti suprema lex was common among medical authors writing on ethics , moll gave it a characteristic , quasi - legal shape . patient relationship was a ( tacit ) contract , with duties and rights for both parties . this contract relationship implied full commitment of the doctor to his patient once he had agreed to take on the treatment , though it also gave a right to the doctor to refuse treatment in the first place except in emergencies . it also gave room for the self - determination of the patient in questions of his or her health , but simultaneously implied an expectation of compliance by the patient with the agreed treatment , at least as far as the patient s personal circumstances permitted it . this basic concept ran through the whole of moll s work on medical ethics , giving it some coherence , despite a strong tendency towards long - winded and overly detailed discussion and excessive casuistry . these latter problems were also observed by reviewers and probably limited the book s success . while two russian editions of medical ethics appeared in 1903 and 1904 , there was no further german edition a marked contrast to the success of moll s books on hypnosis and on sexology . perhaps over - ambitious in its comprehensiveness , moll s medical ethics not only covered the doctor patient relationship , or as moll called it , the relations between doctor and client , but discussed in detail the various forms of practice , as a house doctor , specialist , panel doctor , hospital doctor , country doctor , etc . ; dangerous or morally problematic actions of the doctor , such as abortion or risky surgical interventions ; economic aspects of medical practice , including the health insurance system ; the doctor s professional and private conduct ; the role of public and personal hygiene ; the doctor as an expert for the authorities , courts and insurance organisations ; the ethics of medical science and research on animal and human subjects ; and finally , questions of medical education . this scope was not dissimilar to pagel s book on doctors duties ; the latter , however , had managed to limit his ein kleiner katechismus fr angehende praktiker [ a brief catechism for incoming practitioners ] , as he subtitled it , to a mere ninety - seven pages . pagel covered , in his text , the setting up and running of a medical practice ; the doctor s conduct in society , towards patients and colleagues , including behaviour during consultations ; medical societies and professional discipline ; the relationships to pharmacists , midwives and other healers ; town and country practice , as well as the roles of poor law physician , panel doctor and officer of health ; and the doctor s fees and book - keeping . unlike moll s approach to the doctor patient relationship , pagel s was overtly paternalistic : the doctor had to be , or at least appear to be , this view was in line with that of many other medical practitioners of his generation . in the following sections , i will illustrate moll s particular ethical argumentation with the examples of deceiving patients , euthanasia and abortion . moll was aware of philosophical positions such as those of immanuel kant ( 17241804 ) and johann gottlieb fichte ( 17621814 ) , who had both argued that truthfulness was an unconditional duty in any situation , but he rejected this point of view as unsuitable for the demands of medical practice . according to moll , the doctor had to distinguish whether he had only been asked for his expert opinion on a medical question , or whether he was also in charge of the patient s care . in the former case , an exception could only be made if the prognosis was so dire that suicide of the patient had to be feared . in the second scenario , when the doctor had taken on a mandate for the patient s care , the decision about truthfulness or deception depended on the best interests of the patient . here , deception could be permissible ; for example , a patient with hysteric paralysis who had put hope in a course of magneto - therapy should be encouraged by the doctor in order to make use of the suggestive effect of such a harmless treatment . in dangerous diseases , this might also extend to misleading the relatives of the patient , who might otherwise reveal the seriousness of the condition through their words or behaviour . in this context , moll addressed particularly the question of truth - telling in incurably ill patients . tactful disclosure of the dire prognosis seemed justified in the patient s interest , if the patient had to sort out his or her personal affairs eg . by drawing up a last will or if the patient was religious ( catholic ) and would want to be given the last rites . if the doctor had merely been asked for his expert opinion , disclosure of the prognosis was rather unproblematic in these two situations . if , however , the doctor was also responsible for the patient s treatment , and perhaps , as the house doctor , for the health of the patient s family , he carefully had to weigh the potential damage caused by telling the truth against the potential benefits . moll advised the doctor in this case to involve a third person to communicate the bad prognosis , which would affect the patient s psychological condition less and would keep the doctor in his role as a source of hope and support . moll s discussion of the problem of truth - telling thus gave careful attention to what the specific contract with the patient and his / her family implied . the demands of the contract eventually determined the ethical decision . with his application of his contract theory , it was not , however , radically different from the traditional medical view in this matter , which likewise permitted restrictions on truthfulness vis - - vis the terminally ill . prominent nineteenth - century medical authors , such as christoph wilhelm hufeland ( 17621836 ) and karl friedrich heinrich marx ( 17961877 ) , had warned that telling the truth in this situation might seriously harm , even this silence of the doctors could , as karen nolte has recently shown , conflict with the intentions of nurses belonging to religious orders , who wished to inform incurably ill patients about their imminent end , so that they could be spiritually prepared for death . typical of the attitude of doctors was the advice given by the viennese surgeon robert gersuny ( 18441924 ) in his deontological booklet arzt und patient : winke fr beide [ doctor and patient : hints for both ] ( 1884 ) to be very restrictive in giving information to patients with serious illnesses . diagnoses such as tuberculosis and cancer should , in his view , not be mentioned because they would deprive the patient of hope . the doctor should even exercise caution in speaking with the relatives in such cases , because they might pass the information on to the patient or become unable to be carers as a result of their own sense of hopelessness . similarly , the munich surgeon albert krecke ( 18631932 ) claimed that a cancer patient gains nothing by knowing the nature of his malady . a doctor acts in obedience to the highest dictates of humanity if he conceals the true nature of the complaint from his patient , whilst at the same time endeavouring to effect a radical cure of the gruesome disease. in his memoirs , published in 1936 , moll portrayed his position as one of absolute truthfulness towards his patients a claim that clearly contradicted the advice that he had given in his medical ethics . within his discussion of the doctor patient relationship , moll also addressed the then controversially discussed question of euthanasia . from the perspective of social darwinism , racial hygiene and eugenics , the berlin physician alfred ploetz ( 18601940 ) had propagated , in 1895 , his vision of a society that would practise active euthanasia with morphine of weak and disabled newborns , and that would refrain from caring for the sick , the blind and the deaf - mute in order to avoid counteracting natural selection . a few years earlier , the philosopher friedrich nietzsche ( 18441900 ) , in his gtzendmmerung [ twilight of the idols ] written in 1888 had denounced the sick as parasites on society , who from a certain stage of illness that made them entirely dependent on doctors and other practitioners should be treated with social contempt . doctors , he suggested , should then be the agents of this contempt not offering prescriptions , but instead a daily dose of disgust at their patients. also in 1895 , the notorious booklet das recht auf den tod [ the right to death ] by adolf jost had been published . jost , a student of philosophy , mathematics and physics at the university of gttingen , argued here that in some cases of incurable physical or mental illness , death was desirable both from the patient s and from society s perspective . taking a utilitarian approach , he claimed that the intensity of the individual s suffering and the amount of harm caused to society by the patient s sickness could result in a negative value of the human life. since killing on demand was punishable with not less than three years imprisonment under the german penal code of 1871 ( section 216 ) , jost called for legal reform . a new law would permit , in such cases , voluntary active euthanasia performed by physicians . once this had been established in society , a further step of legal reform might extend also to euthanasia , without consent , of incurably ill mental patients . moll firmly rejected such ideas in his medical ethics . for him , any measures that deliberately shortened a patient s life were inadmissible from the point of view of criminal law as well as of morality . moll used here what we now call a slippery slope argument : if one once admitted such a right to kill the terminally ill , it might also be applied to shorten a patient s life by months and even years , if a painful and socially unproductive remaining lifespan was predicted . life should be the doctor s highest good , and death should be seen by him as the worst evil . moll s argument here was a classical piece of medical ethics , virtually the same as hufeland had used at the beginning of the nineteenth century . as michael stolberg has recently shown , however , there were already individual cases of german medical practitioners performing active life - shortening measures on terminally ill patients around 1800 . although moll very rarely made reference to hippocratic ethics in his work , he pointed to the hippocratic oath s prohibition of giving a deadly poison to a patient , even on demand . moll s position on the question of euthanasia was in line with the prevailing view of the medical profession at his time at least on the level of normative writing and debate . moll rejected traditional techniques of euthanasia , such as pulling away the pillow from underneath the dying person s head or turning the patient on their face . he did , however , concede a generous use of narcotics to mitigate pain , even if they led to unconsciousness . the objection that inducing unconsciousness was incompatible with the dying person s dignity was , in his view , invalid . moll was aware of the fact that high doses of painkillers , such as morphine , could shorten the lifespan of the dying patient , but he regarded this as a case of applying risky treatments , which were permissible according to his contract theory with the patient s consent . there was no clear sense in moll s discussion of this matter of the problematic nature of so - called indirect or double - effect euthanasia . his understanding of the concept of euthanasia was the traditional one , as part of palliative care . in fact , he referred to the berlin professor of clinical medicine johann christian reil ( 17591813 ) , who had described in detail how good nursing care could ease a patient s final hours . still , moll s contract theory also influenced his advice in this area . heroic treatment efforts , such as strong electric stimulation , which would prolong the dying patient s life for just a short time and would only increase his or her pain , should not be undertaken , even if the patient s family urgently asked for them . the contract relationship , moll emphasised , was still with the dying patient , not with the relatives , so the presumed interest of the patient in not suffering even more than necessary in their last moments was paramount . similarly , from his contract - perspective , moll condemned medical experimentation on dying patients as ethically entirely unacceptable and as a shameful act of brutality . the self - determination of the patient or , if this could no longer be exercised , the patient s presumed interest , likewise guided moll s advice in another morally contested issue , the practice of craniotomy or perforation of the fetus when a natural delivery was impossible eg . because of too narrow a pelvis . the alternative , a caesarean section , at that time still carried a relatively high risk of mortality about ten per cent for the mother . craniotomy of the fetus , which inevitably killed it , was seen as a desperate measure for saving the mother s life , although around 1900 it had a similar mortality risk for the mother as a caesarean section . jurists had argued , for example , that the killing of the fetus in this situation could be seen as self - defence or as an act in a state of emergency . moll , however , examined the issue from an ethical point of view . if the woman requested the perforation , the doctor had to follow her wish and carry out the procedure . also , if the woman was unable to express her will due to her condition , or left the decision to the doctor , the craniotomy should be performed . for moll , the fetus was not an independent human being , which meant that its life could not be balanced against the life of the mother . if , however , the mother deliberately wanted to take the risk of a caesarean section , the doctor should also follow her wishes , because self - sacrifice for a high purpose was regarded as ethically permissible . if the mother was dying and unconscious , the caesarean section could also be performed , because the mother s consent to an attempt to save the baby s life could be presumed . moll s emphasis on the wishes of the mother contrasted with that of leading gynaecologists and obstetricians of the time , such as bernhard krnig ( 18631917 ) in freiburg , who wanted to make the decision only on medical grounds , which could mean that a caesarean section was performed against the mother s will . moll s position on the question of the perforation of the fetus also matched his rather liberal views on the morality of earlier abortions . the german penal code , which at the time punished abortion with imprisonment or penal servitude for up to five years ( section 218 ) , was , in his opinion , out of tune with public sentiment . the public did not invariably regard abortion as something unethical , as moll pointed out , especially not if carried out in an early stage of pregnancy . in fact , it has been estimated that in late nineteenth - century germany , between three hundred thousand and five hundred thousand abortions were performed every year . only a small proportion of these cases , less than one thousand per year around the turn of the century ( 18821912 ) , ended with a criminal conviction . moll himself was sympathetic to women who wanted to terminate a pregnancy after rape or if they already had too many children to support . he was also not alone in his view that a doctor should not denounce the woman to the police , if he was called to attend to complications after a botched abortion . patient relationship through a breach of medical confidentiality in this situation , and the serious damage to the woman s reputation , had to be weighed against the value of reporting a crime . in 1911 , moll commented on a case in which a doctor had reported the woman concerned , with her consent , in order to initiate prosecution of the abortionist . both the abortionist and the woman had subsequently been convicted , but the latter had been able to submit a plea for clemency which was supported by the public prosecutor . in moll s view , a case like this was a matter of personal , conscientious decision - making for the doctor . many doctors , it seems , did not report abortions . moll s views on medical confidentiality were differentiated . while he leaned towards protecting patients confidence on the issue of abortion , in 1905 , he successfully supported the defence of a berlin doctor who was accused of a breach of professional secrecy under section 300 of the german penal code for having warned the relatives of a syphilitic patient of the danger of infection . we can conclude from moll s argumentation in the three examined ethical issues of truth - telling , euthanasia and abortion that the self - determination of the patient was a central factor . it competed , to some extent , with the best interest of the patient especially in the case of truth - telling but unlike other writers on medical ethics at the time , such as pagel and scholz , moll was not a paternalist . patient relationship committed him to the well - being and interests of the individual patient , and this included respect for the patient s wishes . in this sense , moll s theory of medical ethics might be seen as a forerunner of the modern concept of patient autonomy , although his practical conclusions were rather moderate and often in line with medical traditions . the patient s consent to treatment , after adequate information and advice , was a crucial element in moll s ethics . this goes some way to explain his position and actions on the issue of clinical experimentation on hospital patients , to which i will turn in the following sections . in his medical ethics , moll provided a summary of about 600 research papers that explicitly or implicitly reported non - therapeutic experimental interventions on human subjects . he had collected these publications from the international literature , as he emphasised : from germany , austria , switzerland , france , italy , england , russia , norway , sweden , denmark , romania , the united states , chile and egypt . many of the examples that moll cited indicated that patients had been harmed , or at least molested or exposed to risks , through experimentation . moreover , in many cases , the human subjects did not seem to have been informed about the nature and implications of the experiment , nor been asked for their consent to being a subject in a trial . typically , the experiments had been carried out on hospital patients or other institutionalised persons , such as orphans or prisoners . while moll s tone in his medical ethics was generally neutral and considered , his outrage about abuses in human experimentation became clearly recognisable at this point : i have observed with increasing surprise that some medics , obsessed by a kind of research mania , have ignored the areas of law and morality in a most problematic manner . for them , the borderline between human beings and animals is blurred for them . the unfortunate sick person who has entrusted herself to their treatment is shamefully betrayed by them , her trust is betrayed , and the human being is degraded to a guinea pig . some of these cases have occurred in clinics whose directors can not talk enough about i have observed with increasing surprise that some medics , obsessed by a kind of research mania , have ignored the areas of law and morality in a most problematic manner . for them , the borderline between human beings and animals is blurred for them . the unfortunate sick person who has entrusted herself to their treatment is shamefully betrayed by them , her trust is betrayed , and the human being is degraded to a guinea pig . some of these cases have occurred in clinics whose directors can not talk enough about there seem to be no national or political borders for this aberration . despite its international dimension , alerted by the scandal surrounding the syphilis experiments of albert neisser , which were discussed in the prussian parliament , the prussian minister for religious , educational and medical affairs [ minister der geistlichen , unterrichts- und medizinal - angelegenheiten ] had commissioned , in march 1899 , an expert report on human experimentation from the ministry s scientific committee for medicine [ wissenschaftliche deputation fr das medizinalwesen ] . neisser himself , as a university professor , was punished with a reprimand and a fine by the royal disciplinary court for civil servants [ kniglicher disziplinarhof fr nicht - richterliche beamte ] because he had failed to obtain consent from the parents or guardians of the minors on whom he had experimented . the syphilis serum injections given to those of his subjects who were prostitutes were , however , regarded by the court as legitimate therapeutic trials , because they might have protected the subjects against the risk of catching syphilis through their occupation . as a kind of immunisation therapy for prostitutes , who could legally be subjected to compulsory medical treatment , these trials did not require consent . this distinction between therapeutic and non - therapeutic trials was central in a directive that the prussian minister issued to the directors of hospitals and clinics on 29 december 1900 . it required information about the risks and consent of the subjects in scientific , non - therapeutic trials , but did not make this a requirement for interventions that served therapeutic , diagnostic or immunisation purposes. moreover , the directive made the hospital or clinic directors personally responsible for the scientific trials , which had to be documented including the compliance with information and consent requirements in the patients files . non - therapeutic experiments on minors and other persons lacking full legal competence were forbidden . moll criticised these regulations in medical ethics , arguing that on the one hand they did not provide enough protection for the human subjects , and that on the other , they went too far in their requirements . they went too far in ruling out scientific experiments on minors ; for example , taking a blood sample from a twenty - year - old within a scientific study would not be allowed in prussia . but the regulations were not stringent enough , as they did not make clear whether the therapeutic , diagnostic or immunisation purposes referred to the individual patient or to all interventions of this type , regardless of whether they might be useful to the individual subject concerned . moll also doubted that simple recording in the patient s file was sufficient to ensure that full information had been given and valid consent obtained . he was aware of the authoritarian milieu in the hospitals and university clinics of the time , in which the mostly lower - class patients were expected to follow doctors orders . many patients would also lack the education to fully comprehend the implications of a proposed trial . moll therefore demanded a guarantor for the proper information and consent of the subjects , and written consent for serious interventions . beyond this critique in his book on medical ethics , moll became personally involved in the issue . with the exception of the cases that were well known through the press , such as those of neisser and strubell , moll deliberately omitted the names of the experimenters in his account of problematic human trials in medical ethics . in a preceding article on the issue of human experimentation , published in november 1899 in the critical political weekly die zukunft his aim was to draw attention to abuses in clinical trials as a general problem , not to individual researchers such as neisser . after publication of this article , however , moll was approached by friedrich althoff ( 18391908 ) , the influential ministerial director in the prussian ministry for religious , educational and medical affairs , who requested to see moll s material on this topic . the request was sent on 30 december 1900 , just one day after the minister s directive on scientific trials had been issued . obviously , the ministry planned investigations , and possibly disciplinary proceedings , against some of the researchers who were resident in prussia . in early january 1901 , moll replied to althoff s letter , stating that he was willing to give access to his material under two conditions : that it was explicitly acknowledged by the ministry that he , moll , had supplied the information upon althoff s request ; and that he was given an assurance that his material was not going to be used in investigations of individual persons . rather , he wanted his material to provide background information for the drawing up of future regulations on human trials . as can be gathered from a memorandum of the ministerial official ludwig elster ( 18561935 ) at the end of february 1901 , moll was subsequently told that the assurance mentioned in his second condition could not officially be granted . moll then continued to refuse permission for access to his material , but agreed that the ministerial official could meet with him privately to look into his documentation , provided he was given the requested assurance by this official . after elster had given moll the assurance , he was able to study the material , a total of ninety - five cases , and to take notes . as elster remarked , however , most cases were totally harmless. the ministry does not seem to have taken much further action on the issue of human experimentation in the following years . as barbara elkeles has found in her research on this matter , only six cases were investigated by the ministry between 1900 and 1913 , but were not pursued any further due to when moll described the affair in his memoirs of 1936 , he indicated that his reluctance to reveal the identity of the experimenters had led to dissonances with the ministry , and he associated these with his failure to obtain an allegedly planned titular professorship at berlin university . the incident had , in fact , drawn the ministry s attention to moll as a person and physician . after moll had published , in march 1901 , another critical article on the issue of human experimentation , this time in the popular illustrated news magazine die woche , elster requested , on behalf of the ministry , a report on moll from the berlin chief of police , specifically on moll s personal circumstances and his standing in medical circles . the report , authored by a medical civil servant in the police department , richard wehmer ( 18541909 ) , was highly ambivalent . good , that he lived unmarried in orderly and prosperous financial circumstances in his berlin flat , and that his recent scientific works had found recognition , but on the other hand , it pointed out that moll had specialised in hypnotism and had fallen out with leading experts over scientific questions ; that he had attacked the work of asylum doctors in the journal die zukunft ; and that he liked to study sexual perversions and had served as an expert in trials against sexual offenders . the report even contained a piece of innuendo , saying that detectives were often seen in his flat , that prostitutes were among his patients , and that there were rumours that something mysterious was going on at his home . the account ended with the observation that moll did not vote in the 1893 general elections , and that he was a member of the progressive party [ fortschrittspartei ] , but that he had never attracted the attention of the police through any political campaigning . the report was filed in the ministry and no further action seems to have been taken . the whole affair revealed a pattern in moll s behaviour : engagement in a public debate on the ethics of medicine ; confident and almost arrogant dealings with the authorities ; and yet also anxious concern for his reputation as well as the reputation of the medical profession . moll was aware of the broader implications of the contemporary debate on human experiments , which included a general lay criticism of scientific medicine and of medical authority , especially as practised in hospital . to the extent that mostly working - class patients were the subjects of clinical experimentation , the issue had also a political dimension . the case of strubell , for example , had been made public by the social democratic newspaper vorwrts . friedrich scholz , in his widely read ethical reflections von aerzten und patienten [ on doctors and patients ] ( first published in 1899 ) , noted complaints about the autocratic attitude of some hospital physicians who regarded the patients that were dependent on them as material and abused them for the sake of science. syphilis inoculations , made out of scientific curiosity and with the knowledge that they would do harm , were in his eyes criminal. moreover , the public attacks against neisser had anti - semitic undertones , something that moll , who had converted from judaism to protestantism in 1895 , as the police report on him noted , is likely to have felt . julius pagel , who , like neisser , was jewish , defended the latter s controversial human trials on syphilis immunisation by explaining their scientific context and medical significance . to a degree , moll s manoeuvring between going public and protecting himself and the profession becomes understandable from a consideration of these various contexts . however , a similar behavioural pattern became apparent again a few years later when moll became involved in the so - called in october 1908 , the medical society of berlin - schneberg [ verein der schneberger aerzte ] began an investigation of medical practitioners referrals of foreign private patients to specialist consultants in berlin hospitals and university clinics . the main target of this investigation was the russian institute for medical consultations in berlin [ russisches institut fr medizinische consultationen zu berlin ] , run by doctors semjon lipliawsky ( born c.1875 ) and siegfried weissbein ( born c.1877 ) , who arranged , for a fee , consultations with leading medical specialists for russian patients visiting the german capital . there were also other such institutes , as well as individual agents in berlin , who provided this service to foreign visitors . allegedly , these middlemen were not only paid by the patients but also by the clinicians consulted . for the medical practitioners of schneberg this would constitute unfair competition for lucrative private patients . through a series of appeals in the berliner rzte - correspondenz ie . the official newsletter of the berlin medical chamber and journal of the local medical professional societies the deputy chairman of the medical society of berlin - schneberg , julius friedemann ( b. 1858 ) , called for information on such practices , in the interest of the professional dignity [ standeswrde ] of the german and especially the berlin medical profession. clearly , the intention was to initiate disciplinary proceedings against consultants who made underhand payments to middlemen for bringing them private patients . moll , as chairman of the so - called committee of fifteen [ fnfzehner - ausschu ] , a group representing the interests of the medical profession in the area of berlin , soon sided with friedemann in this campaign and himself collected the explosive nature of the issue became quickly apparent as ernst von leyden ( 18321910 ) , the doyen of internal medicine and former director of the first medical clinic of the charit - hospital of berlin university , had agreed to be a consultant for a planned outpatient clinic of the russian institute for medical consultations . after friedemann and moll had personally spoken with him , von leyden publicly retracted from this agreement . soon , however , another prominent clinician , karl anton ewald ( 18451915 ) , director of the department for internal medicine at the augusta - hospital in berlin and professor at berlin university , came under suspicion of being involved in the patient trade. such a suspicion was all the more delicate because ewald was , at the time , a candidate in the elections for the council of the prestigious berlin medical society [ berliner medizinische gesellschaft ] . as in the case of von leyden , private conversations took place . on behalf of ewald , hermann senator ( 18341911 ) , the chairman of the berlin medical society and director of the third medical clinic and outpatient clinic of the charit - hospital , followed moll s invitation to speak with him and friedemann about the matter at moll s home . tricked him on this occasion to implicate himself in the dubious practices surrounding russian private patients . the contents of the conversation became public in a libel trial in may 1909 , after senator had been mentioned , in an article in the newspaper berliner zeitung am mittag in march 1909 , as one of those making payments to the russian institute for sending patients to them . as moll later declared as a witness in disciplinary proceedings against ewald , he also had , in early january 1909 , a private conversation about the issue with the latter . before the trial of senator against the editor of the newspaper , however , further revelations about the patient trade and the russian institute had been made in a libel trial of moll against dr albert levin ( b. 1867 ) , a member of the medical society of berlin - schneberg , in march 1909 . levin belonged to the society s committee that had taken on the investigation of this matter . when levin asked moll to give him access to all his collected material on the patient trade issue , moll refused . incensed about this lack of collaboration , levin wrote a letter to moll , in which he accused him of cowardice or of holding back the material for improper reasons , or of actually having no further material at all . initially , moll had taken the view that one should deal discreetly with the patient trade issue , in order to stop abuses but not to expose individual colleagues . however , by the time of the trial against levin his tactics had clearly changed . moll now used the trial to make the conduct of the berlin university professors public , and friedemann became his ally in this enterprise . in his witness statement , friedemann reported that von leyden , senator , and the urologist carl posner ( 18541928 ) , who was an extraordinary professor at berlin university , had admitted to having offered or made payments to middlemen from the russian institute for medical consultations or other agents that had brought them private patients . the judge , keen to limit proceedings to the specific issue of libel , concluded that the payments described had actually happened , that moll had made material on this matter available to friedemann , and that the tone of levin s letter to moll was libellous . the appeal proceedings , held in late may 1909 , shortly after the libel trial of senator against the editor of the berliner zeitung am mittag in which moll appeared as a witness this time , moll s lawyer submitted an extensive motion to take evidence , in which he gave further details about the berlin medical professors relationships with the russian institute . dr weissbein , as one of the russian institute s directors , had a defensive letter read out at the trial , in which he suggested that moll and levin had stage - managed the whole libel case to obtain a public platform for their accusations against the professors and the institute . the trial ended with a settlement : levin acknowledged that moll had had honourable reasons not to release his substantial material , withdrew the accusations that he had made against moll in his letter , and took on the costs for the trial . the details of the trials were not only reported and commented upon in the medical press , such as the berliner rzte - correspondenz and the berliner klinische wochenschrift , but also in newspapers across the political spectrum . the allegations that prominent members of the berlin medical establishment had paid bribes to get private patients provided the right sort of material for a public scandal . as in the neisser case a few years earlier , anti - semitic comments were made . obviously being aware that moll , friedemann , levin and senator , as well as lipliawsky and weissbein , had a jewish background , the deutsche tageszeitung and das reich emphasised that jewish doctors were prominently involved in the affair . when the matter was also mentioned in the lower house of the prussian parliament [ abgeordnetenhaus ] , the ministry for religious , educational and medical affairs promised to investigate the allegations . at their own request , weissbein and friedemann initiated court of honour proceedings against themselves in order to have their conduct vindicated . in the disciplinary proceedings against the three professors , moll was heard four times as a witness by the university judge and government official , geheimer regierungsrat ( senior executive officer ) dr daude . the minister for religious , educational and medical affairs , to whom reports on the hearings were sent , asked daude to obtain more detailed evidence from moll because the latter initially refused to confirm that the accused professors had paid the middlemen specifically for bringing patients . only after daude threatened legal action against moll himself if he refused to give full evidence did moll provide further details that incriminated ewald and posner . in the end , however , none of the involved professors was found guilty . while it was noted that the medical professors concerned had occasionally given money to agents who had brought them patients , the ministry accepted their justification that these payments had just been small tips or small rewards for having acted as interpreters , or for having assisted when the russian patients were seen and examined by the professors . the disciplinary proceedings against senator and ewald were abandoned in november 1909 , as were proceedings against the elderly and ill von leyden which had been initiated only in the previous month . posner , who had regularly paid one specific agent , the russian interpreter and health assistant bernhard rosenberg ( born c.1860 ) had to face a full trial by the royal disciplinary court for civil servants in january 1910 , but was acquitted . with his intervention in this issue of professional conduct moll expert in medical ethics , but clearly at a reputational cost . while his name was now all over the newspapers , and he had turned himself into a champion of the economic interests of berlin s medical practitioners , his relationship to university medicine had suffered further damage ; for example , in the widely reported libel trial of senator against the berliner zeitung am mittag , senator sneeringly called moll and friedemann ... those two gentlemen , who make their appearance as guardians of medical ethics and of collegiality. the young doctor and medical journalist hans lungwitz ( 18811967 ) , who had recently completed his medical training in ewald s department , published a series of polemical articles about the affair in his weekly therapeutische rundschau , in which he ridiculed moll as god s ethicist [ ethiker von gottes gnaden ] and as a medical sherlock holmes who had interrogated the professors in his flat . lungwitz also shared weissbein s suspicion that the moll vs levin libel case had been staged by these two in order to go public with their allegations . as lungwitz claimed , moll s and friedemann s campaign against the berlin professors had damaged the international reputation of german medicine and was anti - german [ deutschfeindlich ] . the latter remark may well have been understood by informed readers as an anti - semitic gesture towards the jewish descent of both friedemann and moll . in principle , moll had repeated his strategy to intervene in an ethical issue , but then to try to protect his reputation and that of the profession by withholding incriminating material until forced to release it to the authorities ; but , as in the issue of human experimentation , such manoeuvring put him in an unfavourable light . guardian of medical ethics [ wchter der medizinischen ethik ] by his medical peers was rather derogatory , and , apparently , after the patient trade affair , moll s continuing membership as a representative in the berlin medical chamber was called into question by some of his colleagues . from my analysis of moll s medical ethics , there appears to be a discrepancy between the appreciation that they might deserve from a present - day , ethical point of view , and the historical impact they actually made . his focus on the contract relationship between doctors and patients gave space to the self - determination of the patient , though he moderated patients wishes with what he thought to be in the patient s best interest . moll can thus be seen , to some extent , as a pioneer of the concept of patient autonomy in medical ethics . carefully discussing conflicts of interest in many areas of medicine , his voluminous text on medical ethics was the most elaborate consideration of the topic at this time . his contemporaries , however , do not seem to have picked up his point about respect for patients wishes . a friendly but bland review in the mnchener medizinische wochenschrift by the hamburg gynaecologist karl jaff ( 18541917 ) asserted that moll s book was a true reflection of the current views on medical ethics and praised his impartial and objective point of view. incoming as well as experienced doctors might learn from it , suggested jaff , but he also expressed his general doubts about the practical usefulness of writings on medical ethics . a similar point was made by the reviewer for the deutsche medizinische wochenschrift , the berlin physician leopold henius , who thought that it was more helpful to seek the advice of an experienced colleague in doubtful cases than to plough through a voluminous handbook . many things in moll s book were not new , claimed henius , and other parts , such as that on the perforation of the fetus , too sophisticated to be convincing. both jaff and henius acknowledged moll s enormous industriousness in writing medical ethics , and , like jaff , henius acknowledged moll s calm , noble and objective tone. but in the end , henius assessment must have been frustrating for moll : on the whole ( ) we have here an industrious work which deserves recognition , and we wish it a large readership , but , for the above - mentioned reasons , we do not expect that it will have it. some reviewers in less prominent medical journals more warmly recommended moll s book to their readership . generally appreciative were also the reviews in the viennese medical periodicals . however , henius prediction seems to have been correct if one considers that there was no further german edition of moll s medical ethics . the reception of the book in the legal profession was perhaps more positive ; for example , melchior stenglein ( 18251903 ) , formerly an official at the german supreme court , praised , in a review in the legal journal der gerichtssaal , the similarity of moll s views to current legal opinion on the issues of euthanasia and consent to surgery . likewise , the berlin lawyer erich sello ( 18521912 ) , a friend of moll s , emphasised , in his review , the many links to legal questions that the book provided . in fact , legal publications on aspects of medical practice , such as consent and confidentiality , regularly cited moll s work on ethics while often ignoring other medical literature . it would be an exaggeration , however , to say that moll s medical ethics was a success , despite its two translations into russian . the editor of the 1904 translation , the marxist medical doctor and writer vikentii veresaev ( 18671945 ) , characterised moll as a cautious , moderate , and prudent philistine , who is devoid of noble purpose and saw parts of the book as illustrating the bourgeois outlook of the modern , ordinary german physician. russian reviewers of medical ethics criticised it for its lack of a foundation in a philosophical system and for its application of the term moll himself , in his memoirs of 1936 , repeatedly claimed that his outrage about abuses in experimentation on hospital patients had made him feel compelled to write medical ethics . in view of his later active engagement in medical politics , as a member of the berlin medical chamber from december 1908 , one may also assume that a more general interest in questions of professional conduct had been a motivating factor . his emphasis on self - determination of the patient might have been a reflection of the specific circumstances of his own practice , in which he saw private patients who paid for the consultations themselves . only in the 1980s and 1990s , when medical ethics and bioethics became increasingly important subjects in german academia , did historians of medicine engage with the contents of moll s book on medical ethics . situation ethics in moll s text , and to his subjective response to the rise of science in medicine , as exemplified by the issue of human experimentation . julius henri schultz , in a short monograph on moll s medical ethics in 1986 , emphasised the latter s closeness to legal thought and philosophical positivism ; and for antonia eben , in her medical doctoral dissertation of 1998 , the his collected materials on human experimentation and his criticism of the prussian ministerial directive of 1900 did not lead to revisions of the regulations , as he had hoped . his activities in the patient trade affair also failed to effect disciplinary punishment for the professors involved . on the other hand , the publicity that his interventions in this matter gained may well have put a temporary stop to the practice of clinicians payments to middlemen . however , a hundred years on , when in august and september 2009 a new patient trade affair went through the german media , nobody remembered that berlin had experienced all this before

in 1902 , albert moll , who at that time ran a private practice for nervous diseases in berlin , published his comprehensive book on medical ethics , rztliche ethik . based on the concept of a contractual relationship between doctor and client , it gave more room to the self - determination of patients than the contemporary , usually rather paternalistic , works of this genre . in the first part of the present paper this is illustrated by examining moll s views and advice on matters such as truthfulness towards patients , euthanasia , and abortion . the second part of this article discusses how moll engaged with the then publicly debated issues of experimentation on hospital patients and the trade of foreign private patients between agents and medical consultants . in both matters moll collected evidence of unethical practices and tried to use it to bring about change without damaging his or the profession s reputation . however , with his tactical manoeuvres , moll made no friends for himself among his colleagues or the authorities ; his book on ethics also met with a generally cool response from the medical profession and seems to have been more appreciated by lawyers than by other doctors .

Introduction Characteristics of Molls Medical Ethics Truth-telling and Deception Euthanasia Perforation of the Fetus Human Experimentation Patient trade Conclusions

PMC3411541

bony fractures are one of the most common reasons for physician visits worldwide . with greater public interest in physical fitness activities to maintain a healthy lifestyle , metatarsal stress fractures are commonly encountered by clinicians . metatarsal stress fractures , which account for nearly 25% of all stress fractures , often heal with nonoperative measures . immobilization of the foot and avoidance of strenuous activity often result in bone healing and return to full functional capacity within 68 weeks of the initial injury . impaired fracture healing in which a patient may fail to form a unionization of bone , or have delayed unionization , is frequently encountered in both inpatient and outpatient settings . patients at risk for impaired fracture healing include elderly patients , diabetics , smokers , patients with osteoporosis , and postmenopausal females due to estrogen deficiency . impaired fracture healing can have devastating consequences in this patient population , and may increase the fracture - related morbidity and mortality . teriparatide , a drug approved for the treatment of osteoporosis in postmenopausal women , has shown promise in the realm of fracture healing . in the initial stages of fracture healing , an increase in bone formation is required . by increasing bone formation through stimulation of osteoblasts this may be useful in treatment of patients who have impaired fracture healing , ultimately affecting quality of life . we present two cases of women with metatarsal fractures who had risk factors for impaired fracture healing , and report the time to fracture healing with the use of teriparatide . a 35-year - old premenopausal caucasian female presented to the emergency department with a complaint of pain in her right foot which occurred abruptly while running . she described the pain as sharp and unremitting , and denied any recent trauma to the affected foot . medical history was significant for a stress fracture of the left second metatarsal two years prior , which took approximately 14 weeks to obtain radiographic evidence of fracture healing with the use of an immobilizing boot . she denied the use of any medications , alcohol , or illicit drugs , but did have a 21 pack - year history of tobacco use . physical examination revealed a well developed female in mild distress . on examination of the right foot radiography of the right foot was consistent with a stress fracture at the base of the fifth metatarsal with associated soft tissue inflammation ( fig . 1 ) . the patient was instructed to wear an immobilizing boot on the right foot and to avoid strenuous activity . ibuprofen was suggested to the patient for analgesia . nearly 6 weeks after sustaining the fracture , the patient returned to the emergency department with continued pain in the right foot despite the use of the immobilizing boot and adequate analgesia . the patient refused further use of the immobilization boot . due to non - compliance with the immobilization boot , she was started on a daily dose of teriparatide , 50,000 iu of vitamin d3 , and 2000 mg of calcium citrate . four weeks into therapy with teriparatide , repeat imaging revealed evidence of bony callus and new bone formation of the right fifth metatarsal , consistent with a healing fracture ( fig . 2 ) . the patient was pain free at this time , and shortly thereafter resumed all prior activities , including running . a 40-year - old caucasian female was seen in the orthopedic clinic with complaints of moderate pain in her left foot . she described the pain as sharp , and worsening with movement of her left foot . medical history was significant for hypothyroidism secondary to hashimoto s disease , celiac disease , and premature ovarian failure . she had no history of osteoporosis but had a previous fracture of the right distal radius , which took approximately 10 weeks to heal as per clinical findings and imaging studies . home medications included vitamin d3 50,000 iu weekly , levothyroxine 175 g daily , and glucosamine / chondroitin sulfate 1500 mg/1200 mg daily . physical examination was significant for tenderness to palpation of the left foot , with the point of maximal tenderness being over the first phalanx . radiography of the left foot confirmed the presence of a first left phalanx stress fracture ( fig . 3 ) , and the patient was instructed to wear an immobilizing boot for at least 6 weeks . because the patient appeared to be reluctant to wear an immobilizing boot for such a long duration , she was also started on teriparatide , 50,000 iu of vitamin d3 daily , and calcium citrate 2000 mg daily . she continued this medication regimen without any additions or changes for a total duration of 4 weeks . one week into therapy with teriparatide , the patient s pain and swelling had subsided and she was able to remove her immobilizing boot . at the end of 4 weeks , repeat imaging revealed a fracture that was well healed with callus formation seen ( fig . a 35-year - old premenopausal caucasian female presented to the emergency department with a complaint of pain in her right foot which occurred abruptly while running . she described the pain as sharp and unremitting , and denied any recent trauma to the affected foot . medical history was significant for a stress fracture of the left second metatarsal two years prior , which took approximately 14 weeks to obtain radiographic evidence of fracture healing with the use of an immobilizing boot . she denied the use of any medications , alcohol , or illicit drugs , but did have a 21 pack - year history of tobacco use . physical examination revealed a well developed female in mild distress . on examination of the right foot radiography of the right foot was consistent with a stress fracture at the base of the fifth metatarsal with associated soft tissue inflammation ( fig . 1 ) . the patient was instructed to wear an immobilizing boot on the right foot and to avoid strenuous activity . ibuprofen was suggested to the patient for analgesia . nearly 6 weeks after sustaining the fracture , the patient returned to the emergency department with continued pain in the right foot despite the use of the immobilizing boot and adequate analgesia . the patient refused further use of the immobilization boot . due to non - compliance with the immobilization boot , she was started on a daily dose of teriparatide , 50,000 iu of vitamin d3 , and 2000 mg of calcium citrate . four weeks into therapy with teriparatide , repeat imaging revealed evidence of bony callus and new bone formation of the right fifth metatarsal , consistent with a healing fracture ( fig . 2 ) . the patient was pain free at this time , and shortly thereafter resumed all prior activities , including running . a 40-year - old caucasian female was seen in the orthopedic clinic with complaints of moderate pain in her left foot . she described the pain as sharp , and worsening with movement of her left foot . medical history was significant for hypothyroidism secondary to hashimoto s disease , celiac disease , and premature ovarian failure . she had no history of osteoporosis but had a previous fracture of the right distal radius , which took approximately 10 weeks to heal as per clinical findings and imaging studies . home medications included vitamin d3 50,000 iu weekly , levothyroxine 175 g daily , and glucosamine / chondroitin sulfate 1500 mg/1200 mg daily . physical examination was significant for tenderness to palpation of the left foot , with the point of maximal tenderness being over the first phalanx . radiography of the left foot confirmed the presence of a first left phalanx stress fracture ( fig . 3 ) , and the patient was instructed to wear an immobilizing boot for at least 6 weeks . because the patient appeared to be reluctant to wear an immobilizing boot for such a long duration , she was also started on teriparatide , 50,000 iu of vitamin d3 daily , and calcium citrate 2000 mg daily . she continued this medication regimen without any additions or changes for a total duration of 4 weeks . one week into therapy with teriparatide , the patient s pain and swelling had subsided and she was able to remove her immobilizing boot . at the end of 4 weeks , repeat imaging revealed a fracture that was well healed with callus formation seen ( fig . the incidence of fractures has been steadily increasing , with an estimated 15.3 million fractures occurring in the us annually.1,2 stress fractures of the metatarsal bones are common , and account for approximately 5% of fractures seen in the primary care setting annually.3 with the treatment of metatarsal stress fractures based primarily on conservative measures such as immobilization , the morbidity associated with such fractures depends largely on the rate of an individual s natural fracture healing time . the first phase involves inflammation and formation of a hematoma at the site of fracture due to disruption of the blood supply at the site of injury.4 in the second phase , chondrogenesis begins and a soft callus forms , which is then mineralized.4 the third phase marks the beginning of osteoblast cells forming woven bone , resulting in formation of a hard callus and union of the fracture.5 in the final phase of fracture healing , remodeling of bone occurs , resulting in the formation of lamellar bone , which is mechanically stronger bone.5 stress fractures of the metatarsal bones are often misdiagnosed initially as a soft tissue injury , because initial radiographs may not show evidence of fracture . 6 if clinical suspicion for a metatarsal stress fracture is high , or diagnosis is confirmed via radiographic imaging , conservative treatment modalities are first - line options . patients with metatarsal stress fractures should refrain from vigorous physical activity , and immobilization of the affected foot is often recommended . in most cases , nonweight - bearing activity and placement of an immobilizing boot leads to decreased pain associated with metatarsal fractures . under normal circumstances , it takes approximately 68 weeks for a metatarsal stress fracture to heal.7 a visible callus is typically seen on radiographs by 6 weeks after the initial injury.6 however , in approximately 10% of patients , impaired fracture healing is encountered , leading to delayed union or nonunion of metatarsal stress fractures . these fractures take longer to heal because of the poor blood supply in the region of the fifth metatarsal . they typically show radiographic evidence of union within 8 weeks with immobilization and use of a rigid boot.8 jones fractures involve the proximal diaphysis , and without surgery , take approximately 20 weeks to heal.8 avoiding weight - bearing is usually recommended for several months , and many patients find it difficult to comply with this . half of these fractures treated with immobilization alone tend to result in refracture or nonunionization of the fracture.9 thus many patients , including athletes , opt for surgical intervention to minimize the healing time . this involves placement of a screw into the canal of the fifth metatarsal , followed by immobilization for at least 2 weeks.10 with surgery , these fractures typically heal within 68 weeks . although the pathophysiology of impaired fracture healing is largely unknown , certain risk factors have been well reported . mechanical causes , such as insufficient immobilization of the fracture , distraction of fracture fragments by fixation devices , or repeated manipulations of a fracture , may lead to delayed or impaired union . nonmechanical risk factors , including patient comorbidities , have also been suggested to result in impaired fracture healing . diabetes , smoking , osteoporosis , and estrogen deficiency have all been identified as possible risk factors for impaired fracture healing.11 in such patient populations , impaired fracture healing may result in significant morbidity and negatively impact quality of life.12 with established risk factors for impaired fracture healing identified , there comes a greater need for therapy that can accelerate fracture healing in patients at high risk for delayed union or nonunion fractures . teriparatide , a recombinant human parathyroid hormone analog ( 134 ) , was approved in 2002 by the food and drug administration for the treatment of osteoporosis in postmenopausal women who are at high risk for fractures.13 the only anabolic agent available for the treatment of osteoporosis , its mechanism of action is unique in comparison with other agents used currently . chronic exposure to endogenous human parathyroid hormone leads to an increase in bone resorption via stimulation of osteoclasts , further exacerbating the condition of osteoporosis.14 teriparatide , because of its ability to reach peak serum concentrations in 30 minutes and return to nondetectable serum levels in a few hours , has the paradoxical effect of stimulating new bone formation by favoring stimulation of osteoblasts over osteoclasts.14 in the natural process of fracture healing , a transient increase in bone formation at the site of fracture occurs . by increasing cortical thickness and bone formation on all bone surfaces , teriparatide may accelerate and augment this process of increased bone formation and fracture healing that happens naturally.4 andreassen et al were the first to report accelerated healing of fractures with teriparatide . the results demonstrated enhanced callus formation and mechanical strength of tibial fractures in rats treated with teriparatide.15,16 in 2006 , manabe et al used teriparatide for fracture healing in cynomolgus monkeys , because their bone remodeling systems are closest to those of humans.17 in the study by manabe et al , the results suggested that by attenuating the degree of mineralization and shrinking the size of the callus , teriparatide may have accelerated the rate of fracture healing.17 although prior case studies regarding the use of teriparatide for accelerated fracture healing in humans exist , the only randomized , double - blind study in humans testing the hypothesis of acceleration of fracture repair was done by aspenberg et al in 2010 . in this study , involving 102 postmenopausal women with fractures of the distal radius , it was concluded that there was a shortened time of fracture healing in a group of patients using 20 g of teriparatide compared with placebo.18 their results suggest that teriparatide may lead to acceleration of fracture healing in humans.18 our cases reported here suggest the possibility of accelerating metatarsal stress fracture healing with the use of teriparatide . 13 taking cost into account , it may be best used to accelerate fracture healing in those patients at high risk for delayed union or nonunion of fractures , or in patients who require a return to baseline activity at a faster rate than is possible with the normal healing process . this includes high - performance athletes , but also includes a large percentage of the workforce where jobs entail manual labor or constant walking throughout the day . for those who have occupations that involve walking throughout the day , patients with jobs entailing manual labor may suffer a greater loss of income than the cost of teriparatide for one month , due to inability to return to work until healing of the metatarsal fracture has occurred . in addition , many employers may not allow for a month away from work , and this could result in loss of employment for the patient . according to data from the centers for disease control published in 1990 , the value of lost wages stemming from health conditions related to both physical and mental impairment as well as costs of medical care related to this reaches over a billion dollars in the us annually.19 therefore , in patients with jobs where the loss of income is greater than the cost of treatment with teriparatide , the drug may be useful in acceleration of fracture healing , allowing earlier return to work . although teriparatide is also indicated for the treatment of osteoporosis associated with use of long - term systemic glucocorticoids in men , there have been no data published on the use of teriparatide in the acceleration of fracture healing in men and premenopausal women . there are ongoing phase ii and phase ii clinical trials investigating the benefits of this medication in these specific patient populations . although more randomized , double - blind , placebo - controlled trials in humans will need to be pursued to investigate further the potential of this drug in fracture healing , clinicians should be aware that teriparatide may become useful in the clinical setting for acceleration of fracture healing .

bone fractures are one of the leading causes of emergency room visits worldwide , with approximately 8 million bony fractures occurring annually in the us alone . although the majority of fractures do not cause significant long - term morbidity and mortality , approximately 10% of these fractures result in impaired fracture healing , drastically affecting quality of life in affected patients . by increasing bone formation , teriparatide , an anabolic agent used in the treatment of postmenopausal osteoporosis , has shown promise in accelerating the rate of fracture healing . we present two patients with impaired healing of metatarsal fractures who were subsequently treated with teriparatide . both patients experienced successful bony union of the fracture after the use of teriparatide . these findings suggest that teriparatide may be useful in the clinical setting for the acceleration of fracture healing , especially in patients who are at risk for impaired fracture healing .

Introduction Case Presentation Case 1 Case 2 Discussion

PMC4018416

it can save lives ( both mother and child ) in the event of serious complications.1 the debate about the ideal cs rate is ongoing and shows no sign of abating.19 whereas in the past the discussion revolved largely around medical issues and whether or not cs could be justified , the question of the woman s right to choose her own mode of delivery without a medical indication has now taken center stage.10,11 this right to cs is an extension of the a child if i want , when i want , and with whom i want view which some see as the rights of women with regard to their own bodies . however , others believe that women who request a cs are victims of overmedicalization or the influence of doctors who can no longer bear the screams and sweat of a delivery room and prefer a more rapid and lucrative cs.10,1214 the woman s right to choose her delivery method has been a matter of debate , mostly in countries with a high cs rate . in other countries , such as those in sub - saharan africa , where the cs rate is less than 2% , improving accessibility is a priority . how can low cs rates be explained in these settings ? however , some authors1518 also point to the importance of social acceptability of cs by women and their families in sub - saharan africa . they see cs not only as an inability or failure of the woman to deliver vaginally15 but also as a threat to the family s financial stability.1921 those involved in maternal health programs need to understand women s perceptions of cs so as to overcome existing barriers ( eg , through reduction of the direct costs of cs or improved transport to a referral hospital ) and see their efforts come to fruition . the objective of this paper was to explore women s experience with and perception of cs in burkina faso and the social and economic implications of cs within the household . we conducted a qualitative study using focus groups and individual semistructured interviews ( supplementary material ) . this study was part of a larger multidisciplinary action research project ( aquasou)22 aiming to improve the quality of and access to emergency obstetric care in the health district of bogodogo , burkina faso . it was based on the coordination and synergy of three complementary approaches , ie , public health , social anthropology , and political and social mobilization . bogodogo district is one of five districts in the center health region and covers a population of approximately half a million people , of whom 67% are from rural areas . the district comprises 41 first - level facilities ( public and private ) which refer complicated cases to the district hospital . the district hospital in bogodogo has 57 beds for pediatrics , surgery , medicine , maternity ( 24 beds ) , an outpatient clinic , a laboratory , and a medical imaging unit ( radiography and ultrasound ) . the operating theater opened on august 1 , 2003 , but emergency obstetric surgery has only been possible for 24 hours on 7 days a week since october 1 , 2004 . admissions to the maternity ward ( operational since november 2001 ) have increased rapidly , doubling in number between 2002 and 2005 ( from 2,053 to 4,182 ) . in 2004 , the hospital performed 193 cs versus 506 in 2005 and 735 in 2009 . the interviews and focus groups were conducted 10 years ago when the hospital was increasing its cs offer but out - of - pocket expenditure was still high . no exemption system was in place in 2003 and 2004 to decrease the financial burden to the household . in 2005 , the district initiated a cost - sharing system to decrease the direct household costs for a cs from 74.000 cfa ( us$136 ) to 25.000 cfa ( us$46 ) . in late 2006 , the government launched a national subsidy for delivery and emergency obstetric care so as to cover 80% of the direct costs , and the direct costs of cs were officially reduced to 11.000 cfa ( us$22 ) . these women s views on cs can be considered as a baseline prior to introduction of the national subsidy . given that some research teams are now evaluating the impact of exemption policies on the perception of cs in west africa , this paper will allow comparison before and after the introduction of an exemption policy for obstetric services . a team of one anthropologist and one health provider ( midwife or theater nurse ) conducted home visits for patients who underwent a cesarean birth at bogodogo district hospital in ouagadougou . a health care provider was added to the research team following a situational analysis conducted during the first year of the aquasou project that demonstrated a lack of health provider knowledge of patients social conditions.23 the objective of the home visits was two - fold : the first was to collect the women s experience of cesarean birth and second to open the eyes of health care providers by observing the women s living conditions and listening to their testimonies and individual perceptions of care . the expected outcome of the second objective was an improvement in relationships between health care providers and patients . only the maternity ward staff took part initially , but operating theater personnel subsequently came on board . thirty - five of 112 women who underwent cs in the operating theater between september 2003 and september 2004 were visited in their homes . one of the anthropologists proposed a home visit to the women before they left the facility , and most agreed after consultation with their husbands . the exact address of the household and ( if applicable ) a mobile phone number were then recorded by the researcher to ensure location of the home . interviews were conducted in moore ( the national language ) and written notes were taken by the interviewer . after each visit , a report was drafted in french by the interviewer and he / she was expected to provide feedback to his / her colleagues during a subsequent team meeting . in february 2004 , five focus groups were conducted with women who attended various maternity wards in the health district of bogodogo ( four urban facilities and one rural facility ) . these focus groups collected data to assess the perceptions of cs by women in the community . home interviews were embedded in the action research and served as an exploratory phase to prepare for user - provider meetings in the second part of the project . each group consisted of about 15 women , which allowed us to look into the subject in greater depth . the composition of the groups was determined during antenatal consultations in the four urban facilities and one rural facility . the activity was proposed to the women , and if they accepted , details , days , and hours of the discussion were given . the first objective was to collect data on women s perception of pregnancy , childbirth , and associated problems , including the different views on social support received during pregnancy , ie , who are the people involved in the financial expense of pregnancy and childbirth . the women were asked whether they were satisfied with the care they received and what they would like to see changed regarding access to and organization of the facilities they used . the second objective was to secure input for the next step , which was organization of user - provider meetings to improve the quality of care . these focus groups allowed the project s promoters to identify the main touchy or problematic issues from the women s perspective to be discussed with the providers , eg , informal payment and lack of communication . individual home visit interviews and discussions in the user focus groups were conducted in moore ( the national language ) , audio - recorded , and transcribed into the french language using microsoft word . a content analysis was undertaken manually according to the analysis methodology of qualitative data.24,25 no software was used . the authors first read all the interviews to identify the different themes , and each interview was then analyzed according to the themes identified during the first round of reading . the results were triangulated by the two social anthropologists ( fo and sz ) . permission to perform the aquasou action research and its different nested studies was obtained from the ministry of health of burkina faso ( ms / sg / dgsp / dsf/27.02.2002 ) and the regional directorate of the health center region . ethical regulations regarding participant consent and treatment of the data were followed according to the tenets of the declaration of helsinki . bogodogo district is one of five districts in the center health region and covers a population of approximately half a million people , of whom 67% are from rural areas . the district comprises 41 first - level facilities ( public and private ) which refer complicated cases to the district hospital . the district hospital in bogodogo has 57 beds for pediatrics , surgery , medicine , maternity ( 24 beds ) , an outpatient clinic , a laboratory , and a medical imaging unit ( radiography and ultrasound ) . the operating theater opened on august 1 , 2003 , but emergency obstetric surgery has only been possible for 24 hours on 7 days a week since october 1 , 2004 . admissions to the maternity ward ( operational since november 2001 ) have increased rapidly , doubling in number between 2002 and 2005 ( from 2,053 to 4,182 ) . in 2004 , the hospital performed 193 cs versus 506 in 2005 and 735 in 2009 . the interviews and focus groups were conducted 10 years ago when the hospital was increasing its cs offer but out - of - pocket expenditure was still high . no exemption system was in place in 2003 and 2004 to decrease the financial burden to the household . in 2005 , the district initiated a cost - sharing system to decrease the direct household costs for a cs from 74.000 cfa ( us$136 ) to 25.000 cfa ( us$46 ) . in late 2006 , the government launched a national subsidy for delivery and emergency obstetric care so as to cover 80% of the direct costs , and the direct costs of cs were officially reduced to 11.000 cfa ( us$22 ) . these women s views on cs can be considered as a baseline prior to introduction of the national subsidy . given that some research teams are now evaluating the impact of exemption policies on the perception of cs in west africa , this paper will allow comparison before and after the introduction of an exemption policy for obstetric services . a team of one anthropologist and one health provider ( midwife or theater nurse ) conducted home visits for patients who underwent a cesarean birth at bogodogo district hospital in ouagadougou . a health care provider was added to the research team following a situational analysis conducted during the first year of the aquasou project that demonstrated a lack of health provider knowledge of patients social conditions.23 the objective of the home visits was two - fold : the first was to collect the women s experience of cesarean birth and second to open the eyes of health care providers by observing the women s living conditions and listening to their testimonies and individual perceptions of care . the expected outcome of the second objective was an improvement in relationships between health care providers and patients . only the maternity ward staff took part initially , but operating theater personnel subsequently came on board . thirty - five of 112 women who underwent cs in the operating theater between september 2003 and september 2004 were visited in their homes . one of the anthropologists proposed a home visit to the women before they left the facility , and most agreed after consultation with their husbands . the exact address of the household and ( if applicable ) a mobile phone number were then recorded by the researcher to ensure location of the home . interviews were conducted in moore ( the national language ) and written notes were taken by the interviewer . after each visit , a report was drafted in french by the interviewer and he / she was expected to provide feedback to his / her colleagues during a subsequent team meeting . in february 2004 , five focus groups were conducted with women who attended various maternity wards in the health district of bogodogo ( four urban facilities and one rural facility ) . these focus groups collected data to assess the perceptions of cs by women in the community . home interviews were embedded in the action research and served as an exploratory phase to prepare for user - provider meetings in the second part of the project . each group consisted of about 15 women , which allowed us to look into the subject in greater depth . the composition of the groups was determined during antenatal consultations in the four urban facilities and one rural facility . the activity was proposed to the women , and if they accepted , details , days , and hours of the discussion were given . the first objective was to collect data on women s perception of pregnancy , childbirth , and associated problems , including the different views on social support received during pregnancy , ie , who are the people involved in the financial expense of pregnancy and childbirth . the women were asked whether they were satisfied with the care they received and what they would like to see changed regarding access to and organization of the facilities they used . the second objective was to secure input for the next step , which was organization of user - provider meetings to improve the quality of care . these focus groups allowed the project s promoters to identify the main touchy or problematic issues from the women s perspective to be discussed with the providers , eg , informal payment and lack of communication . individual home visit interviews and discussions in the user focus groups were conducted in moore ( the national language ) , audio - recorded , and transcribed into the french language using microsoft word . a content analysis was undertaken manually according to the analysis methodology of qualitative data.24,25 no software was used . the authors first read all the interviews to identify the different themes , and each interview was then analyzed according to the themes identified during the first round of reading . permission to perform the aquasou action research and its different nested studies was obtained from the ministry of health of burkina faso ( ms / sg / dgsp / dsf/27.02.2002 ) and the regional directorate of the health center region . ethical regulations regarding participant consent and treatment of the data were followed according to the tenets of the declaration of helsinki . women with a cs who were later interviewed were representative of the patients hospitalized for cs in the district hospital ( bogodogo district [ 73% ] and other districts [ 27% ] ) , and all originated from urban or semiurban areas . their average age was 27 ( range , 2233 ) years , all were married , and had an average of two children at the time of interview . regarding employment , 38% were employed in the formal sector ( work with an employment contract ) . women in the focus group originated from urban ( 80% ) or rural ( 20% ) areas , and were aged 1840 years . most had primary schooling , and were housewives working in the informal sector ( street sellers ) . almost all women interviewed mentioned the unpredictability of health problems during pregnancy , and felt that no two pregnancies are alike . . you can be pregnant and it hurts , it hurts all the way until the delivery . [ member of the patient focus group linked to an urban maternity ward ] childbirths , like pregnancies , are all different , with some being quick and easy , while others are difficult , with women suffering a prolonged labor . the statement that childbirth is war is found in popular representations of childbirth in nigeria,26 but might just as well be a claim of women in burkina faso . the method of delivery is also a source of anxiety : you fear that you are between life and death ; some people die in childbirth , or you can give birth and not be healthy . in addition , you get frightened if you believe you may have to undergo an operation . [ member of a patient focus group linked to an urban maternity ward ] a 31-year - old woman , who had studied accountancy and underwent cs described her fears when she learnt she had to have the operation : i had pain in my lower abdomen , and i was in the maternity ward . the midwife asked me to do an ultrasound so she could know the presentation of the baby [ ] . when i went to the ultrasound , the guy told me what it was going to be ( an operation ) . he told me no , because it was too low , the baby was stuck and had no room to turn . [ ] people tell so many things , there is a mystery around it . [ a woman interviewed at home ] the women s stories generally referred to fears of medical procedures , including cs , as reflected in the statement : once a cs , always a cs ! , highlighting the fear of repetition . another aspect often mentioned by the women was a lack of information during their antenatal consultations about the possibility of a cesarean birth . the interviewees regularly referred to their fear of poor and slow healing of the scar after a cs and the resulting physical side effects , eg , back pain and fatigue . there was intense fear associated with the risk of dying during childbirth , which is probably higher in women who know women who have died during childbirth . it was serious , she arrived there in the morning , and at 10 pm she had still not given birth . they told her to push , she did not want to push , she did not push , she tightened her buttocks , until she was referred to the hospital . they brought her inside , they came out to hand over the baby , they returned inside . when they returned we saw that they were taking her body to the morgue . seeing [ member of a patient focus group linked to an urban maternity ward ] for others , fear of cs was related to hearing other people s horror stories and tales of serious consequences , such as paralysis of the lower limbs . fear of childbirth , and more specifically of cs , is inseparable from the medical , social , and financial implications facing women : if you re going to give birth , you re scared because you do not know how it will happen . you can get a prescription but the money turns out to be a problem [ ] [ member of a patient focus group linked to an urban maternity ward ] men are expected to meet the obstetric care expenses.27,28 very few women know the cost of their cs . this fact does not imply negligence on the part of the women , but reflects the distribution of tasks within marriage . , men may think it indiscrete if a woman asks this type of question . [ ] when you give birth , except your husband , no one else will come and pay the expenses . even if your husband has no money , he will borrow and has to reimburse later . [ ] if you have given birth , the man must do anything to get the money and come and pay . [ member of a patient focus group linked to an urban maternity ward ] families are rarely prepared to cover the additional costs of childbirth . in fact , the urgency solves the problem ( when things get hot , we find it [ ] ) . however , there seems to be a gap between the social norm and reality . in urban areas and in the context of economic crisis , negotiations and arrangements between men and women about the costs of pregnancy and childbirth are not uncommon . among men , there are those who are able to pay the costs of delivery , those who do not want to pay , and a large group who do not have the means to pay . in the latter case the strategy adopted by the woman is to ensure that the members of her entourage ( family members and caregivers ) do not find out that she is the one who pays . for the woman , it is an act of saving her husband s reputation and showing that he is capable of meeting the costs of pregnancy and childbirth . for example , as i know that my husband did not have the means , i gave him money on the day i was coming out of hospital , take it , go and pay and we ll get out , it was my husband who paid , [ women laugh ] . [ member of patient focus group linked to an urban maternity ward ] a teacher explained , for example , that she had covered the costs of her three pregnancies since her husband lost his job . even for the health care providers , the rule is that the man pays the costs of delivery . exchanges between caregivers and the patient s companions are limited to giving prescriptions and claiming consultation fees . in poorer settings , it is difficult to prepare financially for childbirth . other than the profits from a small business , women try to save on the money their husbands give them to buy condiments , but do not tell them about these savings . , men do not feel concerned by the problems faced by their wives during pregnancy . in addition to the woman s psychological anxiety , pregnancy and childbirth seem to add tensions to her relationship with her partner . the man seems to reach a critical point where his reputation in his close circle is at stake , and rests on his capacity to pay hospitalization bills . motherhood thus becomes a threat to the social status of both the man and the woman . fear or refusal of certain medical procedures ( including emergency referral to the hospital ) is mainly due to concern about incurring higher costs in a complex medical environment where patients receive little information.29 communication or the lack of it was a recurrent theme in the various interviews conducted at home and in the focus groups . in addition to this lack of information , the interactions between health care providers and users seem underpinned by a breakdown of communication . for example , some women said that they were not informed of the decision to perform a cs in the delivery room . she did not have the chance to see them and said she had not been informed about the cs : they put me on a stretcher in the maternity ward to bring me to the operating theater . [ young woman , mother of one child , during interview conducted at home in february 2004 ] in many cases , information given by staff is not understood because it is too vague . one of the objectives of having a health care provider on the team of interviewers during the home visits was to improve this communication and help providers to realize that the information given can have a significant impact in the weeks following a cesarean birth . for example , rosalie , a 25-year - old woman who was told that she needed an operation because her baby was too large ( her medical file stated generally contracted pelvis ) , said that she would ensure that her next child was smaller . she told the interviewers when she was interviewed at home that she would avoid eating food that boosted her baby s growth . another woman who was told to avoid soaking the wound in water after a cs interpreted this as meaning that she could not wash herself . during the home visit , several days after her return home , the caregiver noticed the smell and physical appearance of this young mother . the home visit provided the opportunity to show the mother how to wash herself . in order to facilitate free discussion , initially , many patients were surprised or scared when midwives and anthropologists visited them in their homes . a caregiver visiting a patient at home to listen to her story was unheard of in the local history of burkina faso . one woman , whose husband the team wanted to interview , later confided the reason for her reluctance : i was scared and wondered what my husband had done for the team wanting to see him ! however , after some initial surprise , when families realized that they had done nothing wrong and that the health care provider was open and attentive , there was a high demand from mothers for information on personal hygiene , eating habits , and resumption of sexual activity after cs . almost all women interviewed mentioned the unpredictability of health problems during pregnancy , and felt that no two pregnancies are alike . . you can be pregnant and it hurts , it hurts all the way until the delivery . [ member of the patient focus group linked to an urban maternity ward ] childbirths , like pregnancies , are all different , with some being quick and easy , while others are difficult , with women suffering a prolonged labor . the statement that childbirth is war is found in popular representations of childbirth in nigeria,26 but might just as well be a claim of women in burkina faso . the method of delivery is also a source of anxiety : you fear that you are between life and death ; some people die in childbirth , or you can give birth and not be healthy . in addition , you get frightened if you believe you may have to undergo an operation . [ member of a patient focus group linked to an urban maternity ward ] a 31-year - old woman , who had studied accountancy and underwent cs described her fears when she learnt she had to have the operation : i had pain in my lower abdomen , and i was in the maternity ward . the midwife asked me to do an ultrasound so she could know the presentation of the baby [ ] . when i went to the ultrasound , the guy told me what it was going to be ( an operation ) . he told me no , because it was too low , the baby was stuck and had no room to turn . [ ] people tell so many things , there is a mystery around it . [ a woman interviewed at home ] the women s stories generally referred to fears of medical procedures , including cs , as reflected in the statement : once a cs , always a cs ! , highlighting the fear of repetition . another aspect often mentioned by the women was a lack of information during their antenatal consultations about the possibility of a cesarean birth . the interviewees regularly referred to their fear of poor and slow healing of the scar after a cs and the resulting physical side effects , eg , back pain and fatigue . there was intense fear associated with the risk of dying during childbirth , which is probably higher in women who know women who have died during childbirth . it was serious , she arrived there in the morning , and at 10 pm she had still not given birth . they told her to push , she did not want to push , she did not push , she tightened her buttocks , until she was referred to the hospital . they brought her inside , they came out to hand over the baby , they returned inside . when they returned we saw that they were taking her body to the morgue . seeing [ member of a patient focus group linked to an urban maternity ward ] for others , fear of cs was related to hearing other people s horror stories and tales of serious consequences , such as paralysis of the lower limbs . fear of childbirth , and more specifically of cs , is inseparable from the medical , social , and financial implications facing women : if you re going to give birth , you re scared because you do not know how it will happen . you can get a prescription but the money turns out to be a problem [ ] [ member of a patient focus group linked to an urban maternity ward ] men are expected to meet the obstetric care expenses.27,28 very few women know the cost of their cs . this fact does not imply negligence on the part of the women , but reflects the distribution of tasks within marriage . , men may think it indiscrete if a woman asks this type of question . [ ] when you give birth , except your husband , no one else will come and pay the expenses . even if your husband has no money , he will borrow and has to reimburse later . [ ] if you have given birth , the man must do anything to get the money and come and pay . [ member of a patient focus group linked to an urban maternity ward ] families are rarely prepared to cover the additional costs of childbirth . in fact , the urgency solves the problem ( when things get hot , we find it [ ] ) . however , there seems to be a gap between the social norm and reality . in urban areas and in the context of economic crisis , negotiations and arrangements between men and women about the costs of pregnancy and childbirth are not uncommon . among men , there are those who are able to pay the costs of delivery , those who do not want to pay , and a large group who do not have the means to pay . in the latter case the strategy adopted by the woman is to ensure that the members of her entourage ( family members and caregivers ) do not find out that she is the one who pays . for the woman , it is an act of saving her husband s reputation and showing that he is capable of meeting the costs of pregnancy and childbirth . for example , as i know that my husband did not have the means , i gave him money on the day i was coming out of hospital , take it , go and pay and we ll get out , it was my husband who paid , [ women laugh ] . [ member of patient focus group linked to an urban maternity ward ] a teacher explained , for example , that she had covered the costs of her three pregnancies since her husband lost his job . even for the health care providers , exchanges between caregivers and the patient s companions are limited to giving prescriptions and claiming consultation fees . in poorer settings , it is difficult to prepare financially for childbirth . other than the profits from a small business , women try to save on the money their husbands give them to buy condiments , but do not tell them about these savings . , men do not feel concerned by the problems faced by their wives during pregnancy . in addition to the woman s psychological anxiety , pregnancy and childbirth seem to add tensions to her relationship with her partner . the man seems to reach a critical point where his reputation in his close circle is at stake , and rests on his capacity to pay hospitalization bills . motherhood thus becomes a threat to the social status of both the man and the woman . fear or refusal of certain medical procedures ( including emergency referral to the hospital ) is mainly due to concern about incurring higher costs in a complex medical environment where patients receive little information.29 communication or the lack of it was a recurrent theme in the various interviews conducted at home and in the focus groups . in addition to this lack of information , the interactions between health care providers and users seem underpinned by a breakdown of communication . for example , some women said that they were not informed of the decision to perform a cs in the delivery room . she did not have the chance to see them and said she had not been informed about the cs : they put me on a stretcher in the maternity ward to bring me to the operating theater . [ young woman , mother of one child , during interview conducted at home in february 2004 ] in many cases , information given by staff is not understood because it is too vague . one of the objectives of having a health care provider on the team of interviewers during the home visits was to improve this communication and help providers to realize that the information given can have a significant impact in the weeks following a cesarean birth . for example , rosalie , a 25-year - old woman who was told that she needed an operation because her baby was too large ( her medical file stated generally contracted pelvis ) , said that she would ensure that her next child was smaller . she told the interviewers when she was interviewed at home that she would avoid eating food that boosted her baby s growth . another woman who was told to avoid soaking the wound in water after a cs interpreted this as meaning that she could not wash herself . during the home visit , several days after her return home , the home visit provided the opportunity to show the mother how to wash herself . in order to facilitate free discussion , initially , many patients were surprised or scared when midwives and anthropologists visited them in their homes . a caregiver visiting a patient at home to listen to her story was unheard of in the local history of burkina faso . one woman , whose husband the team wanted to interview , later confided the reason for her reluctance : i was scared and wondered what my husband had done for the team wanting to see him ! however , after some initial surprise , when families realized that they had done nothing wrong and that the health care provider was open and attentive , there was a high demand from mothers for information on personal hygiene , eating habits , and resumption of sexual activity after cs . several papers from nigeria , a country that , like burkina faso , has a low rate of cs , have reported women s experiences of cesarean birth.15,17,18 in a study of 413 women in nigeria reported by aziken et al , high costs ( 19.8% ) ranked third in the list of reasons for refusing cs , after fear of dying ( 31.7% ) and fear of pain ( 29.1% ) . the feeling of failure followed in fourth place ( 7.2%).15 many women reported general mental distress after cs . it is noteworthy that , in nigeria , women who can not deliver vaginally are believed to have not prayed enough , to have been unfaithful to their husbands , or to be witches.15 in another nigerian study including 6,224 patients , ezechi et al reported that only 33.3% of women believed that cs was carried out for a medical indication.17 other reasons given were that hospitals and staff want to make a profit ( 42.7% ) or that young doctors want to practice performing the procedure ( 29.3% ) . in a study of cs in 145 women from ghana , women who preferred vaginal deliveries said that vaginal is best , with no long - term pain , less risk of dying , and less cost.16 women who preferred cs described it as a quick and easy delivery method accompanied by fewer labor pains . there were no significant associations between choice of delivery mode and the individual characteristics of the women ( ie , age , marital status , education , and place of residence ) . women who underwent a cs wanted to avoid the pain of labor and childbirth , and women who chose to deliver vaginally wanted to avoid post - cs pain . fear of dying during or following a cs is real in many african countries , where we see an increased mortality rate even in women undergoing elective cs without risk factors.18,30 mazzoni et al did a systematic review to explore women s preference for cs , and found an overall pooled preference for cs of 15.6% . in a subgroup meta - analysis by region worldwide , the highest preference for cs was found in the americas , comprising the usa , canada , and latin america ( 21.3% , 95% confidence interval 16.426.7),31 and within the american subgroups , the preference for cs was higher in latin america than in the usa or canada . unfortunately , the african region was under - represented in this meta - analysis , with only one study from south africa meeting the inclusion criteria . an exploration of women s preferences for cs in the different regions of africa would be interesting . if we refer to soulet s definition : the vulnerability is understood as endogenous to the characteristics of the social system , interacting with the system properties and those individuals who are members . but those who can not [ ] actively participate in the continuous production of the collective life , become highly vulnerable.32 an african woman who undergoes a cs runs a greater risk of dying than her latin american or asian counterparts , according to a major world health organization study on maternal health.11,30,33 supply problems with essential drugs and poor quality services result in low quality care and can lead to fatal anesthetic accidents . cs carried out in cases of prolonged labor and the resulting infections leave the cs scar vulnerable . the risk of uterine rupture increases in a subsequent pregnancy , and may be fatal if it occurs at home or in a small health center distant from the hospital . in the severe maternal morbidity in west africa ( moma ) study , cs increased the risk of uterine rupture during subsequent pregnancies by eleven - fold . in settings of poor access to emergency obstetric care , a third of women who suffered a uterine rupture died ( 33.3% ) , while half lost their child ( 52%).34 care is expensive and not covered by social security systems . a cs may leave a family in debt , and can endanger a couple s relationship when the husband blames the wife for impoverishment of the family . he may even choose another woman to bear his future children for fear that the present one may need more cesarean births.17,21,35 in some communities , a woman may feel rejected by other women if she is not able to deliver vaginally . a study by ezechi et al reported that 26.8% of patients in nigeria who had a cs said that they would rather die than have another.17 the reasons given for refusing a second intervention were feelings of failure ( 81.2% ) and the financial implications ( 66.5% ) . this study emphasizes the importance of showing that one is strong enough to deliver vaginally . the aversion towards cs is deeply rooted in culture and tradition , and the level of education in this sample of 6,224 women had very little influence on their opinion regarding the issue . ezechi et al described how one woman was rejected by her in - laws after a cs . she was accused of not knowing how to care for the child because she had not experienced the pain of childbirth . during the next pregnancy , she remained brave as long as possible in a small mission clinic but was transferred to the emergency services of the hospital with uterine rupture . several papers from nigeria , a country that , like burkina faso , has a low rate of cs , have reported women s experiences of cesarean birth.15,17,18 in a study of 413 women in nigeria reported by aziken et al , high costs ( 19.8% ) ranked third in the list of reasons for refusing cs , after fear of dying ( 31.7% ) and fear of pain ( 29.1% ) . the feeling of failure followed in fourth place ( 7.2%).15 many women reported general mental distress after cs . it is noteworthy that , in nigeria , women who can not deliver vaginally are believed to have not prayed enough , to have been unfaithful to their husbands , or to be witches.15 in another nigerian study including 6,224 patients , ezechi et al reported that only 33.3% of women believed that cs was carried out for a medical indication.17 other reasons given were that hospitals and staff want to make a profit ( 42.7% ) or that young doctors want to practice performing the procedure ( 29.3% ) . in a study of cs in 145 women from ghana , women who preferred vaginal deliveries said that vaginal is best , with no long - term pain , less risk of dying , and less cost.16 women who preferred cs described it as a quick and easy delivery method accompanied by fewer labor pains . there were no significant associations between choice of delivery mode and the individual characteristics of the women ( ie , age , marital status , education , and place of residence ) . women who underwent a cs wanted to avoid the pain of labor and childbirth , and women who chose to deliver vaginally wanted to avoid post - cs pain . fear of dying during or following a cs is real in many african countries , where we see an increased mortality rate even in women undergoing elective cs without risk factors.18,30 mazzoni et al did a systematic review to explore women s preference for cs , and found an overall pooled preference for cs of 15.6% . in a subgroup meta - analysis by region worldwide , the highest preference for cs was found in the americas , comprising the usa , canada , and latin america ( 21.3% , 95% confidence interval 16.426.7),31 and within the american subgroups , the preference for cs was higher in latin america than in the usa or canada . unfortunately , the african region was under - represented in this meta - analysis , with only one study from south africa meeting the inclusion criteria . an exploration of women s preferences for cs in the different regions of africa would be interesting . if we refer to soulet s definition : the vulnerability is understood as endogenous to the characteristics of the social system , interacting with the system properties and those individuals who are members . but those who can not [ ] actively participate in the continuous production of the collective life , become highly vulnerable.32 an african woman who undergoes a cs runs a greater risk of dying than her latin american or asian counterparts , according to a major world health organization study on maternal health.11,30,33 supply problems with essential drugs and poor quality services result in low quality care and can lead to fatal anesthetic accidents . cs carried out in cases of prolonged labor and the resulting infections leave the cs scar vulnerable . the risk of uterine rupture increases in a subsequent pregnancy , and may be fatal if it occurs at home or in a small health center distant from the hospital . in the severe maternal morbidity in west africa ( moma ) study , cs increased the risk of uterine rupture during subsequent pregnancies by eleven - fold . in settings of poor access to emergency obstetric care , a third of women who suffered a uterine rupture died ( 33.3% ) , while half lost their child ( 52%).34 care is expensive and not covered by social security systems . a cs may leave a family in debt , and can endanger a couple s relationship when the husband blames the wife for impoverishment of the family . he may even choose another woman to bear his future children for fear that the present one may need more cesarean births.17,21,35 in some communities , a woman may feel rejected by other women if she is not able to deliver vaginally . a study by ezechi et al reported that 26.8% of patients in nigeria who had a cs said that they would rather die than have another.17 the reasons given for refusing a second intervention were feelings of failure ( 81.2% ) and the financial implications ( 66.5% ) . this study emphasizes the importance of showing that one is strong enough to deliver vaginally . the aversion towards cs is deeply rooted in culture and tradition , and the level of education in this sample of 6,224 women had very little influence on their opinion regarding the issue . ezechi et al described how one woman was rejected by her in - laws after a cs . she was accused of not knowing how to care for the child because she had not experienced the pain of childbirth . during the next pregnancy , she remained brave as long as possible in a small mission clinic but was transferred to the emergency services of the hospital with uterine rupture . cultural perceptions associate vaginal birth with femininity . a cesarean birth is not valued by women or their families because it is seen as a failure to reach woman - mother status . given that this status exists only through the eyes of others , the sense of failure acquires its full meaning indirectly via discourse with family or community members . . the cost of cs can be quite substantial for urban and rural households . even if exemption schemes exist , the costs remain important for the family and especially for the man , who assumes social responsibility for health care spending . finally , both parties are left with questions about the need for such an intervention in the future . popular perceptions are also related to the quality of care that women receive for their cs . testimonies of their fears point to a crisis of confidence with regard to the procedure and the impact it can have on women s health . fear of dying and fear of side effects reveal the level of distrust in health care services . poor quality of care and the economic burden of cs place women in a multifaceted situation of vulnerability within their families . cs has a medical , emotional , social , and economic impact for poor african women . impact of cs can not be underestimated and the intervention can not be performed without a medical indication in such a context of substandard care . maternal health program managers need to understand women s perceptions of cs so as to overcome existing barriers to this life - saving procedure . we encourage researchers to conduct similar research among users of maternity wards and women who have undergone cs to see if the increased affordability of cs has changed their experiences and perceptions of the procedure . for women with cesarean section , bogodogo district , ouagadougou number of children profession spouse s level of education address / telephone number notion of a difficult pregnancy how was this pregnancy ? if so , when did this happen ? where ? can you tell us a little about how it happened ? explain the criteria of choice for the medical facility have you attended the discussion sessions ( causeries ) given by the midwife during pregnancy ? what did the midwives say during these sessions ? during the antenatal consultation , what did caregivers tell you personally ? do you have any idea when the pain started ? who did you discuss it with ? what care did you receive , if any ? how did you come to maternity ward ? who decided to send you ? who accompanied you to the maternity ward ? who helped you to give birth cost and financial players of pregnancy and childbirth for the antenatal consultation , how much did you pay ? how much did you pay for delivery ? knowledge of the health facility did you know about the existence of the maternity ward ? for the next delivery , would you choose to give birth in the maternity ward ?

backgroundthis paper explores women s experience and perception of cesarean birth in burkina faso and its social and economic implications within the household.methodsfive focus groups comprising mothers or pregnant women were conducted among residents of bogodogo health district in ouagadougou to assess the perceptions of cesarean section ( cs ) by women in the community . in addition , 35 individual semistructured interviews were held at the homes of women who had just undergone cs in the referral hospital , and were conducted by an anthropologist and a midwife.resultshome visits to women with cs identified common fears about the procedure , such as once you have had a cs , you will always have to deliver by cs . the central and recurring theme in the interviews was communication between patients and care providers , ie , women were often not informed of the imminence of cs in the delivery room . information given by health care professionals was often either not explicit enough or not understood . the women received insufficient information about postoperative personal hygiene , diet , resumption of sexual activity , and contraception . overall , analysis of the experiences of women who had undergone cs highlighted feelings of guilt in the aftermath of cs . other concerns included the feeling of not being a good mother who can give birth normally , alongside concerns about needing a cs in future pregnancies , the high costs that this might incur for their households , general fatigue , and possible medical complications after surgery.conclusionpoor quality of care and the economic burden of cs place women in a multifaceted situation of vulnerability within the family . cs has a medical , emotional , social , and economic impact on poor african women that can not be ignored . managers of maternal health programs need to understand women s perceptions of cs so as to overcome existing barriers to this life - saving procedure .

Introduction Materials and methods Study setting Individual semistructured interviews Focus groups with women Results Fear of cesarean birth: its unpredictability and consequences Social and economic consequences of cesarean birth Lack of communication about CS by health care providers Discussion Aversion to cesarean birth Physical, medical, economic, and social vulnerability Conclusion Supplementary material

PMC3105556

based on the concept of emotional stability as defined in the literature , 250 items were developed covering initially the following 6 dimensions of emotional stability : pessimism vs. optimismanxiety vs. calmaggression vs. tolerance.dependence vs. autonomy.emotions vs. logicapathy vs. empathy pessimism vs. optimism aggression vs. tolerance . the items were given to the judges , who were qualified psychologists with minimum 10 years of experience in the field of personality assessment . out of these , 90 items were such where the judges could not reach a consensus , therefore these items were eliminated and only 160 items were retained for the initial trial . besides this , the judges felt that there was an overlapping of items in the last two subscales i.e. emotions vs. logic and apathy vs. empathy . hence the items of these subscales were merged together and the scale had only 5 dimensions . pessimists are gloomy and depressed , disappointed with their existence and at odds with the world . they have low self - esteem , are introverts , have feelings of guilt , interpersonal dependency , and remain passive in social situations . on the other hand optimists are generally cheerful and positive in their outlook . they are satisfied with themselves , find life rewarding , and are at peace with the world . they show persistence in seeking goals in spite of setbacks and obstacles , operating from the hope of success rather than fear of failure . they perceive failures as being due to manageable circumstances rather than a personal flaw . example : do you seem to have more than your share of bad luck ? . people who are apathetic are detached , shrewd , worldly and expedient and harbor self- interest in their dealings with other people . people who are empathic in nature consider other 's feelings along with related factors in the process of making intelligent decisions . example : does it worry you if someone is annoyed with you for a mistake , which you have actually not committed ? the dependent person lacks self - reliance , thinks of himself as a helpless pawn of fate , is pushed around by other people and events and shows a high degree of authoritarian submission ( the unquestioning obedience to institutional power ) . the persons high on autonomy enjoy a great deal of freedom and independence , make their own decisions , view themselves as a master of their own fate and take realistic actions to solve their own problems . example : do you place your trust in supernatural powers such as god or fate to see you through safely ? anxious persons are easily upset by things that go wrong and are inclined to worry unreasonably about things that may or may not happen . people who are calm are placid , serene and resistant to irrational fears and anxieties . because of this ability they can stay calm under pressure . example : do you often feel restless as though you want something but do not really know what ? aggressive individuals are given to the direct or indirect expression of anger , for example , behavior such as temper tantrums , fighting , violent arguments and sarcasm , or participation in adventurous activities like mountaineering , car rallies etc . they take no nonsense from anyone and feel compelled to return fire or get back at anyone who transgresses against them . tolerant individuals are gentle , even tempered , with no personal conflicts and are not given to violence either direct or indirect . an answer sheet was developed , wherein the subjects had 5 alternatives to answer each question i.e. always , never. since the test was a combination of both positive and negative items the scores for positive items ranged from 5 to1 and for negative items it ranged from 1 to 5 . pessimists are gloomy and depressed , disappointed with their existence and at odds with the world . they have low self - esteem , are introverts , have feelings of guilt , interpersonal dependency , and remain passive in social situations . on the other hand optimists are generally cheerful and positive in their outlook . they are satisfied with themselves , find life rewarding , and are at peace with the world . they show persistence in seeking goals in spite of setbacks and obstacles , operating from the hope of success rather than fear of failure . example : do you seem to have more than your share of bad luck ? . people who are apathetic are detached , shrewd , worldly and expedient and harbor self- interest in their dealings with other people . people who are empathic in nature consider other 's feelings along with related factors in the process of making intelligent decisions . example : does it worry you if someone is annoyed with you for a mistake , which you have actually not committed ? the dependent person lacks self - reliance , thinks of himself as a helpless pawn of fate , is pushed around by other people and events and shows a high degree of authoritarian submission ( the unquestioning obedience to institutional power ) . the persons high on autonomy enjoy a great deal of freedom and independence , make their own decisions , view themselves as a master of their own fate and take realistic actions to solve their own problems . example : do you place your trust in supernatural powers such as god or fate to see you through safely ? anxious persons are easily upset by things that go wrong and are inclined to worry unreasonably about things that may or may not happen people who are calm are placid , serene and resistant to irrational fears and anxieties . because of this ability they can stay calm under pressure . example : do you often feel restless as though you want something but do not really know what ? aggressive individuals are given to the direct or indirect expression of anger , for example , behavior such as temper tantrums , fighting , violent arguments and sarcasm , or participation in adventurous activities like mountaineering , car rallies etc . they take no nonsense from anyone and feel compelled to return fire or get back at anyone who transgresses against them . tolerant individuals are gentle , even tempered , with no personal conflicts and are not given to violence either direct or indirect . an answer sheet was developed , wherein the subjects had 5 alternatives to answer each question i.e. always , never. since the test was a combination of both positive and negative items the scores for positive items ranged from 5 to1 and for negative items it ranged from 1 to 5 . based on stratified random sampling method , the scale was administered on 200 college students in the age range of 17 to 21 years . item analysis was carried out by calculating the t values for each item using allen 's , formula . the t values for 160 items ranged from .94 to 9.73 . out of these a total of 50 items with highest the above mentioned scale is a 50 item self - administered 5 point rating scale comprising of 10 items from each of the abovementioned dimensions . the scores range from 5 to 1 for positive items and from 1 to 5 for negative items . hence the minimum score on the scale can be 50 and the maximum score can be 250 . cronbach 's alpha was computed as an index of internal consistency for the 50 items of the scale on a sample of 400 college students in the age range of 17 to 21 years . content validity was seen through collective agreement among the judges as well as empirically through item analysis . construct validity of the scale was measured in terms of inter - correlation between subscales of emotional stability scale and total scale . correlation matrix in table 1 reveals that the inter - correlation among various subscales ranged from .135 between aggression vs. tolerance and apathy vs. empathy to .503 between dependence vs. autonomy and pessimism vs. optimism . all the subscales showed significantly positive correlation with the total score on the test ranging from .33 ( aggression vs. tolerance ) to .63 ( anxiety vs. calm ) . inter - correlation between emotional stability subscales and total scale the norms are given in the form of cumulative percentages based on the frequency distributions of the total score . based on the obtained frequencies as shown in table 2 , 181 can be taken as minimum cut off score for a normal individual . in terms of mean and standard deviations 12.79 ) , which means the scores in the range of 174 to 200 can be considered as normal . a 50 item 5 point rating scale is developed based on the psychometric principles of test development to measure various dimensions of emotional stability . however further research in the area can be taken up to develop the norms for various age groups .

background : emotional stability remains the central theme in personality studies . the concept of stable emotional behavior at any level is that which reflects the fruits of normal emotional development . the study aims at development of an emotional stability scale.materials and methods : based on available literature the components of emotional stability were identified and 250 items were developed , covering each component . two - stage elimination of items was carried out , i.e. through judges opinions and item analysis.results:fifty items with highest t values covering 5 dimensions of emotional stability viz pessimism vs. optimism , anxiety vs. calm , aggression vs. tolerance . , dependence vs. autonomy . , apathy vs. empathy were retained in the final scale . reliability as checked by cronbach 's alpha was .81 and by split half method it was .79 . content validity and construct validity were checked . norms are given in the form of cumulative percentages.conclusion:based on the psychometric principles a 50 item , self - administered 5 point lickert type rating scale was developed for measurement of emotional stability .

MATERIALS AND METHODS Pessimism vs. optimism Apathy vs. empathy Dependence vs. autonomy Anxiety vs. calm Aggression vs. tolerance RESULTS AND DISCUSSION CONCLUSION

PMC3933263

one in four people in the world is affected by mental or neurological disorders at some point in their live . about 450 million people currently suffer from such conditions , placing mental disorders among the leading causes of ill - health and disability worldwide . according to the world health organization ( who ) , the vast majority of countries allocate less than 2% of their health budgets to mental health , leading to a treatment gap of more than 75% in many low and middle income countries . the poor often bear the greater burden of mental disorders , both in terms of the risk in having a mental disorder and the lack of access to treatment . constant exposure to severely stressful events , dangerous living conditions , exploitation , and poor health in general contribute to the greater vulnerability of the population . the lack of access to affordable treatment makes the course of the illness more severe and debilitating , leading to a vicious circle of poverty and mental health disorders that is rarely broken . often referred to as the common cold of mental disorders , both anxiety and depression are debilitating conditions that greatly impair psychological , social , and emotional well - being . more than 20% of the adult population suffer from these conditions at sometime during their life . anxiety disorders are common mental diseases of the central nervous system ( cns ) that include heterogeneous phenomena such as panic disorder , phobias , obsessive - convulsive disorders , generalized anxiety disorders , and posttraumatic stress disorders while depressive disorders are illnesses that involve the body , mood , and thought . anxiety and depressive disorders have many causes : on one hand those related to environment , mode of life , modernization of habits , industrialization , worries , stressing professional situations , and economical and financial crisis . on the other hand , we have intrinsic causes that can be genetic , some diseases like cardiac and respiratory insufficiencies and malfunctioning of the thyroid gland . for many years , anxiety and depression were considered as two different mental diseases , with the benzodiazepines used as drugs of choice for acute anxiety states and the amine uptake inhibitors to treat depression . meanwhile the clinicians seem to have become less sure of the original mutually exclusive classification of the two diseases , with margin between anxiety and depression becoming blurred [ 4 , 6 , 7 ] . in the treatment of anxiety disorders , the benzodiazepines are now slowly replaced by antidepressants , which are not only efficacious in depression but also in acute and long - term treatment for major anxiety disorders . the different drugs used act on gaba receptors ( gabaa ) as agonist at the receptors , increasing calcium influx . in a general manner , gaba acts to reduce the firing of the dopaminergic neurons . however almost all pharmacological treatments used to diminish these disorders have adverse side effects including sedation , sleep disturbance , and sexual dysfunctioning and are not accessible to all due to poverty . there is a great interest in fundamental and clinical research to explore new therapeutic targets and new molecules acting on the central nervous system . at the same time , there is a growing interest of people across the country to the use of alternative medicine , particularly in the acute phase of neurological disorders . however , the evidence to recommend the use of herbal medicines in the treatment of these illnesses is still insufficient . there are reports about the popular use of tapinanthus dodoneifolius ( dc ) danser ( loranthaceae ) also called agelanthus dodoneifolius ( polh and wiens ) in treating hypertension and malaria . in the far north region of cameroon , it is used in the treatment of diabetes and cancer . during an ethnobotanical survey in this region we also found that the stem bark of t. dodoneifolius was systematically used by more than two thirds of traditional healers in the treatment of epilepsy . these observations indicate that this plant may certainly have some therapeutic effects on the cns . nevertheless , there is no scientific evidence about potential effects of tapinanthus dodoneifolius on the neurological disorders . the main objective of the present study was then to evaluate the neuroprotective effects and memory improvement properties of tae on animal models of anxiety and depression . animals were adult albino swiss male mice ( 12 weeks , 27 2 g ) . they were purchased from lanavet ( laboratoire national vtrinaire , garoua ) and housed in groups of 5 in polypropylene cages with wood shavings as bedding , under a natural 12 h/12 h light / dark cycle ( lights on at 7:00 a.m. ) and controlled temperature ( 23 2c ) . the animals had free access to water and food , with the exception of 1 h before and during the experiments . the animals were not specifically handled prior to the experiments , with the exception of handling during necessary animal care ( cleaning the cages ) and drug administration ( weighing , tail marking , and drug administration ) . the stem bark of tapinanthus dodoneifolius used in our experiments was collected in october 2012 , from mokon in the far north region of cameroon and identified by mrs . hamawa youkouda , agroforestrist , and froumsia moksia , botanists , at the department of agriculture , livestock and by - products of the higher institute of sahel ( iss ) of the university of maroua . this was later authenticated at the national school of fauna , garoua , where the voucher specimen already existed under the number hefgn 7406 . the stem bark of the plant was chopped into small pieces and shade dried in the laboratory at room temperature for three weeks until all the water evaporate . one hundred gram of dried , ground material was extracted in 1000 ml of boiled distilled water and allowed to infuse for 60 minutes . the extract was later filtered using a whatman gf / c ( 90 mm ) paper and evaporated to dryness in an oven ( 50c ) . the yield of the extract was 3.6 w% . tae ( 25 , 50 , and 100 mg / kg ) , diazepam ( 2 mg / kg ; roche , france ) , and imipramine ( 10 mg / kg , sigma , italy ) as well as vehicle ( 10 ml / kg ) were administered intraperitoneally ( i.p . ) . tae dose was set based on a preliminary ethnopharmacological survey , as administered to human being . preliminary phytochemical screening was carried out according to classic laboratory methods as described by harborne . in this study , the mice were assigned to independent groups ( 5 in each group ) , except in the y - maze test where there were 6 animals per group . the procedures used in the present study were in accordance with the guide for the care and use of laboratory animals eighth edition ( copyright 2011 by the national academy of sciences ) . the experiments were performed in the morning ( 812 h ) , and the illumination level was 200 lux in the experimental room . the elevated plus - maze apparatus consists of four arms ( 25 5 cm ) elevated 40 cm above the floor , with each arm positioned at 90 relative to the adjacent arms . open and closed arms were connected via a central area ( 5 5 cm ) to form a plus sign . mice were randomly assigned to experimental groups : negative control ( normal saline 10 ml / kg , i.p . ) , positive control ( diazepam 2.0 mg / kg , i.p . ) , and tae ( 25 , 50 , and 100 mg / kg , i.p . ) . testing commenced 30 minutes after administration of drug by placing a mouse on the central platform of the maze , facing an open arm . the number of entries into and the time spent on each of the two types of arms were recorded during 5 minutes [ 14 , 15 ] . we also recorded in the apparatus behavioral parameters such as head - dips ( dipping the head below the open arm of the epm , with all four paws on an open arm ) , stretch attend postures ( frequency with which the animal demonstrated forward elongation of the head and shoulders followed by retraction to the original position ) , numbers of grooming ( duration of time the animal spent licking or scratching itself while stationary ) and rearing ( frequency with which the mice stood on their hind legs in the maze ) , and time in the central platform . the plus - maze was thoroughly cleaned with 70% ethanol between tests . the open field arena was made of white polywood ( 72 cm 72 cm 36 cm ) and divided into twelve squares of equal areas . mice were carried to the test room in their home cages and were handled by the base of their tails at all times . the five groups of animals received the various treatments as described above 30 minutes prior to the test . each animal was placed into the center of the open field and allowed to explore the apparatus for 5 minutes . after the 5 minutes test , mice were returned in their home cages and the open field cleaned with 70% ethanol and permitted to dry between tests . the observed parameters were ambulations ( the number of squares crossed with all four paws ) , numbers of grooming and rearing , and time spent at the center and at the border if the arena . the apparatus consisted of a transparent glass cylinder ( 30 cm high 20 cm diameter ) filled to a 20 cm depth with water maintained at 25 2c . in the pretest , mice were placed in the cylinder for 15 min , 24 h prior to the 5-min swimming test . animals were distributed 30 minutes prior to the swimming test as in the epm test , but diazepam was replaced by imipramine at 10 mg / kg . for the 6-minutes swimming test , the following behavioral responses were recorded by a trained observer during the last four minutes : swimming time , defined as movement time throughout the swim chamber , which included crossing into another quadrant , and immobility time considered when the mouse made no further attempts to escape except the movements necessary to keep its head above the water . increases in active responses , such as climbing or swimming , and reduction in immobility , were considered as behavioral profiles consistent with an antidepressant - like action . y - maze test is widely used to assess exploratory behaviors , learning , and memory function in rodents [ 19 , 20 ] . the y - maze apparatus consisted of three identical arms ( 33 11 12 cm each ) in which the arms are symmetrically separated at 120. animals were randomly divided into five groups of 5 or 6 animals : negative control ( 10 ml / kg of normal saline ) ; tae ( 25 , 50 , and 100 mg / kg ) ; and positive control ( diazepam 2.0 mg / kg ) . because we were interested in the cognitive and not in the neuroendocrine changes induced by running - wheel activity , we tried to design our experiment in such a way that stress levels are not minimized . y - maze learning can also be stressful to the mice because of the new environment . no habituation session was undertaken , and by so doing , stress was associated with this task due to the fact that the mice were not allowed to voluntarily enter the apparatus but were placed by the experimenter on an arm of the apparatus . nonhabituated mice were gently placed at the end of one arm and were allowed to freely explore the y - maze during 8 min . the number of arm visits ; sequence of arm visits ; rearing frequency ; grooming frequency ; and the number of lines crossed were recorded for each mouse by a trained observer . the number of maximum spontaneous alternation behaviors was then calculated by subtracting two units from the total number of arms entry , and the percent of spontaneous alternation was calculated as ( actual alternations / maximum alternations ) 100 [ 21 , 22 ] . spontaneous alternation behavior is considered to reflect spatial working memory , which is a form of short - term memory while the total number of arm entries was considered to reflect spontaneous locomotor activity . one way anova followed by newman - keuls multiple comparison test was performed using graphpad prism version 5.00 for windows , graphpad software , san diego california usa , http://www.graphpad.com/. a probability level of 0.05 or less was accepted as significant . pearson 's correlation coefficient and regression analysis were used to evaluate the connection between the working memory errors and some parameters like locomotion , grooming and rearing in the y - maze test . the phytochemical screening revealed that tae contains proteins , alkaloids , glycosides , saponins , tannins , phenols , and flavonoids . in the epm , tae at the doses used significantly ( f(3.28 ) = 5.30 , p < 0.010 ) reduced the number of entries in closed arms in a dose - dependent manner , as compared to the control , with the higher activity at the higher dose , that is , 100 mg / kg ( 3.50 2.06 versus 9.80 2.92 for the control group ) . we also observe that diazepam brought about a highly significant ( f(5.31 ) = 30.67 , p < 0.0006 ) decrease in the number of entries in closed arms and an increase in the open arms ( figure 1 ) . figure 2 shows that in the epm animals of the control group spent more time in the closed arm . when treated with the plant extract at the doses of 25 and 50 mg / kg , no remarkable changes were noticed . the highest dose of tae ( 100 mg / kg ) significantly increased ( f ( 5.31 ) = 7.30 , p < 0.030 ) the time spent by the animals in the open arm compared to the control , with a concomitant decrease of the time spent at the closed arm . this was an index of the anxiolytic effect of the extract at this dose . diazepam as the reference drug also exhibited this pharmacological activity with a more important effect . as shown in figure 3 , the rearing , grooming , and head dipping behaviors were high in control animals in the epm . but when the animals were pretreated with the different doses of tae , the number of grooming significantly ( f(3.28 ) = 4.62 , p < 0.02 ) decreased in a dose - dependent manner with the highest effect obtained at the dose of 100 mg / kg . interestingly , the inhibitory activity of the extract on the grooming behavior was more pronounced than that of diazepam ( 2 mg / kg ) , the reference drug . similarly , the rearing which is a form of vertical locomotor activity also significantly ( f(3.28 ) = 5.70 , p < 0.009 ) decreased with the maximum effect at the dose of 50 mg / kg . the head dipping behavior in the elevated plus maze test was also significantly ( f(3.28 ) = 8.39 , p < 0.002 ) inhibited by the pretreatment of the animal with the extract ( figure 3 ) . when placed in the open field arena , the control animals exhibited ambulatory activities marked by the number of lines crossed ( 94.40 17.35 ) . with the tae pretreatment , the number of lines crossed severely dropped to 24 3.4 and 23 9.7 , respectively , at the doses of 50 and 100 mg / kg ( figure 4 ) . however , the inhibitory effect of diazepam on the locomotion was more pronounced than that of the plant extract . the saline - treated animals passed all the time at the border of the field , spending about 4 min and 66 s of the 5 min test session at that zone . with the tae treatment , a nonsignificant decrease in the time spent at the border of the maze was noticed ( results not shown ) . the results of this test revealed that the swimming time of the animals treated with tae increased in a dose - dependant manner . however , the significant response was obtained only at the highest dose ( 100 mg / kg , ( f(5.31 ) = 10.63 , p < the immobility time increased with the dose of 25 mg / kg and drops with doses of 50 mg / kg and 100 mg / kg . the standard drug in this test , imipramine , also decreased significantly the immobility time ( figure 5 ) . after a single administration of tae , there was an increase in the percentage of spontaneous alternation in animals treated with the intermediate dose ( 66.71% 9.51 ; 50 mg / kg ) of the plant extract , when compared to control group ( 49.80% 8.42 ) , suggesting effects on short - term memory . this increase in the percentage of spontaneous alternations was significant ( f(2.92 ) = 1.93 , p < 0.05 ) ( figure 6 ) . the number of lines crossed in the treated animals decreased in a dose - dependent manner as compared to the saline treated animal ( control ) . the effect obtained at a dose of 50 mg / kg was comparable to that of the standard drug diazepam ( 2 mg / kg ) . tae also significantly reduced the grooming ( f(3.23 ) = 12.73 , p < 0.0002 ) and the rearing ( f(3.23 ) = 9.69 , p < 0.0007 ) behaviors of animals in this task . when linear regression was determined , negative correlation between spontaneous alternation versus number of entries in the maze ( n = 5 , r = 0.745 , p = 0.2678 ) ( figure 8(a ) ) , between spontaneous alternation versus grooming time ( n = 5 , r = 0.716 , p = 0.358 ) ( figure 8(b ) ) however a positive but very low and therefore not significant correlation between spontaneous alternation versus number of rearing in the maze ( n = 7 , r = 0.237 , p = 0.2678 ) was noted ( figure 8(c ) ) . in the present study , the anxiolytic , and antidepressant - like effects of the acute administration of tae were studied in different animal models of anxiety and depression . the effect of this extract on memory improvement was later investigated using y - maze paradigm . the plant extract was first studied using the elevated plus maze which is considered to be an etiologically valid animal model of anxiety because it makes use of natural stimuli . anxiety - like behavior may be represented by an avoidance of the open arm and immobility of an animal placed in the elevated plus maze . in accordance with previously published reports , anxiolytic substances like diazepam increased the percentage of open arm entries and the time spent by animals in the open arms [ 2426 ] . in this work , treatment of mice with tae lead to a dose - dependent and significant ( p < 0.01 ) decrease in the number of entries in closed arms . the time spent in open arms increased but was significant only at 100 mg / kg ( p < 0.05 ) . diazepam , a benzodiazepine anxiolytic drug used as standard drug , also significantly ( p < 0.001 ) increased the open arm exploration . it is quite important to notice that in the epm , some stress parameters like rearing , grooming , and head dipping were significantly reduced when the animals were pretreated with the extract ( p < 0.05 ) or with diazepam ( p < 0.01 ) , when compared to control mice . the increase in time spent in the open arms in the epm is an indicator of reduction of anxiety in rodents . however , in this study , the reduction of the time spent in the close arm was not systematically followed by an increase of the time in the open arm . the anxiolytic effects of benzodiazepine drugs are usually accompanied by decreased ambulatory activity and sedation . taken together , these results highly suggested that the action of tae may be more sedative than anxiolytic . the oft was also used for the investigation of the anxiolytic state of the mice . general activity in the oft measures various behavioral parameters , among those related to emotional , exploratory , and motor behaviors . the first exposure of the animal to the open field has a more marked emotional component than the remaining aspects of exposure . the results obtained with this animal model showed that diazepam ( 2 mg / kg ) , tae at all the doses used , significantly decreased the number of lines crossed by mice ( ambulatory activity ) . moreover , the time spent at the center was also slightly increased . this may indicate that the plant possesses substances that may have sedative or anxiolytic properties . on the other hand , the reduced locomotion pattern of the mice in the oft confirmed the result obtained with the epm and strongly suggested that this plant may produce sedative effects . it is well known that benzodiazepines have sedative and ataxic side effects [ 25 , 30 , 31 ] . this was entirely confirmed in our study , using 2 mg / kg of diazepam . however , it is noteworthy that a reduction in spontaneous activity can be due to a variety of causes other than sedation , such as motor impairment or muscle relaxation . therefore , other behavioral evaluation , such as prolongation of pentobarbital - induced sleep are needed to confirm the sedative effect of tae . moreover since in the forced swimming test the result of the extract was an increase in swimming time , we tend to exclude the muscle relaxation amongst the causes of the reduction of spontaneous locomotion of mice . these findings raised the possibility that the anxiolytic or sedative and other central depressant effects of the extract may be exerted by different phytoconstituents possibly acting through different receptor subtypes or having different affinity for the relevant receptors . nevertheless , the reduction of anxiety in the two animal models used in our study may either be related to the action of the tae on benzodiazepine sites of gabaa receptor complex . the tae may bind to the gamma subunit of gaba - a receptor , increasing the gaba activity in the brain , through an increase in chloride ion conductance and inhibition of the action potential . this proposed mechanism may explain the powerful sedation and the anxiolytic effect of the extract as it is well known that benzodiazepines have this pharmacological profile . however an antagonist on 5-ht13 receptors can also be hypothesis [ 32 , 33 ] . flavonoids with anxiolytic activity have been described in many plant species used in folk medicine such as passiflora caerulea . the tlc phytochemistry of t. dodoneifolius carried out by wahab et al . revealed the presence of terpenoids and terpenoid - related compounds . these substances have proven their efficiency in animal models of anxiety , pain , depression , and epilepsy . the forced swimming test is a well - known behavioral test in rodents that predicts the clinical efficacy of many types of antidepressants and investigates the mechanism underlying their action . various antidepressant medications including serotonin reuptake inhibitors ( ssri ) , tricyclic antidepressants , or nmda receptor antagonists reverse the immobility posture and promote the occurrence of escape - related behavior . the immobility displayed by rodents when subjected to unavoidable stress such as forced swimming is thought to reflect a state of despair or lowered mood , which are thought to reflect depressive disorders in humans . rodents when forced to swim in a cylinder from which they can not escape will , after an initial period of vigorous activity , display a characteristic immobile posture which can be readily identified and is said to reflect a state of despair . moreover , it has some drawbacks represented by the possibility of obtaining some false positive or negative responses . drugs that enhance motor activity may give a false positive effect in the forced swimming test . therefore , this test would not be as a good test for antidepressants such as nomifensine , amineptine , and any plant extract exhibiting myostimulant activities , since these agents increase motor activity . in this study , the single administration of 100 mg / kg of tae provoked significant reduction of the immobility time of mice subjected to forced swimming as compared to the control group . the result was quite comparable to that of imipramine , the tricyclic antidepressant drug used as standard at the same dose of 10 mg / kg , indicating that the extract possesses antidepressant activity on the central nervous system . to discount the possibility of false positive response as shown in the elevated plus maze and in the open field tests , the extract did not increase spontaneous motor activity in mice but had a significant inhibition on the locomotory activity . putting these results together , we can strongly suggest that the antidepressant activity of the tae has no link with any skeletal muscle stimulation . depression is commonly accepted to be a disorder due to disturbances in neurotransmitters function , particularly serotonin , noradrenalin , and dopamine . reduction in brain serotonin has been reported to be one of the most important etiological factors for genesis of depression , and the most widely used antidepressants namely ssris , increase extracellular availability of serotonin [ 18 , 41 , 42 ] . thus tae may exert its antidepressant effect through one or some of these central nervous neurotransmitters acting on glutaminergic receptors , gabaergic receptor , or serotonergic pathways . at this level , it is not possible to give the exact mechanism of action by which t. dodoneifolius acts . however we can hypothesize according to our results that the antidepressant effect of tae appears to be related to enhancement of central noradrenergic and/or serotonergic neurotransmissions . this type of maze tests has been used to assess the spatial learning behavior of mice by many scientists [ 44 , 45 ] . it is also used to determine the effect of different drugs on learning and memory process . it is known worldwide that rodents particularly have exploratory behavior in the maze and this ability comes from their evolutionary history . in fact rodents are small burrowing animals that have spent millennia digging and finding their way around underground tunnels . in this behavioral paradigm , the spontaneous alternation behavior recorded is considered to reflect spatial working memory , which in turn is a form of short - term memory . in the present study , animals treated with a single dose ( 50 and 100 mg / kg ) of tae showed an improvement of the memory compared to control group . however , this improvement was significant only at the dose of 50 mg / kg . it is important to note that the process of memory is really very complex and in our study , the plant extract was acutely given once . it will thus be important and maybe interesting to know the pattern of the results of this test if the extract is given chronically for one or two weeks . in summary , the current work demonstrated that acute administration of the tae produced anxiolytic and antidepressant effects with a sedative side effect on mice and it slightly improves short - term memory . we speculated that the antidepressant effect of tae observed is not due to the inhibition of the locomotory activity . although the action mechanisms of this extract still to be studied , our results bring the pharmacological evidence of the traditional use of this plant for the treatment of some neurological disorders .

we evaluated the anxiolytic and antidepressant effects of the aqueous extract of the bark of tapinanthus dodoneifolius ( tae ) ( danser ) ( 25 , 50 , and 100 mg / kg ) , using open field , elevated plus maze , and forced swimming tests . effect of tae was compared to standard drugs diazepam ( 2 mg / kg ) and imipramine ( 10 mg / kg ) . additionally , the same doses of tae were evaluated on rat 's memory using y - maze task . results showed a significant ( p < 0.05 ; 100 mg / kg ) increase in the percentage of open arm entry and the time spent in the open arms in the elevated plus maze , suggesting an anxiolytic activity of the extract . in a dose - dependant manner , tae at 25 mg / kg significantly ( p < 0.05 ) decreased the number of lines crossed and the rearing behavior in the open field test , suggesting its possible sedative activity . in the forced swimming test , the immobility time of the animal was significantly reduced ( p < 0.05 ) by tae ( 100 mg / kg ) , compared to control , and this effect was quite comparable to that of imipramine . in the y - maze paradigm , tae at 50 mg / kg caused a significant increase in the spontaneous alternations but with a significant decrease in exploratory behavioral pattern . taking these results together , tae improved the spatial memory and showed anxiolytic , antidepressant , and sedative activities . the present results support the anxiolytic and antidepressant activities of tae and , to our knowledge , for the first time , demonstrate its enhancing effect on memory .

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

PMC3110481

in the western cultural tradition the mind tends to be viewed as separated from the body and , in accordance with this tradition , the sciences of the mind try to understand the mind with no reference to the body . in the last few decades , however , this has changed . the cumulative and fast advances of the sciences of the body ( neurosciences , evolutionary biology , genetics , the biological sciences more generally ) make all attempts at studying the mind while ignoring the body less and less plausible . in fact , the idea that the mind is embodied and that to understand the mind it is necessary to take the body into consideration is being accepted by an increasing number of researchers and constitutes the premise of many important current investigations ( barsalou , 1999 , 2008 ; robbins and aydede , 2009 ) . the embodied view of the mind has led to a recognition of the importance of the actions with which the organism responds to the stimuli in determining how the world is represented in the organism 's mind , in contrast to the traditional emphasis on mental representations as either entirely abstract or derived only from sensory input . this action - based view of the mind underlies a number of important ideas such as the grounding of symbols in the interactions of the organism with the physical environment ( harnad , 1990 ) , the mental ( neural ) simulation of actions as a crucial component of all sorts of understanding ( gallese et al . , 1996 ; rizzolatti and craighero , 2004 ) , the mental representation of objects in terms not of their sensory properties but of the actions that the objects make possible ( affordances ; gibson , 1977 ) , the action - based nature of categories ( borghi et al . , 2002 ; di ferdinando and parisi , 2004 ) . the embodied view of the mind is also reflected in computational models which reproduce the part of the body more directly linked to the mind , i.e. , the brain ( artificial neural networks ) and , more recently , the entire body of the organism ( robotics ) , and which have abandoned the disembodied view of the mind which is at the basis of artificial intelligence and of conceptions of the mind as symbol manipulation . however , although the study of the mind can greatly benefit from an embodied conception of the mind , it still has to free itself from another tradition of western culture which constitutes an obstacle to a complete understanding of the mind : the mind tends to be identified with cognition , that is , with knowing , reasoning , deciding , and acting . the other half of the mind is its emotional half and , although the two halves of the mind continuously interact and behavior is a result of both halves , no satisfactory account of the mind can be provided if the science of the mind is only cognitive science . grounded cognition , and the mental representation of objects in terms of the actions with which organisms respond to them . but organisms , including humans , do not only have knowledge , goals , and the capacity to act . they also have motivations and emotional states which play a crucial role in their behavior . current embodied views of the mind tend to be concerned with the cognitive half of the mind but an embodied account of the mind must be extended to the other half of the mind , its emotional half and , in fact , some psychologists and neuroscientists are trying to extend the embodied conception of the mind to emotions ( see , for example , gallese , 2008 ; freina et al . even if one assumes that the mind generally does not contain anything which is unrelated to sensory input and motor output ( which is what embodied theories assume ) , the cognitive and the emotional halves of the mind may not function in the same way . input to the brain can be input from the environment but also input from inside the body , and output from the brain can be external motor output but also changes in the internal organs and systems of the body , and these different sensory inputs and motor outputs may have different characteristics and consequences . this is why we need models that capture both the cognitive half and the emotional half of the mind and their interactions . these models should explicitly indicate both similarities and differences between embodied cognition and embodied emotion . this also applies to the study of the mind through the construction of computational models or robots . robots are the most appropriate tools for exploring embodied theories of the mind because , although in a very simplified form , they reproduce the body of organisms and the physical organ that controls the organisms behavior ( neuro - robots ) , and this is true for both physically realized robots and for robots which are simulated in a computer . however , current robots mostly try to reproduce the cognitive half of the mind but they ignore its emotional half . the robots displace themselves in the environment , move their arms and reach for objects , turn their eyes and their face , but they do not have emotions . some current robots produce postures and movements of their bodies ( mostly , the face ) that in humans express emotions and they can recognize the expressed emotions of humans as a purely perceptual task , but they can not be said to really have emotions and to really understand the emotions of others ( picard , 2000 , 2003 ; breazeal , 2002 ; adolphs , 2005 ; canamero , 2005 ; dautenhahn et al . , 2009 ; robinson and el kaliouby , 2009 ; cf . ( for an attempt at understanding the functional role of emotions in behavior , see ziemke , 2008 . ) . the cognitive half of the mind is the result of the interactions of the brain with the external environment or of processes self - generated inside the organism 's brain ( mental life ) . current robots have artificial brains which interact with the external environment and , in some cases , can even self - generate inputs and respond to these self - generated inputs ( mirolli and parisi , 2006 , 2009 ; parisi , 2007 ) . but current robotics is an external robotics : robots reproduce the external morphology of an organism 's body , the organism 's sensory and motor organs , and the interactions of the organism 's brain with the external environment . in contrast , the emotional half of the mind is the result of the interactions of the organism 's brain with the organism 's body and with the organs and systems that are inside the body . if we want to construct robots that can be said to really have emotions , what is needed is an internal robotics , that is , robots that have internal organs and systems with which the robot 's brain can interact ( parisi , 2004 ) . only an internal robotics can help us to better understand the emotional half of the mind and to construct a complete embodied theory of the mind . . theories in psychology tend to be expressed verbally but verbally expressed theories have limitations because words often have different meanings for different people and because verbally expressed theories may be unable to generate specific , detailed , and non - controversial predictions . the theory is used to construct a robot and therefore , in a sense , it can be directly observed and it can contain no ambiguity because otherwise the robot can not be constructed . furthermore , the theory generates many specific , detailed , and uncontroversial predictions which are the behaviors exhibited by the robot . these predictions can be empirically validated by comparing them with all sorts of empirical facts : the results of behavioral experiments , data on the ecology and past evolutionary history of the organism , and data on the organism 's body and brain . as we have said , robotic models are especially appropriate for formulating embodied theories of the mind because , by definition , a robot has a body and the robot 's behavior clearly depends on its having a body . furthermore , since the brain is part of the body , to be consistent robots should be neuro - robots , that is , robots whose behavior is controlled by a system that resembles the structure and functioning of the brain , i.e. , an artificial neural network . this has the advantage that it becomes possible to examine the internal representations contained in the robot 's brain ( the patterns of activation and successions of patterns of activation in the robot 's neural network ) and to determine if they are embodied or non - embodied representations , i.e. , if they reflect the robot 's actions and the reactions of the robot 's internal organs and systems to sensory input rather than the sensory input . emotional robots which are not controlled by neural networks but by symbolic systems such as those of breazeal and brooks , 2005 . ) what we will do in this paper is describe a number of simple robots that may help us to construct an entire theory of mind as made up of a cognitive half and an emotional half . the first step toward the construction of robots which have both a cognitive and an emotional mind is to construct robots that have many different motivations which can not all be satisfied at the same time and therefore the robots have to decide at any given time which motivation to pursue with their behavior . their behavior is complex : for example , they may approach an object with their legs or wheels , reach the object with their arm , grasp the object with their hand , and put the object in their mouth . but behind this behavior there is a single motivation , say , the motivation to eat . in contrast , a robot 's behavior may be very simple but the robot has many independent motivations . an example is a robot that has both a motivation to eat and a motivation to drink . the behavior which is needed to satisfy the motivation to eat or the motivation to drink may be very simple but the robot has to decide which motivation to satisfy if the two motivations can not be both satisfied at the same time . some current robots do have more than one motivation but it is their user which decides which motivation should control their behavior at any given time , i.e. , what they must do , and in this sense current robots are not really autonomous . ( for attempts at constructing robots that are motivationally autonomous or that take motivational decisions , cf . brooks , 1986 ; maes , 1990 , although the last two references are to symbolically controlled robots . the difference between neural network and rule - based approaches to motivations and motivational decisions is discussed in seth , 1998 , 2007 . ) they have many different and largely independent motivations and one of the most important aspects of their behavior is that they have to autonomously decide which motivation to pursue at any given time . in fact , the behavior of real organisms has two levels of functioning , the strategic or motivational level and the tactical or cognitive level ( ruini et al . , 2010 ) . at the strategic level the organism has to decide which one of its many different motivations will control its behavior at any particular time . at the tactical level , the organism has to execute the behavior which is appropriate to satisfy the motivation which has been decided at the strategic level . imagine an organism which lives in an environment containing both food and water and which to survive has to both eat and drink . food and water are located in different places in the environment so that , at any given time , the organism has to decide whether to approach and reach food , and satisfy its motivation to eat , or to approach and reach water , and satisfy its motivation to drink . once one of the two different motivations has been chosen at the strategic level , the tactical level of functioning of the organism generates the appropriate behavior which allows the organism to satisfy the motivation . we assume that the decision is based on a simple mechanism . at any given time each motivation has an intensity which may vary from time to time , and the organism decides to pursue the motivation which currently has the highest intensity . we use the verb to decide but what we are talking about is implicitly expressed motivations and a purely physical mechanism that compares different motivational intensities , although in complex and verbal animals such as humans , the decision mechanism may involve talking to oneself , making explicit predictions , and producing explicit evaluations . the intensity of the different motivations is determined by two classes of factors : ( 1 ) the intrinsic intensity of the different motivations , which may depend on the overall adaptive pattern of the organism ( e.g. , the organism 's body needs more food than water ) or on the properties of the organism 's environment ( e.g. , food is less abundant than water in the organism 's environment ) , and ( 2 ) the current stimuli arriving to the organism 's sensors from the external environment ( e.g. , the organism presently sees food rather than water ) or from the organism 's own body [ e.g. , the current level of nutrients ( hunger ) is lower than the current level of water ( thirst ) ] or , in more complex animals such as humans , the stimuli which are self - generated ( predicted , imagined , remembered ) in the organism 's brain . once one motivation has been chosen at the strategic level , the organism produces the behavior that satisfies , or should satisfy , the motivation . notice that since the intensity of the different motivations can change very rapidly because of the arrival of new stimuli and for other reasons , the organism should be able to shift very quickly from pursuing one motivation to pursuing another motivation , and this may happen even if the first motivation has not been satisfied or entirely satisfied . that organisms function at two levels , the motivational and the cognitive level , is indicated by the fact that an individual may be good at the motivational level but not very good at the cognitive level while the opposite may be true for another individual . an individual may be good at choosing to satisfy its hunger rather than its thirst because its body needs energy rather than water but then it may not be very good at finding food . in contrast , another individual may be very good at finding both food and water but it makes the wrong motivational decisions , or is slow at deciding or , even more critically , is unable to decide and does nothing . notice that to stay alive and possibly reproduce an individual should be sufficiently good at both levels since both being unable to decide correctly and rapidly and being unable to do what is necessary to satisfy the motivation decided at the motivational level may reduce the organism 's chances of surviving and reproducing or the organism 's well - being . this indicates that a crucial component of behavior is that the organism 's motivational decision mechanism must function effectively and efficiently . it must take the correct motivational decisions and it must take these decisions at the appropriate time , which in many cases means quickly . emotions are states of the organism 's body that allow the motivational decision mechanism to function more effectively and more efficiently . emotional states influence the current intensity of the organism 's motivations in such a way that the organism functions better at the strategic level . the sight of a potential mate might cause an emotional state in the organism that increases the probability that the organism will pursue the motivation to mate rather than other motivations . the sight of a present danger may induce an emotional state that increases the probability that the organism will stop pursuing other motivations and try to avoid the danger , and will do this fast . in more complex organisms such as humans , even the thought of a mate or of a danger may increase the importance of the motivation to mate or the motivation to avoid the danger , with the consequence that the organism will be less likely to pursue other motivations . in the next section we describe some simple robots that have to decide among different motivations and we show that if the robots have emotions their motivational decisions are more correct and efficient . we have constructed a robot which lives in an environment containing both food and a predator ( parisi and petrosino , 2010 ; ruini et al . , 2010 ) . to remain alive the robot has both to eat the food and to avoid being killed by the predator . the predator is not always present but when it appears the robot has to cease looking for food and escape from the predator . the neural network controlling the robot 's behavior has sensory input units encoding the presence and location of both food and predator , motor output units encoding the movements that allow the robot to displace itself in the environment , and an intermediate layer of internal units . to eat the robot has to approach and reach the food elements while to avoid being killed by the predator the robot has to avoid physical contact with the predator . the connection weights of the robot 's neural network are developed using a genetic algorithm with a population of robots that reproduce selectively and with the constant addition of random variations to the inherited connection weights ( mitchell , 1998 ) . they have a circular body with two wheels allowing the robot to displace itself in the environment and sensory organs allowing the robot to perceive what is in front of the robot within a given distance from the robot . the robots neural network has the architecture we have already described . in the other population we add to the robot 's neural network an emotional circuit made up of a certain number of units which receive activation from the sensory units encoding the presence of the predator and send their activation to either the internal units or the output units and therefore influence the robot 's behavior . these emotional units have special properties compared to the other internal units of the robots neural network . they have no bias , they have an activation threshold , and an emotional unit is not only active in the particular cycle in which activation arrives to the emotional unit from the input units but its activation may persist in subsequent cycles . the unit 's activation threshold and the parameters that control how the activation of a unit persists in subsequent cycles all have evolved values . ( for a more detailed description of the emotional circuit and of the results of the simulations , see parisi and petrosino , 2010 . ) the results of the simulations show that the robots with the emotional circuit reach higher level of performance ( they live longer ) than the robots which do not have the emotional circuit in their brain . if we look at the robots behavior , we see that they immediately cease looking for food and fly away when the predator appears , thereby reducing the probability of being reached and killed by the predator . the robots without the emotional circuit are less fast at shifting from the motivation to eat to the motivation to avoid being killed by the predator , and this leads to shorter lives . in other simulations the robots have to make other motivational decisions : they have to decide whether to eat or drink , whether to eat or look for a mate , whether to eat or take care of their offspring , or whether to eat or rest when their body incurs some physical damage that can be healed by resting . the neural network of the robots has sensory units , motor units , and an intermediate layer of internal units and , in addition , it has internal inputs units encoding the current level of energy inside their body ( hunger sensors ) and , for the robots that have to both eat and drink , also internal input units encoding the level of water ( thirst sensors ) , while the robots which can incur physical damage have internal input units encoding the presence of physical damage ( pain sensors ) . all these robots have to take motivational decisions , and their fitness depends on both their ability to take the appropriate motivational decisions with the required rapidity and their ability to produce the behavior which satisfies the motivation which has been chosen . the results of the simulations indicate that for all robots the possession of an emotional circuit allows them to take better and more rapid motivational decisions so that their fitness is higher than the fitness of the robots lacking the emotional circuit . the robots we have described respond to the input from the external environment and/or from within their body not only with actions but also with emotional states , i.e. , states of their emotional circuit , that make their motivational decisions more effective and efficient and therefore their behavior more fit . the emotional circuit of these robots is very simple . the emotional units receive activation from the external environment ( e.g. , the sight of a predator ) and/or from inside the body ( e.g. , the current level of energy in the robot 's body ) and they send their activation to the internal units or to the motor units of the robot 's neural network , thereby influencing the robot 's behavior . the equivalents of our robots emotional units send their activation to various internal organs and systems such as the heart , the gut , the hormonal system , to the muscles of the face , and to other parts of the body , and receive activation from all these parts of the body , and it is this activation which influences the organism 's motivational decisions and therefore the organism 's behavior . in fact , as originally proposed by james and lange ( 1922 ) , and recently elaborated by damasio ( 1994 , 2004 ) and ledoux ( 1996 , 2000 ) , felt emotional states are largely the product of these interactions . however , although very simple , our robots can be said to have emotions and to implement an embodied theory of the other half of the mind . they live alone in their environment , and predators , mates , and offspring are not robots themselves but are only objects which are present in the environment and are perceived by the robots . many animals , and especially humans , are social , which means that they live with conspecifics and a large portion of their adaptive pattern consists in being able to interact appropriately with conspecifics . what is the relation of emotions to sociality ? many motivations in social animals can be satisfied only with the participations of conspecifics , i.e. , they are social motivations . for example , in species that reproduce sexually , an individual can not satisfy its motivation to have offspring without the participation of an individual of the opposite sex . since emotional states help the strategic level of functioning of the organism to take better motivational decisions , social animals tend to have social emotional states that allow them to take better motivational decisions when these decisions concern social motivations . the other way in which emotions and sociality are related is that emotional states are associated with postures and movements of the organism 's body which can be perceived by other individuals and therefore can inform these individuals about the emotional states of the organism . examples of social motivations are the motivation to mate with another individual , the motivation to take care of one 's offspring , and the motivation to be helped or to avoid being damaged by another individual . all these motivations enter into the motivational decisions of the organism which has to decide which motivation to pursue at any given time . hence , social organisms tend to have social emotional states which regulate the current intensity of their social motivations and positively influence their motivational decisions that have to take into account social motivations . it may not be too difficult to construct robots that have social emotions . in some recently completed simulations , the robots can be either female or male and to reproduce a robot has to mate with a robot of the opposite sex . the robots live in an environment with both food and other robots of the two sexes and to leave their genes to the next generation they have both to eat to remain alive and to approach and reach a robot of the opposite sex to reproduce . female and male robots have different colors so that they can be recognized as females or males by conspecifics . after reproductively mating with a male robot , a female robot can not reproduce for a certain number of time steps and during this non - reproductive period the female robot changes its color and this change of color is perceived by the other robots . a pregnant female knows its current state because its neural network has internal input units encoding the pregnant state of its body . the results show that while the average number of offspring is necessarily identical for females and males , males have more reproductive variability than females , that is , there are males with many offspring and males with very few or no offspring while females all have more or less the same number of offspring . furthermore , the robots alternate appropriately between looking for food and looking for mates , with reproductive females behaving differently toward males compared to non - reproductive females , and males approaching only reproductive females . the next step is to add an emotional circuit to the neural network of these robots and to see if in this case too the possession of the emotional circuit leads to better performance . what would make the states of this circuit social emotional states is that , unlike the emotional states of the robots described in the preceding section , the emotional states of these robots would be activated by the sight of a conspecific and they would allow a robot to take better motivational decisions about what to do socially . the emotional states of the robots we have just described are social only in the sense that they are associated with social motivations but they are not social in the sense that they are expressed , that is , they are communicated to other individuals . what appears to be really important to understand the role of emotions in sociality is to construct robots that express their emotional states and in this manner cause other individuals to know their emotional states and to be influenced in their behavior by this knowledge . as we have said , current emotional robots express emotions that they do not have , that is , that do not play any functional role in their behavior . to construct robots that express emotional states which they really have it is necessary to link the emotional circuit of the robots we have described in the preceding section to postures and movements of the robot 's body that can be perceived by other robots . in the robots described in the preceding section , the emotional circuit influences the manner in which the robot responds to both external and internal inputs but it has no interactions with the rest of the robot 's body . as we have said , this is not so in real organisms , in which the emotional circuit of the brain sends its activation to other parts of the body and these parts of the body respond by sending activation to the brain . as we have also said , some of the parts of the body activated by the emotional circuit are external and therefore their state or change of state ( postures and movements ) can be perceived by another individual . in this way an individual may know the emotional states of another individual by observing the postures and movements of the body of the other individual that result from the activations sent to the body by its emotional units . why should the emotional states of an individual be reflected ( expressed ) in the postures and movements of the individual 's body and therefore be accessible to the sensors of another individual ? why should the other individual be able to understand the emotional states of the individual by perceiving the postures and movements of its body ? postures and movements of the body may be simply a by - product of having emotional states , with no specific adaptive value . but the richness of the expression of emotions in some animals , and especially humans , seems to indicate that expressing one 's emotions has adaptive value and has evolved for this reason . this adaptive value is informing other individuals of one 's emotional states . the adaptive value appears to consist in two things : predictability and manipulation . for the other individual it may have adaptive value to know the emotional states of the first individual by perceiving the postures and movements of its body . adapting to the environment is to a large extent to be able to predict the future state of the environment given its present state in order to prepare for the future state . for social animals an important component of their adaptive environment is constituted by conspecifics in that many of the motivations of an individual can only be satisfied with the participation of other individuals . therefore , for a social individual it is important to be able to predict the behavior of other individuals , and knowing the emotional states of other individuals is a powerful predictor of their behavior . this may explain why social animals ( of some complexity ) tend to attend to and to be able to understand the emotional states of other individuals as these emotional states are expressed by the postures and movements of certain parts of their body . but why should an individual care to express its emotional states through the postures and movements of its body so that another individual can know them ? the explanation in this case is not in terms of predictability but in terms of manipulation , where manipulation is behaving in such a way that the other individual will behave in some desired manner . in other words , an individual will express its emotional states because this will induce another individual which is informed of such emotional states to behave in ways which are desirable for the first individual . both functions of the expression of emotional states can be simulated with robots possessing an emotional circuit which interacts with the robot 's body so that specific states of the circuit will cause specific postures and movements in the robot 's body which can be perceived by other robots . this will allow the first robot to let its emotional state be known by other robots and therefore to influence their behavior and will allow the other robots to predict the behavior of the first robot . being able to predict x is an important component of ( or perhaps the same thing as ) understanding x. what is for individual a to understand individual b ? the embodied theory of sociality says that a understands b by simulating b 's perceived actions in its own brain ( gallese , 2010 ) . but the behavior of b does not only consist in doing actions but also in expressing emotions , and sociality implies understanding not only b 's actions but also b 's expressed emotions . hence , a more complete theory of sociality proposes that a understands b not only by observing b 's actions and simulating these actions in its own brain but also by observing b 's expressed emotions and simulating these emotions in its own brain body . ( remember that , while the cognitive half of the mind is a product of the brain , the emotional half is a product of the interaction between the brain and the body . ) there is an accumulating experimental literature on the role of expressed emotions in sociality . ( 2007 ) have shown that how objects are affectively evaluated by an individual is influenced by the gaze and emotional expression of another individual , and ferri et al . ( 2010 ) that the emotional expression of another individual may affect the goal - directed behavior of the observer . these are among the experimental results which our robots that have emotions should be able to replicate . an extension of the embodied view of mind to language proposes , and tries to show with experiments , that we understand nouns by internally representing in our brain the action with which we respond to the object designated by the noun and we understand verbs by simulating in our brain the action designated by the verb ( fischer and zwaan , 2008 ) . however , if the mind has two halves , the cognitive and the emotional half , an embodied conception of language should give an account of both the cognitive and emotional meanings of words . what we do in this section is describe some simple robots that ( begin to ) have language , and to suggest how words can evoke in these robots not only a cognitive meaning but also an emotional meaning . most words are sounds , or phono - articulatory movements that produce acoustic sounds , which co - vary with specific objects ( nouns ) or actions ( verbs ) . the cognitive orientation of most current theories of the mind can also be seen in the privilege accorded to the cognitive component of the meanings of words rather than to their emotional component , where the cognitive component refers to the perceptual properties of the object designed by a noun or to the action designed by a verb while the emotional component refers to the emotional states evoked by the noun or verb . but if we want to construct robots that can be said to have language , it will be necessary that their words evoke emotional states , not only cognitive meanings , in other robots . ( for a more detailed description of the model , see parisi , 2010 . for robotic models of nouns and verbs , see cangelosi and parisi , 2001 . ) our robots live in an environment with other robots and their neural network is made up of two sub - networks , the non - linguistic sub - network ( nol ) and the linguistic sub - network ( l ) . nol is made of input units encoding non - linguistic sensory input ( perceived objects and perceived actions of another robot ) and output units encoding non - linguistic actions ( e.g. , reaching and grasping an object ) . l has input units encoding linguistic sounds ( produced by another robot ) and output units encoding phono - articulatory movements that produce linguistic sounds . both nol and l have a layer of internal units which connects the input units to the output units . in addition , the two internal layers also have horizontal connections linking the internal units of nol to those of l , and vice versa . given these horizontal connections , perceiving an object can lead to executing a non - linguistic action but also to executing a phono - articulatory action , i.e. , producing the word which designates the object . and hearing a word may lead to reproducing the word by executing the appropriate movements of one 's phono - articulatory organs ( imitating the sound of the word ) or to executing a non - linguistic action . when the robot learns language ( which , in children , begins at 1 year of age ) , the robots learns the appropriate connection weights for these horizontal connections . what is the role of these connection weights ? in the robot 's social experience one specific linguistic sound tends to co - vary with one specific object or action , and vice versa . the robot incorporates these co - variations in the connection weights of its neural network so that when the robot hears a linguistic sound and activation spreads from the internal layer of l to the internal layer of nol , the pattern of activation evoked in the internal units of nol is more or less the same pattern of activation evoked by perceiving the object or action which co - varies with the linguistic sound . when the robot perceives an object or action , activation spreads from the internal units of nol to the internal units of l , which causes the robot to produce the sound which in its experience co - varies with that object or action . in other words , the robot is able to both produce and understand language . a crucial assumption of the model we have described is that there is no entity in the robot 's neural network which can be called the meaning of a word . if we assume that the internal units of nol have internal connections linking the units of the same layer , when an activation pattern is evoked in the internal units of nol , this activation pattern will evoke a second activation pattern in the same or other units , then a third , and so on , so that the initial activation pattern is only the first step of a process which has no natural end and no fixed boundaries and is influenced by a number of factors such as the linguistic and non - linguistic context in which the word is being experienced by the robot , inter - individual differences among the robots , the frequency with which a word has been experienced by the robot , and others . notice that the internal layer of nol is not a specialized semantic module but is the entire brain ( minus the l sub - network ) . this implies that the spreading of activation in nol can invade the emotional circuit of the robot 's neural network and in this manner it can trigger emotional states in the robot . words have emotional meaning if a word co - varies in the robot 's experience with non - linguistic experiences that cause emotional states in the robot . this of course is only a first step toward a robotic account of the emotional meaning of words . here is a list of interesting questions that this account should be able to answer . why , while all words have a cognitive meaning , only some words appear to have an emotional meaning ? why the cognitive meaning of words appears to be more well - defined , articulated , and specific than their emotional meaning ? words often appear to have emotional meaning not in isolation but in the context of other words , i.e. , in sentences ( havas et al . , 2007 ) . why this is so ? in some experiments it has been shown that abstract words tend have more emotional meaning than concrete words ( kousta et al . , 2009a ) other experiments indicate that emotional words are processed more rapidly than non - emotional words ( kousta et al . , 2009b ) . theories expressed as robots have two important advantages compared to verbally expressed theories : they are unambiguous because they refer to things that can observed and measured and they generate many detailed and non - controversial empirical predictions , which are the behaviors of the robot and what happens in the robot 's brain . we have described some robotic models that begin to address the question of how to develop a theory of mind that takes into consideration both the cognitive and the emotional halves of the mind . we have shown that if we add an emotional circuit to the neural network that controls a robot 's behavior , the robot 's behavior becomes more effective because the emotional circuit allows the robot to take more correct and faster motivational decisions . unlike current emotional robots , robots that possess this circuit can be said to actually have emotions in that one can show that the circuit plays a clear functional role in the robot 's behavior . we have then addressed the question of how emotions are related to sociality and we have distinguished two aspects of this relation . robots can have social motivations , that is , motivations that can only satisfied with the participation of conspecifics , and emotions can be said to be social if they cause the robot to make better motivational decisions when the choice set includes social motivations . the other link between emotions and sociality is the expression of emotions . the emotional circuit allows the brain ( neural network ) to interact not only with what is inside the body but also with the external body , causing postures and movements of some parts of the body ( especially the face ) that can be perceived by other robots . we have advanced the hypothesis that this expression of emotions has two adaptive advantages : it allows a robot that expresses its emotions to manipulate the behavior of other robots that perceive the expressed emotions and it allows the robot which perceives the emotions expressed by another robot to predict and anticipate the behavior of the other robot . finally , we have briefly discussed which type of neural network should control the behavior of robots that have language and how words can have both cognitive and emotional meanings for a robot . the robotic models we have described are very simple and very tentative and one still has to show that they are able to explain ( reproduce ) the constantly accumulating empirical facts about brain and behavior that involve the other half of the mind . ( for some attempts at simulating the brain involved in emotions , see rolls and treves , 1998 ; rolls , 1999 ; avila - garcia and canamero , 2004 . ) furthermore , we have said that current emotional robots do not actually have emotions because emotions do not play any clearly identifiable functional or adaptive role in their behavior , but the results obtained with our robotic models should be compared with those of alternative models , both robotic and non - robotic . an important test of the models will be their ability to reproduce not only the results of experiments but also other empirical facts such as inter - individual differences in the emotional half of the mind , i.e. , differences in personality and character rather than in cognitive ability , the pathologies of the emotional half of the mind , i.e. , psychiatric and psychological disturbances rather than neurological ones ( stein and ludik , 2008 ) , and the expression of the emotional half of the mind in the production and appreciation of works of art . the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

embodied theories of mind tend to be theories of the cognitive half of the mind and to ignore its emotional half while a complete theory of the mind should account for both halves . robots are a new way of expressing theories of the mind which are less ambiguous and more capable to generate specific and non - controversial predictions than verbally expressed theories . we outline a simple robotic model of emotional states as states of a sub - part of the neural network controlling the robot 's behavior which has specific properties and which allows the robot to make faster and more correct motivational decisions , and we describe possible extensions of the model to account for social emotional states and for the expression of emotions that , unlike those of current emotional robots , are really felt by the robot in that they play a well - identified functional role in the robot 's behavior .

Introduction The Strategic and the Tactical Level of Functioning of Organisms Robots that Have Emotions Emotions and Sociality Emotions and Language Summary Conflict of Interest Statement

PMC4180634

world health organization ( who ) has recommended artemisinin class of compounds as promising novel antimalarials for further clinical development . several semisynthetic derivatives of artemisinin , the active ingredient of chinese herb qing hao ( artemisia annua ) used traditionally for treating fevers , have been used increasingly over past two decades . discovery of artemisinin , as the active principle of chinese traditional drug artemisia annua , has opened new possibilities in malaria chemotherapy . artemisinin and its more potent derivatives ( artemether , artemotil , and artesunic acid , etc . ) these drugs are fast acting and are well suited for treatment of cerebral malaria caused by p. falciparum ( drug sensitive as well as drug resistant species ) . peroxide group , present in form of 1,2,4-trioxane , is essential for antimalarial activity of these drugs . the endoperoxide sesquiterpene lactone moiety of this class of compounds is found to be indispensable for the erythorocytic schizontocidal activity and reacts with intraparasitic heme and forms free radicals . these free radicals appear to damage intracellular targets and perform their antimalarial activity [ 24 ] . however , the existing artemisinin class of drugs is to be improved regarding efficacy , neurotoxicity , stability , and pharmacokinetic ( pk ) behaviors . synthesis of a large number of structurally simple trioxanes have been reported , several of which have shown promising in vivo antimalarial activity . central drug research institute ( cdri ) developed a promising antimalarial compound , 97/63 ( racemate ) ( figure 1(a ) ) , in its drug discovery program . 97/63 showed good potency against malarial parasites , in vitro as well as in vivo . it possesses a 1,2,4-trioxane nucleus similar to endoperoxide lactone of artemisinins , essential for antimalarial activity [ 79 ] . several synthetic trioxanes , simplified analogs of artemisnin retaining the crucial endoperoxide bridge , have been reported but only a few have entered successfully in clinical trials till date . firstly , 97/63 was synthesized which showed promising antimalarial activity but , due to its poor bioavailabilty , it was resynthesized as a hemisuccinate derivative and coded as 97/78 which is currently into clinical trials phase i. the 1,2,4-trioxane nucleus present in antimalarial 97/63 is responsible for its pharmacological activity [ 711 ] . in continuation of drug development program after successful completion of pharmacological activities , the preclinical development was undertaken . so , the present study describes pharmacokinetic behavior of 97/63 after single oral and intravenous dose administration in male rats . characterization of preclinical pharmacokinetic studies in animal models is required for toxicological and preclinical studies and also for extrapolation of pharmacokinetics and pharmacodynamics in humans . analytical method was developed and validated in terms of sensitivity , specificity , accuracy , precision , and recovery . the study samples were analyzed using validated hplc - uv assay developed for quantification of 97/63 in rat serum . pure reference standards ( purity > 99% ) of 97/63 and 99/411 ( figure 1(b ) ) used as internal standard ( is ) were obtained from medicinal and process chemistry division , cdri , lucknow , india . acetonitrile , hplc grade , was purchased from thomas baker ( chemicals ) limited ( mumbai , india ) . analytical grade potassium dihydrogen orthophosphate for buffer preparation was supplied by sd fine - chem limited , mumbai , india . orthophosphoric acid ( analytical grade ) for ph adjustment of the buffer was purchased from qualigens fine chemicals , mumbai , india . for sample extraction , n - hexane ultrapure water of 18.2 m cm was obtained from a milli - q plus pf system . drug - free pool of serum was obtained from male rats ( sprague - dawley , sd ) which were obtained from laboratory animal services division , cdri . animals were cared for in accordance with the guidelines laid by local ethical committee for animal experimentation and the principles of the guide for care and use of laboratory animals were followed ( department of health education and welfare , number [ nih ] 85 - 32 ) . hplc system consisted of a pump ( lc-10atvp with scl 10avp system controller , shimadzu , japan ) with quaternary flow control valve system ( fcv-10alvp ) and a degasser ( dgu-14a ) to pump the mobile phase . the detection of 97/63 and is was performed using spd-10a uv - vis detector at 244 nm . chromatographic separation was performed isocratically on spheri-5 , rp-18 column ( 100 4.6 mm i.d . 5 mm ) ( pierce chemicals co. , rockford , il , usa ) coupled with a guard column ( 30 4.6 mm i.d . , 5 mm ) packed with the same material . the mobile phase composed of acetonitrile : phosphate buffer ( 10 mm , ph 4.0 ) ( 70 : 30% v / v ) at flow rate 1 ml min . phosphate buffer was prepared by dissolving 1.36 g of potassium dihydrogen orthophosphate in one liter triple - distilled water and its ph 4.0 was adjusted with 40% orthophosphoric acid . phosphate buffer was filtered with 0.22 m cellulose membrane under vacuum and degassed for 20 min in sonicator ( bransonic cleaning co. , usa ) before use . data was analysed using class - vp software ( shimadzu , japan ) running on compaq pc . hplc system was equilibrated for approximately 30 min at flow rate 1 ml min before the commencement of analysis . the chromatographic run time was 10 min and injection volume was optimized to 20 l . a vortex - mixer ( thermolyne , usa ) , model svc-220h speed vac concentrator ( savant , ny , usa ) and model k130 centrifuge ( bhg hermle ) , was used for sample preparation . serum samples were stored at 60c in ultra freeze u41085 , ultra low freezer ( new brunswick scientific , usa ) . stock solution ( 1 mg ml ) of 97/63 and is was prepared by dissolving separately 10 mg of compounds in 10 ml acetonitrile . working stock solutions of strengths 10 g ml and 50 g ml were prepared by transferring 100 l and 500 l of stock solution into volumetric flask and final volume was made up to 10 ml with acetonitrile . these solutions were serially diluted in mobile phase by adding 10 l ml of working stocks to get 10 , 25 , 50 , 100 , 250 , and 500 ng ml concentrations . spiking solution of is was prepared by diluting 10 l of mother stock with 1990 l of acetonitrile to yield solution of 5 g / ml . these analytical standards were used to determine hplc system reproducibility and absolute recovery for 97/63 from biomatrix used . calibration standards ( cs ) samples of 97/63 from 10500 ng ml in rat serum were prepared by adding appropriate volumes of working stocks in 200 l of pooled rat serum so that the volume ratio of organic phase added was less than 2.5% . qc samples at low ( 10 ng ml ) , medium ( 100 ng ml ) , and high ( 500 ng ml ) concentrations were used for the method validation program . prior to hplc analysis these cs and qc samples were processed according to method outlined in the following paragraph . cs , qc , and test samples were prepared separately using a simple and efficient two - step liquid - liquid extraction process using n - hexane . to the blank or spiked serum ( 200 l ) , 10 l of is was added to each sample to get 250 ng / ml of 99/411 and 3 ml of n - hexane was added . this mixture was vortexed for 3 min followed by centrifugation at 2200 g , at 4c for 10 min . the organic layer was transferred by snap freezing the aqueous layer in liquid nitrogen into a clean tube and evaporated to dryness in speed vac concentrator . the dry residue was reconstituted in 200 l mobile phase and injected for analysis . method development involved use of different columns ( cyano and rp-18 ) to check noninterference in the region of compound of interest with endogenous substances , drug metabolite , and so forth in the determination of concentration . use of different buffers with different ph range ( ph 47 ) was checked in different compositions of various organic solvents . hplc - uv method was validated for five days at three different concentrations ( 10 , 100 , and 500 ng ml as low , medium , and high qc resp . ) in five replicates . the method developed was validated in terms of hplc system sensitivity , specificity , linearity , absolute recovery , accuracy , precision , freeze - thaw ( f - t ) cycle stability , long - term stability at 60c , dry residual stability , and autosampler stability . the lower limit of detection ( lod ) for 97/63 was the quantity in the serum after the sample cleanup corresponding to three times the baseline noise ( s / n > 3 ) . the limit of quantitation ( loq ) is the concentration of sample that can be quantified with less than 20% variation in precision . so matrix effect was evaluated by peak area ratio of the analytes dissolved in the supernatant of processed blank serum to that of analytical standard of same concentration . moreover highly sensitive methods could be affected by carryover , visible in lc - uv detection . in this study carryover was evaluated by analyzing the mobile phase after plasma samples with the highest analyte concentration 2000 ng ml . linearity for cs ( n = 6 ) in five replicates for five days was assessed by subjecting the peak area of spiked concentrations to linear regression analysis ( y = mx + c ) with 1/x weighing factor showed the best fit for the calibration curve . the choice of proper calibration method depends on the residuals obtained and the coefficient of correlation [ 14 , 15 ] . the recovery of 97/63 prior to analysis was determined at qc samples of low ( 10 ng ml ) , medium ( 100 ng ml ) , and high ( 500 ng ml ) concentrations . the extraction recoveries at three qc levels were determined by comparing peak areas obtained from plasma samples with those found by direct injection of standard solutions prepared in mobile phase of same concentrations . accuracy was determined by injection of calibration standards and qc standards in five replicates on five different days ( n = 75 , five each of low , medium , and high concentration ) . the interday and intraday accuracy was determined by calculating % bias from the theoretical concentration using equation ( % bias = ( observed concentration nominal concentration)/nominal concentration 100 ) of quality control samples . the interday and intraday precision was determined by subjecting the data to one - way analysis of variance ( anova ) in terms of relative standard deviation ( % rsd ) . long - term stability of 97/63 was carried by preparing qc samples at low , medium , and high concentrations in three replicates for four different days and stored at 60c . these sets of samples were analyzed after 0 , 7 , 15 , and 30 days of storage and their concentrations were read from the respective calibration standard curve . f - t stability of 97/63 in serum samples was determined by preparing qc of strengths low ( 10 ng ml ) , medium ( 100 ng ml ) , and high ( 500 ng ml ) in three replicates for four different days . one set of three concentrations was analyzed on day of preparation ( no f - t cycle ) before storing the remaining sets at 60c . other sets were analyzed after 1 , 2 , and 3 f - t cycles . thawing was achieved by keeping the sealed tubes at room temperatures for 30 min . the effect of storage on dry residue stability was determined at qc 10 ng ml ( low ) , 100 ng ml ( medium ) , and 500 ng ml ( high ) concentration levels . all the samples ( three replicates of three concentrations 4 sets ) were prepared and dry residue was stored in glass tubes sealed with aluminum foil and then with parafilm . one set in triplicate was reconstituted on day 1 with mobile phase and analyzed on the same day ( without storage ) . the remaining sets were similarly analyzed on days 2 , 4 , and 8 and results are expressed as % deviation . after sample preparation , stability of 97/63 in the autoinjector was evaluated for 4 h ; the maximum duration for which the sample may have to wait in the autoinjector tray for pending analysis and results are expressed in % deviation with initial injection ( t = 0 h ) . male sprague - dawley ( sd ) rats weighing 250 25 g for this study were obtained from institutional laboratory animal division , cdri , lucknow , prior to study . rats were given standard pellets for feeding and filtered water was given ad libitum and they were housed in well ventilated cages which were kept at room temperature on a regular 12-h light - dark cycle . animals were kept in accordance with the guidelines of local ethical committee ( ethical approval 58/08/pkm / iaec ) for animal experimentation and principles of the guide for care and use of laboratory animals were followed ( department of health education and welfare , number [ nih ] 85 - 32 ) . oral and iv study was carried out in two sets ( i and ii ) . each set contained two groups , that is , groups a and b. pk was carried out by dosing 97/63 in two groups ( a and b ) containing four rats in each group for sparse sampling . young and healthy male sd rats weighing 250 25 g ( obtained from laboratory animal division of cdri ) were housed in well ventilated cages and kept at room temperature 25 2c on a regular 12-h light - dark cycle . animals were cared for in accordance with the principles of guide for care and use of laboratory animals . animals in oral study group were fasted overnight to minimize the effects of food on pharmacokinetic profile of 97/63 . intravenous dose was administered as a single bolus injection through tail vein using a 26 g needle after dilating the tail vein . an oral dose of 72 mg kg and intravenous dose of 18 mg kg as suggested by the efficacy studies were , therefore , used in the study . the intravenous formulation for single dose , 18 mg kg , was a solution of 97/63 in dmso . the oral formulation for single dose , 72 mg kg , was also a solution of 97/63 in dmso . the formulations were subjected to quality control ( qc ) checks to ensure their strength and content uniformity prior to use . the strength of formulation for intravenous and oral administration was 18 mg ml and 36 mg ml . compound 97/63 formulated in dmso was administered to overnight fasted rats by oral and intravenous route . the dose volume factor for intravenous administration was 1 ml kg , with 250 g rat receiving 0.25 ml , and for oral administration it was 2 ml kg , with 250 g rat receiving 0.5 ml of formulation . the total volume of blood withdrawn within 24 hours was less than 5% of blood volume . blood samples were collected in glass tubes at 0.083 , 0.25 , 0.5 , 0.75 , 1 , 1.5 , 2 , 3 , 4 , 6 , 8 , 10 , 18 , 24 , 48 , and 60 h after oral dose . blood samples were stored at room temperature for 30 min to separate the serum . serum was separated by centrifugation at 1500 g for 10 min and stored at 60c until analysis . curve fitting and estimation of pharmacokinetic parameters of 97/63 were carried out using winnonlin ( 5.1 ) software program ( pharsight corp , usa ) . the data were tested using gauss - newton algorithm , with noncompartment , one compartment , and two - compartment models after applying different weighting factor to the observed values of the concentrations . decision on the suitability of a particular compartment model in explaining the data was undertaken based on the akaike or schwarz information criterion , correlation coefficient , and scatter of residuals . data are reported as means sd and statistical analysis was performed using one way analysis of variance ( anova ) at 95% confidence interval ( ci ) . the successful analysis of analytes in biological fluids using hplc relies on the optimization of sample preparation , chromatographic separation , and postcolumn detection . each of these steps was carefully optimized for developing sensitive , selective , and reproducible assay of 97/63 in rat serum . the effect of molar strength and nature of buffers in mobile phase on peak shape of 97/63 was studied . buffers like ammonium acetate and potassium dihydrogen orthophosphate were used for the preparation of mobile phase . the use of buffer is particularly important in separating polar compounds on reversed phase column , since a properly chosen ph can alter the retention time of the eluting peaks . the absence of buffer salt resulted in increased retention time , peak broadening , and hence lower sensitivity . appropriate ratio of buffer and organic solvent leads to better separation with decreased retention time , sharpening of peak , and adequate separation from the inherent serum components . different columns ( cyano and rp-18 ) of varying length were used to check the peak shape and retention time . the isocratic elution with 70 : 30 acetonitrile : phosphate buffer at 1 ml min on spheri-5 , rp-18 column showed no interference with any endogenous impurity . the retention time of 97/63 and is was 6.38 0.25 and 8.21 0.07 min , respectively . the hplc system reproducibility was checked with five replicate injections of each analytical standard and the % bias and % rsd were between 12.51 to 12.36 at all concentrations . the variations in the peak areas of 97/63 were maximal at 10 ng ml ( 3.9% ) and were less than 2.0% at all other concentration levels indicating that the system yields reproducible data . the results showed that the variations were within the acceptable limit of p > 0.05 in one - way anova . sample clean - up techniques were also optimized to get rid of interfering endogenous substances without sacrificing the recovery of 97/63 and is . extraction solvents like n - hexane , diethyl ether , ethyl acetate , and different composition of isopropyl alcohol in n - hexane were used . the hplc chromatogram ( figure 2 ) shows no endogenous peak interferes with peaks of interest . the recovery of 97/63 and is from spiked serum sample was calculated by comparing the peak area with those obtained from mobile phase standards . the absolute recoveries of 97/63 over the range 10500 ng ml from serum are summarized in table 1 . the peak area of 97/63 in rat serum varied linearly with concentration over the range 10500 ng ml . the calibration model was selected on individual calibration data by linear regression with or without intercept and weighing factor . the linear regression equation was y = 5.228x 3.869 and the regression coefficient for standard calibration curve was r = 0.999 . the % accuracy and % rsd at three concentrations in five replicates for 97/63 are presented in table 2 for complete validation in rat serum . the result showed that the analytical method was accurate as the intraday and interday variations ( 13.42 to 14.00 ) were within the acceptable limit 20% for low and 15% for all other concentrations . similarly the % rsd for different qc samples was between 14.8 and 4.71 , well within the acceptable limit . 97/63 was found to be stable over a period of 30 days in normal serum when stored at 60c ( figure 3(a ) ) . the analyte was found to be stable after three f - t cycles ( figure 3(b ) ) and the dry residue after extraction was also found to be stable over a period of 8 days when stored at 60c ( figure 3(c ) ) . the percent deviation calculated was within 20% at low concentration and within 15% for all other concentrations . the reinjection reproducibility was established to determine if an analytical run could be reanalyzed in case of unexpected delay in analysis . the same set of qc samples was repeated after one analysis with 4 h gap in between the samples which were stored at 4c and in all cases the deviations were less than 10% . following oral dose administration in male rats , the levels of 97/63 showed biexponential decay up to 48 h. a two - compartment 1st - order pharmacokinetic model without lag time and first - order elimination rate was considered to be the best fit to explain the generated data . mean serum concentration - time profile of 97/63 after single oral dose of 97/63 in male rats is shown in figure 4(a ) . table 3 summarizes the pharmacokinetic parameters of 97/63 after oral administration at 72 mg kg to male rats . the cmax , tmax , and auc0 were found to be 229.24 64.26 ng ml , 1 0.7 h , and 1268.97 27.04 ngh ml , respectively . the distribution ( t1/2 ) and elimination ( t1/2 ) half - life were found to be 0.84 0.14 and 10.61 0.2 h , respectively . serum concentration - time profile of 97/63 in male rats after intravenous administration at 18 mg kg dose has been depicted in figure 4(b ) . the initial concentration ( c0 ) and auc0 were found to be 1799.99 330.24 ng ml and 2025.75 574.3 ngh ml , respectively . the distribution and elimination half - life were found to be 0.45 0.08 and 10.57 0.16 h , respectively . no abnormal clinical symptoms were noted following oral and intravenous administration of 97/63 in male rats . compound analysis by hplc - uv method is a traditional technique for quantification and quantitation as well as easily available at affordable cost . a well developed and validated hplc - uv method is more effective as compared to lc - ms method which is a costly affair . so various parameters responsible for development of a new assay to quantify drugs by hplc were optimized . the chromatographic conditions such as molarity and ph of buffer and type of columns were optimized . the results suggested that the bioanalytical method is accurate , as the bias is within the acceptance limits of 20% of the theoretical value at lloq and 15% at all other concentration levels . the precision around the mean value never exceeded 15% at any of the concentrations studied . data from quality control samples revealed that the proposed method shows adequate specificity , sensitivity , accuracy , and precision . no significant matrix suppression and/or enhancement as well as no endogenous peak interference were observed with peaks of interest . the absolute recoveries of 97/63 over the range 10500 ng ml as well as is from serum were more than 74% . the result showed that the analytical method was accurate and precise over the linearity concentration range . 97/63 was subjected to long - term , f - t cycle , dry residual , reinjection stability studies and the compound showed good stability . following oral and intravenous dose administration in male rats , the levels of 97/63 could be monitored in serum from the first dose sampling point , that is , 5 min . after oral administration 97/63 was rapidly absorbed from the gastrointestinal tract , attaining maximum serum level concentration ( cmax ) at 1 h after oral dose administration . a dominant phase characterized much of the profile and exhibited clear log - linear behavior from ~6 to ~18 h after dose administration . the absorption rate constant ( ka ) was 0.82 h. no metabolite peaks were observed in rat serum . the observed rapid absorption of the compound after administering a single oral dose ( 72 mg kg ) may also be due to the action of cosolvent dmso which is known to have penetration enhancing property . the low bioavailability may be due to first - pass metabolism of drug which might be excreted through the bile or variable rate of gastric emptying and motility of the gastrointestinal tract along the alimentary canal . metabolism in intestinal membrane during course of absorption as well as in liver is of major concern with the oral administration of many drugs , since it can reduce the systemic bioavailability of the drug . during gastrointestinal transit , the drug gets exposed to various ph conditions , gut flora , and enzymes due to which its systematic bioavailability may be reduced . prodrug concepts at present are established tools for better fabrication of the physicochemical , biopharmaceutical , or pharmacokinetic properties of pharmacologically active agents . many times prodrug concept application at early stages of drug discovery program are fruitful rather than as a technique of last report due to high percentage of enhanced and successful uptake of drug by suitable prodrug formation [ 20 , 21 ] . prodrugs provide a rationale and opportunities to reach target physicochemical , pharmacokinetic , and pharmacodynamic properties . they can be designed to overcome pharmaceutical , pharmacokinetic , or pharmacodynamic barriers such as insufficient chemical stability , poor solubility , unacceptable taste or odor , irritation or pain , insufficient oral absorption , inadequate blood - brain barrier permeability , marked presystemic metabolism , and toxicity [ 22 , 23 ] . with these concepts early stage development of prodrugs of 97/63 was undertaken and in due course of time different derivatives of 97/63 were synthesized . among derivatised 97/63 compounds , the present bioanalytical assay was found to be specific , accurate , and precise over the linearity range of 10500 ng ml . liquid - liquid extraction method for sample preparation was efficient which shows extraction recovery of 97/63 from serum more than 74% . there were no stability problems for 97/63 during storage and sample processing hence fulfilling the criteria for bioanalytical methods . the relevance of pharmacokinetics in drug development has been well recognized and systematic interpretation of disposition behavior is of considerable use in reducing the expenditure and time involved in drug development besides optimization of drug therapy . thus , a suitably validated method in different species can lead to rapid pharmacokinetic studies . the observed low bioavailability ( 15.66% ) and high variability of 97/63 can be attributed to lower log p value ( < 3.5 ) which indicates poor absorption of the compound in the aqueous medium and less lipophillic behavior and hence produrg 97/78 was synthesized as hemisuccinate derivative of 97/63 which enhanced the solubility and absorption manyfold .

single dose pharmacokinetics study of 97/63 ( ind191710 , 2004 ) , a trioxane antimalarial developed by central drug research institute , lucknow , india , was studied in rats following intravenous and oral administration . serum samples were analysed by hplc - uv assay . separation was achieved on a rp-18 column attached with a guard using acetonitrile : phosphate buffer ( 70 : 30% v / v ) with uv detector at wavelength 244 nm . serum samples were extracted with n - hexane . two - compartment model without lag time and first - order elimination rate was considered to be the best fit to explain the generated oral and intravenous data . method was sensitive with limit of quantification of 10 ng ml1 . recovery was > 74% . terminal half - life and area under curve ( auc ) after administering single oral ( 72 mg kg1 ) and intravenous ( 18 mg kg1 ) doses were 10.61 h , 10.57 h , and 1268.97 ng h ml1 , 2025.75 ng h ml1 , respectively . after oral dose , 97/63 was rapidly absorbed attaining maximum concentration 229.24 ng ml1 at 1 h. bioavailability of 97/63 was ~16% . the lower bioavailability of drug may be due to poor solubility and first - pass metabolism and can be improved by prodrug formation of 97/63 .

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

PMC1160141

the construction of long dna oligonucleotides , such as synthetic genes , is useful for a number of reasons . for protein expression , it is possible to improve the yield of a desired protein by optimizing codon usage to reflect the relative codon abundance in the host organism used for protein production . long oligodeoxynucleotides can also be used to produce large amounts of rna for structural or biochemical studies by in vitro transcription methods ( 1 ) . many rna molecules studied to - date are under 100 nt in length and the dna oligonucleotides needed for their production are readily synthesized and are commercially available ( up to 100150 nt long ) . as larger rna molecules are studied , or if methods such as the use of cis-3 and/or 5 ribozymes to facilitate rna production ( 24 ) or if rna - based affinity tags to aid in rna purification ( 5 ) are used , it is not possible to purchase dna oligonucleotides of sufficient length to encode the desired rna product . to produce these long dna molecules ( over 120150 nt ) it is necessary to use techniques originally developed for gene synthesis , such as assembly pcr . pcr methods can be used to construct a synthetic gene through the use of a two - step pcr method referred to as assembly pcr ( figure 1 ) ( 6,7 ) . in the first step of assembly pcr , multiple oligodeoxynucleotides that contain overlapping regions anneal , and the dna polymerase extends the primers and fills in the regions between the primers . a range of products of different lengths results from the different possible combinations of annealing that involve less than all the oligodeoxynucleotides . in the second pcr step a pair of primers is introduced that is specific for the full - length oligodeoxynucleotide and the full - length product is selectively amplified from the mixture . the code for assembly pcr oligo maker is written in java version 1.4.1 available from . we have attempted to make this program compatible with most browsers , however , if there is any difficulty running the program we recommend downloading the latest java runtime environment available from sun microsystems . the program was tested on the windows ( 98 , 2000 , xp ) , linux ( fedora core 3 ) , sgi ( irix 6.5 ) and mac ( osx 10.3 ) operating systems and is functional using virtually any web browser . the default method employed by the program for calculating dna melting temperatures uses the nearest neighbor method employing the unified dna dna hybridization thermodynamic parameters of allawi and santalucia ( 8) with a monovalent cation correction to the entropy term as outlined by santalucia ( 9 ) . the default sodium ion ( monovalent cation ) concentration is 50 mm , while the default dna concentration is 0.5 m . alternately , the user can choose to calculate melt temperatures using the method of wallace et al . ( 10 ) where melting temperature ( tm ) = 2(a + t ) + 4(g + c ) . vent dna polymerase was purchased from new england biolabs ( neb ) . upon receipt , the oligodeoxynucleotides for the first step of assembly pcr were diluted to 7 m ( 0.125 g/l ) with double distilled water , while the oligodeoxynucleotides for the second pcr step were diluted to 42 m ( 0.25 g/l ) . for the first pcr reaction 4 l of each oligo , 4 l of 5 mm dntps , 10 l of 10 thermopol buffer ( neb ) , 1.5 l of vent dna polymerase ( 2000 u / ml ) and 68.5 l of double distilled water were combined . this mixture was then subjected to 8 cycles of amplification at 94c ( 1.5 min ) , 54c ( 2 min ) and 72c ( 3 min ) . during the first cycle after the last cycle was completed an additional 5 min 72c elongation step was performed . for the second pcr reaction , 1 l of the crude mixture from the first pcr reaction was mixed with 4 l of each primer , 4 l of 5 mm dntps , 10 l of 10 thermopol buffer ( neb ) , 1.5 l of vent dna polymerase and 75.5 l of double distilled water . this mixture each cycle consisted of a 30 s 94c step , a 2 min 54c step and a 1.5 min 72c step . before the first cycle the pcr amplified dna and a sample of the puc18 vector were then digested with the restriction enzymes hindiii and ecori ( neb ) . the pcr amplified dna was then ligated into puc18 using t4 dna ligase ( neb ) and transformed into dh5 escherichia coli cells . the assembly pcr oligo maker program is designed to be flexible and easy to use . the user can input either an rna sequence or a dna sequence and can alter a number of settings used by the program during the determination of the oligodeoxynucleotide sequences . the user can input the target oligonucleotide sequence in either the 5 to 3 or 3 to 5 directions . for either rna or dna input , the user is queried as to whether the top17 sequence ( 1 ) for t7 rna polymerase priming should be added or not . we will note that for highest rna production the sequence following the t7 promoter should code for two or three guanosine nucleotides ( 1 ) . if rna is used as input , the corresponding dna sequence to be produced is determined and provided as output . the simplest use of the program therefore is to determine the dna sequence needed to produce a particular rna molecule . at this point , the target dna sequence to be produced is known and the program computes the necessary oligodeoxynucleotides for both steps of the two - step assembly pcr synthesis method . by default , the program works by dividing the target dna into an even number of segments with a user - defined maximum length . the user can define the maximum length of the calculated oligodeoxynucleotides to be 40 , 50 , 60 or 70 nt long . the length of overlap between the different oligodeoxynucleotides is optimized so that the overlapping region matches a target tm . the target tm value employed is a user defined variable with choices available between 50 and 60c , the default value is 55c . a consistent tm value among the different oligodeoxynucleotides helps contributes to successful pcr reactions . a key feature to ensure the success of assembly pcr is the necessity that the oligodeoxynucleotides only anneal where desired . if an oligodeoxynucleotides can anneal in multiple ways , in either an intramolecular or intermolecular fashion , the subsequent pcr reactions would produce a host of undesired products . the assembly pcr oligo maker program tests the oligodeoxynucleotides calculated for both pcr reactions for regions of overlap outside of the desired location . if a stretch of nucleotides is found to overlap at a region outside of the desired site , the program calculates a tm value for this region of overlap . if the tm value of the overlapping region detected is greater than a user defined tm threshold a warning appears notifying the user that an undesired overlapping region was detected the user can then make changes to the sequence of the desired pcr product or ignore this warning and proceed to attempt the pcr reaction despite the presence of an unwanted overlap region . alternately , upon detection of an undesired region of overlap the user can choose to calculate oligodeoxynucleotides with a smaller or greater maximum length in order to try and break up the region of unwanted overlap . the default tm value for triggering a warning for the presence of an undesired overlap is 40c and is user variable between 30 and 45c . the output from the program is available in the main window ( figure 2 ) from which the user can cut and paste text in order to save the output . the output consists of all the necessary oligodeoxynucleotides to perform both of the pcr reactions . alternately , the user can view a summary of the input and output in a single page available from it is also possible to cut and paste text from this page to a file . also present in the results window is a view of how the different oligodeoxynucleotides in the assembly pcr reaction align with each other . initially , a dna oligonucleotide target was used as input , and the oligodeoxynucleotides determined by the program were analyzed to check that the correct dna product would result from the two - step pcr dna synthesis method . next , the ability of the program to find regions of overlap outside of the desired region was confirmed . a target dna oligonucleotide containing a repeat region was used as input . depending on where the repeat region was in relation to where the target dna product was divided , the program was able to detect it , and the calculated tm of the unwanted overlap region was reported . finally , the program was experimentally verified by using the oligodeoxynucleotides determined by the program for the two - step assembly pcr construction of a dna molecule that is to be used to produce an rna molecule . the desired rna product is a 191-nt molecule consisting of 5 and 3 cis hammerhead ribzoymes and a core 20-nt region that forms a hairpin structure and is having its structure studied in our lab by nuclear magnetic resonance methods . when using the default value of 70 for the maximum oligonucleotide length , the program broke this 191-nt dna molecule into four segments for the first pcr reaction and produced the two oligodeoxynucleotide molecules for the second pcr reaction ( figure 3b ) . as shown by gel analyses ( figure 3d ) the first pcr reaction produces a diffuse band or smear , while the desired full length product results from the second pcr reaction . this behavior is consistent with previous reports of assembly pcr gene construction ( 6,11,12 ) . the product of the second pcr reaction was cloned into the puc18 plasmid and the correctness of its sequence was verified by dna sequencing . the program we present here for determining the oligodeoxynucleotides needed for the assembly pcr construction of artificial genes has several advantages over similar applications that are currently available . these advantages are in addition to the ability of assembly pcr oligo maker to handle rna as an input and add the top17 sequence to determine the dna sequence needed for rna production . while this is a trivial process , the use of assembly pcr oligo maker does remove the human element from this task that can inadvertently introduce sequence errors into the final product . the other programs available that aid in oligodeoxynucleotide design for the assembly pcr construction of long dna molecules include dna works ( 11 ) , gene2oligo ( 12 ) and dnabuilder ( ) . the major advantage of using assembly pcr oligo maker is that the user has the option of determining the maximum length of oligodeoxynucleotides for assembly pcr that are created by the program . the user thus has the option of choosing to create fewer but longer oligodeoxynucleotides than determined by other programs . working with fewer oligodeoxynucleotides means that the process of dissolving and diluting them and setting up the assembly pcr reaction is simplified and that fewer opportunities for human error are available during the experiment . this function is shown in the example in figure 3a where the 191-nt dna target is broken into four oligodeoxynucleotides of length 56 , 64 , 64 and 59 nt for a total of 243 nt . for this calculation , a maximum length of 70 ( the default value ) was chosen as the maximum oligonucleotide length calculated . when the same target is used as input for dnabuilder the output was 6 oligodeoxynucleotides with a total of 306 nt ; dnaworks had an output of 10 oligodeoxynucleotides comprising a total of 371 nt ; and using this sequence as input gene2oligo produced an output of 13 oligodeoxynucleotides made up of a total of 440 nt . this is almost twice the number of nucleotides as obtained from assembly pcr oligo maker . in assembly pcr oligo maker the user has the option to choose to calculate more and shorter oligodeoxynucleotides if desired . typically , the use of shorter oligodeoxynucleotides may be desired due to concerns that the user may have about using the longer oligodeoxynucleotides in a pcr reaction without performing additional purification steps , such as page purification of the longer oligodeoxynucleotides . we will note that we have had no trouble using oligodeoxynucleotides 6065 nt in length in the assembly pcr reaction using them as provided by the manufacturer , without employing any additional purification steps . an additional advantage of our assembly pcr oligo maker program is its flexibility in handling the input sequence . assembly pcr oligo maker is able to correctly handle input containing spaces in the sequence , as you would include if working with the sequence in units of three nucleotides . the program also allows input in both the 3 to 5 direction as well as the 5 to 3 direction . our program is readily accessible on the web and can be accessed with any web browser and does not depend on the computer operating system used . finally , the output of assembly pcr oligo maker is very clear , it lists the oligodeoxynucleotides needed for the assembly pcr reaction and the two oligodeoxynucleotides needed for the second pcr reaction as well as provide the predicted tm values . all the oligodeoxynucleotides in the output are listed 5 to 3 which is in a form that is suitable for cutting and pasting the sequences into an email or web form for ordering oligodeoxynucleotides from vendors of users ' choice . in summary , we have created a program that is freely available on the internet to aid in the design of oligodeoxynucleotides for the construction of long dna molecules . the program can assist in constructing any dna molecule , but has a focus on creating dna templates for rna production . the sequences reported by assembly pcr oligo maker were experimentally verified by the successful construction of a 191-nt dna molecule . the assembly pcr method for constructing long dna molecules . ( a ) in the first pcr step a pool of oligodeoxynucleotides anneal and are ( b ) elongated to produce a full - length dna molecule . in addition to the full - length product , a host of shorter molecules also results . ( c ) in the second pcr step the desired full - length molecule is selectively amplified from the mixture using primers specific for the desired full - length product . the oligodeoxynucleotide sequences for the first pcr step are found in the assembly oligo box , and the two oligodeoxynucleotide sequences for the second pcr step are found in the two flanking primers boxes . user controlled settings and information about the program are accessible under the file and help menus . the construction of a 191-nt dna molecule using the oligodeoxynucleotides determined by the assembly pcr oligo maker program . ( c ) diagram showing how the four oligodeoxynucleotides anneal to produce the full - length dsdna product ( d ) agarose gel showing the results of the first ( lane 2 ) and second ( lane 3 ) pcr steps .

we describe a computer program , assembly pcr oligo maker , created to automate the design of oligodeoxynucleotides for the pcr - based construction of long dna molecules . this program is freely available at and has been specifically designed to aid in the construction of dna molecules that are to be used for the production of rna molecules by in vitro synthesis with t7 rna polymerase . the input for assembly pcr oligo maker is either the desired dna sequence to be made or an rna sequence . if rna is the input , the program first determines the dna sequence necessary to produce the desired rna molecule . the program then determines the sequences of all the oligodeoxynucleotides necessary for a two - step assembly pcr - based synthesis of the desired dna molecule . the oligodeoxynucleotide sequences outputted are designed to have a uniform melt temperature and are checked for regions of overlap outside of the desired priming regions necessary for the pcr reaction . the validity of the program was verified experimentally by synthesizing a 191-nt long dna molecule using the dna sequences suggested by the program .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION Figures and Tables

PMC2483485

a pigment is a coloured organic or inorganic solid that is usually insoluble in its application media . pigments are used when extreme resistance to light , weather or temperature is needed , e.g. in automotive paints and other outdoor applications . due to its insolubility , a pigment will usually retain its crystal structure throughout the preparation and application process . various physico - chemical properties of pigments , including colour , thermal stability , particle shape , light fastness and fluorescence , are dependent on the crystal packing of the pigment molecules . despite the commercial significance of non - azo organic pigments , only few full crystal structures of these materials have been reported , because single crystals are hard to obtain . the crystallites are often so small that they lead to considerable line broadening in x - ray powder diffraction . single - crystal growth by sublimation methods , on the other hand , rarely produces crystals of suitable quality for conventional x - ray single - crystal structure determination . as a result , the important crystallographic features , controlling the crystal chemistry of such materials , have remained unknown for many pigments . the purpose of this research is to study the feasibility of obtaining the crystal structures of the polymorphs of pigments with the aid of crystal structure prediction ( csp ) tools in combination with limited experimental data . a similar approach was reported by erk ( 2001 ) and schmidt et al . one can obtain a good impression of the state - of - the - art in crystal structure prediction by looking at the results of csp tests organized in recent years by the cambridge crystallographic data centre ( see day et al . blind tests in 1999 , 2001 and 2004 indicated that certain progress has been made in the ab initio prediction of structures of small ( not more than 25 atoms ) rigid molecules containing only c , h , n and o atoms . as an example of an insoluble pigment with poor crystal quality , unsubstituted linear trans - quinacridone , often called quinacridone ( qa ) , a parent compound of various related pigments ( fig . 1 ) , was chosen . a review on various quinacridone - related compounds has been given by lincke ( 2000 ) . in patent reports , at least six polymorphic forms of quinacridone have been reported on the basis of x - ray powder data ( reidinger & struve , 1955 ; struve , 1955 ; manger & struve , 1957 ; filho & oliveira , 1992 ) . , 2002 ) is available from the cambridge structural database ( csd ) , while for the polymorph only the cell parameters , space group and a powder pattern have been published ( paulus et al . , 1989 ; struve , 1955 ) . the structure of the polymorph , derived through calculations and reported in the csd ( lincke & finzel , 1996 ) , is likely to be incorrect , as discussed later . crystal structure prediction of quinacridone polymorphs has been mentioned previously ( leusen , 1996 ) by accelrys ( formerly molecular simulations ltd ) as an example of the use of the cerius polymorph predictor , but the details of the prediction and the resulting structures remained unpublished . the pure and polymorphs of qa were provided by agfa - gevaert nv and used as received . the powder of -qa was obtained by slow water dilution of a concentrated h2so4 solution of qa . for that , a solution of -qa in concentrated sulfuric acid was placed in one test tube and water in a second one . the tubes were connected with each other and the water tube was heated to increase the water vapour pressure in the system . after the precipitation of a dark violet powder in the sulfuric acid solution , the mixture was filtered and the powder of -qa was collected and dried . x - ray powder diffraction ( xrpd ) patterns of the samples were obtained using a philips pw 1710 diffractometer by scanning the samples over 2 angles ranging from 2 to 35 , using cu k radiation with wavelength 1.5418 . crystal structure prediction was conducted using the accelrys polymorph predictor ( pp ) module in the cerius package ( accelrys , 2002 ) . in short , this program generates randomly a number of crystal packings in a chosen space group . the structures that are highest in rank are expected to have the highest probability of being found experimentally . for more details on the theory and background of the prediction procedures implemented in the pp , refer to the work of gdanitz et al . the molecule of quinacridone was first drawn in the three - dimensional sketcher and then energetically minimized in the dreiding 2.21 force field . electrostatic potential derived ( espd ) point charges were assigned to the atoms in the molecule . fock calculations at the given molecular geometry using the quantum mechanics program gaussian94 ( frisch et al . , 1998 ) with the 6 - 31 g * basis set . before starting the prediction procedure , the geometry of the molecule was optimized again in the force field , but this did not contribute any significant changes to the geometry . the dreiding 2.21 force field was also used to minimize the energy of the crystal structures in the final step of the pp procedure . the calculation of the van der waals and coulomb interactions was performed using the ewald summation method ; the dielectric constant r was kept constant ( unlike the default cerius setting of distance proportionality ) . rietveld refinement was performed using the dbws-9006 program ( wiles & young , 1981 ) , implemented in cerius . the refinement was performed in the range of 2 from 2 to 35. only the scale factor , background , cell parameters and the peak profile parameters were varied ; the fractional atomic coordinates were kept fixed at this stage . the atomic coordinates were optimized by lattice energy minimization with fixed unit - cell parameters between the rietveld steps . ab initio structure prediction is usually conducted in the space groups most frequently found among organic solids . 18 space groups together account for more than 93% of the molecular crystals reported ( baur & kassner , 1992 ) . however , since the space groups of quinacridone polymorphs are already known from the literature [ for -qa ( although questionable ) and for and -qa ] , we used this knowledge to limit the choice of the space groups . is not recognized during the pp prediction process , the prediction should be done in a space group that is a subgroup of the desired space group ; the inversion centre is expected to be part of the symmetry elements of the space group . for the present case , the subgroup should contain all symmetry elements of the desired group , excluding the inversion centre . therefore , we conducted the search in the space groups , and only . it is not necessary to search in all three subgroups : with a sufficient number of trials all structures will be found among the structures of each of these subgroups . for practical reasons ( to reduce the calculation time ) , the search was conducted in all three subgroups and the results were subsequently merged . then , the clustering algorithm was applied to remove duplicate structures which resulted in total of 497 structures . minimization of the total potential energy was then performed on the remaining structures , now also relaxing the rigid - body constraints . after the prediction process , an additional step was performed to find the actual symmetry ( with the find symmetry tool ) followed by a further energy minimization of the crystal structures in the space group found if it differed from the initial space group . the latter step is necessary to reach the optimal structure in the space group found , as the atomic coordinates obtained by imposing the new symmetry may not correspond exactly to an energy minimum , in particular in the case of intramolecular symmetry , where small deviations in bond lengths lead to a substantial increase in energy . as the prediction procedure contains a monte carlo stage , it can give different results for different runs . three runs were enough to obtain reproducible results . in order to evaluate the polymorph prediction results , x - ray powder diffraction patterns of the predicted structures were calculated and compared with the experimental ones . due to the limited amount of predicted structures , it was possible to conduct the comparison manually for every structure . the quality of the experimental data was not good enough for indexing the patterns and deducing the structure , but sufficient to evaluate the pp results . the structures with powder patterns closest to the experimental ones were subjected to the rietveld refinement . the prediction , as a result of three consecutive runs , ended with 103 structures ranging in lattice energy from 53 to 28 kcal mol ( 1 cal = 4.184 j ) . we used density as a parameter to present the results in a clearer way and to distinguish structures with the same energy . a higher density might suggest a more stable structure , but this indicator is not as strong as low lattice energies . table 1 gives the information of the first 15 low - energy structures sorted with respect to the lattice energy . the last column shows the attribution of the structures to known polymorphs on the basis of the xrpd pattern . structures in bold are considered to be close enough to the experimental structures to serve as input for the rietveld refinement . . 2 . table 2 gives the crystallographic data of the refined structures and published experimental data on - and -qa for comparison . we only consider the three widely accepted polymorphs ( , , ) , which have distinct powder patterns . additional crystal phases mentioned in patents were reported on the basis of powder patterns of poor quality , giving rise to quite some uncertainty concerning their existence . moreover , one of these phases , designated as ( deuschel et al . , 1963 ) , was proven to be -qa . variations in the crystallite size and shape caused , in this case , the differences in the powder patterns ( potts et al . , 1994 ) . the structure of the polymorph , determined by single - crystal x - ray measurements , is known from the csd ( reference code qnacrd04 ) . the prediction of this structure served as a test to evaluate the quality of the force field and polymorph prediction in general . structures 1 and 2 in table 1 have powder patterns similar to the experimental one ( fig . the difference between the structures is so small that they can be considered to represent the same structure . 3 . each quinacridone molecule forms hydrogen bonds with four neighbours and they are packed in a criss - cross manner in ribbons along . rietveld refinement performed on both structures resulted in r factors of 18.5 and 14.1% ( structures 1 and 2 , respectively ) and nearly the same final structure . in table 2 , we cite data for the second structure , as it showed a better rietveld fit . as a test , the experimental structure qnacrd04 was refined against our experimental powder data in the same way as it was for the predicted structure . the r factor of the fit was 10.8% , a value which is an indication for the order of the accuracy of fits for the other predicted structures , given the limited quality of the experimental powder diffraction pattern . in spite of the commercial significance of -qa and many attempts to grow single crystals , its structure is still unknown in the open literature . at present , only the cell parameters , the space group and a sketchy powder pattern are available ( paulus et al . , 1989 ; struve , 1955 ) . according to these data , -qa has a monoclinic structure , space group and cell parameters as listed in table 2 . the polymorph prediction resulted in several structures for -qa ; all fit the experimental powder pattern well ( fig . 5 ) . however , none of the predicted structures reproduces the small peak at 14 in the experimental pattern , which can be attributed to an impurity . structure 4 , designated here as 1 , is monoclinic , space group , and has a long c axis ( 31 ) . each molecule forms hydrogen bonds with two neighbours , forming ribbons that are arranged in two directions , along and ( fig . the cell parameters of this structure fit well to the data reported in the literature ( table 2 ) . structure 5 , which is labelled as 2 , has the same molecular packing and a similar unit cell as structure 4 , though their cell lengths do not correspond . 7 . the distances between the planes of the molecules in the stacks in these two structures , 1 and 2 , are 2.86 and 2.89 , and the angles between molecules in different stacks are 60.2 and 60.3 , respectively . this may give the impression that the structures are defined in different settings of the space group and that they can be adjusted to have the same cell parameters . however , the unique axis 3.988 in the 1 structure clearly differs from its value of 5.720 in the 2 structure . structure 14 has nearly the same arrangement of molecules and cell parameters as structure 13 , but in space group , which is quite uncommon for a molecule with inversion symmetry . the unit - cell axes lengths of 3 are comparable with the two previously mentioned structures , but the unique axes differ ( table 1 ) . the crystal structures of 3 resemble that of 1 and 2 with the difference that the stacks of hydrogen - bonded molecules are arranged in a nearly parallel way , along ( fig . the hydrogen - bond patterns are similar for all the structures ; each molecule has four hydrogen bonds to two neighbouring molecules and the resulting ribbons are packed in stacks . rietveld refinement of the 1 and 2 structures against the experimental powder pattern resulted in both cases in a reasonable fit with r values of 13.0 and 12.6% , respectively . the corresponding peak in the experimental pattern is broad and makes it impossible to distinguish between these two structures . it is notable that both refined structures are very close in the energy density plot ( fig . both structures of 3 undergo cell compression during the rietveld refinement due to the shortening of the long axis . the energy minimization performed between the rietveld steps forces molecules to leave the nearly parallel arrangement of stacks ( see fig . 9 ) . the refinement converges at r values of 19.7% and 20.6% for the structures 13 and 14 , respectively , and leads to nearly the same structure in both cases . therefore , only the crystallographic data for the refined structure 13 are listed in table 2 . as a result of the rietveld refinement , 1 and 2 show better similarity with the experimental powder pattern , and the published data favour the 1 structure . however , considering that r exp of the structure reported by paulus et al . ( 1989 ) is not known and that the energies of all three structures mentioned here are within the error of the prediction ( a few kcal mol ) , each of them remains a candidate for the experimental structure . it has been derived from the -qa structure by systematic variation of atomic coordinates ( considering chemical bonding constraints ) in order to fit the experimental powder pattern of -qa ( lincke & finzel , 1996 ) . the only difference with the structure is the angle between the molecules in the criss - cross arrangement . using the dreiding force field , we found that this structure converts during minimization readily to the structure of the polymorph ; this makes the qnacrd03 structure questionable . several structures , with numbers 812 in table 1 , are predicted by the pp to have powder patterns similar to the experimental one ( fig . structures 811 are triclinic , space group . each quinacridone molecule shares hydrogen bonds with two neighbours , forming ribbons similar to those in -qa , along . these are packed in such a manner that all molecules in the structure lie parallel to one another ( fig . structure 12 is monoclinic , space group , and it has a different packing motif . crystallization of centrosymmetric molecules in a non - centrosymmetric space group is , however , very rare . the quality of the experimental powder pattern was not high enough to obtain an acceptable fit of the patterns . structures 10 and 11 , as being closest to the experimental powder pattern , were used further for the rietveld refinement . the refinement converged at r values of 19.6% and 21.4% , respectively , and produced the structures at substantially much higher energy as compared with the initial values ( fig . rietveld refinement of the structure 12 resulted in a relatively low r value of 14.3% . visual inspection of the resulting xrpd pattern showed , however , that the structure 12 fitted the experimental pattern badly , mainly due to the mismatch of the peaks in the area 2530. the low r value is misleading in this case . structure 10 , showing the best rietveld fit among all five structures for the polymorph , can be regarded as the closest to the experimental structure . it is debatable if such a high r value is acceptable to confirm the similarity of the predicted structure and the experimental one . a higher quality powder pattern , at least , is necessary to arrive at a better fit . what are the other structures in table 1 ? showing no similarity with the known experimental powder patterns , they have molecular packing and hydrogen - bond patterns that are similar to the polymorphs discussed above . structure 3 has a criss - cross molecular motif , similar to that of -qa , but its powder pattern is very different from the experimental one ( fig . structures 6 and 7 , as 1-qa , consist of hydrogen - bonded ribbons running in two directions . structure 15 contains squeezed layers of molecular ribbons which resemble the packing of the 2 structure after the rietveld refinement . an important question is how certain one can be of the results of the polymorph prediction approach outlined in this paper . the confidence in the results heavily depends on the quality of the force field and the atomic charges used . the dreiding force field ( mayo et al . , 1990 ) , a generic force field , can be applied to a wide range of heteroatomic organic molecules with reasonable accuracy . it shows the best results when used with the charges derived from high - level quantum mechanics calculations ( espd charges ) . it models hydrogen bonds in crystal structures reasonably well . in certain cases , for example hydrogen bonds in acids , the force - field description of the structures in the present case appears to be quite adequate , since all three polymorphs were found within 3 kcal mol . moreover , -qa , usually mentioned in the literature as a stable polymorph at room temperature , is found to have the lowest lattice energy . if calculated lattice energies are close to the sublimation enthalpies , this can serve as another validation of the force field used . lattice energy for the structures listed in table 1 is 5053 kcal mol . the sublimation enthalpy of quinacridone , as for many other pigments , is hard to measure due to the difficulties in precise measurements at high sublimation temperature . a number of promising developments have been reported in the area of force - field development , such as a better description of the electrostatics by means of a multipole expansion ( van eijck et al . , 2001 ) , using a large number of charged cells to describe the charge density ( gavezzotti , 2002 , 2003 ) , or developing a tailor - made potential for the compound under investigation from quantum chemical calculations ( day & price , 2003 ) . as current progress in csp showed , the problem in predicting is not the generation of all the possible structures ( for that , present algorithms are good enough ) , but rather the ranking and choosing the right structures from the list . in the case of pure ab initio prediction , we combine ab initio csp with experimental xrpd patterns to increase the chance of arriving at the right structure . the differences arise from the limited prediction power of the force field and from the bad quality of the experimental diffraction pattern . an absolute match of the powder patterns in such cases is mostly regarded as a pure coincidence and is rather rare . thus , the effectiveness of powder pattern comparison , in order to choose the right structures from the list of predicted ones , varies from case to case . to get an impression of the deviation from the experimental pattern , it is interesting to compare powder patterns for -qa . 12 shows the experimental powder pattern together with four calculated ones , namely calculated from the csd structure qnacrd04 before and after energy minimization in the force field , the powder pattern of structure 2 in the list of predicted structures ( minimized ) , and that of structure 2 after rietveld refinement against the experimental data . in this figure it is remarkable that the patterns ( c ) and ( d ) are identical . this means that the structure predicted by the pp is the same as the experimental structure minimized using the same force field and , therefore , it is probable that these two structures are also identical . 12 also demonstrates how the effect of the force field leading to the difference between patterns ( b ) and ( c ) is eliminated by rietveld refinement . three peaks at 1215 are overlapping in the experimental pattern ( a ) and distinct in the one calculated from the csd structure ( b ) . due to the inaccuracy of the force field , minimization of the energy results in changes in the structure ( fig . 13 ) and the three peaks merge into two in pattern ( c ) . after the rietveld refinement three peaks are resolved again in the pattern ( e ) . these five structures are assumed to be the same , but their powder patterns differ to a certain extent . this difference should be kept in mind when comparing other predicted structures with the experimental powder patterns . rietveld refinement is often used to complete the prediction procedure and to refine the trial structures against powder data . full rietveld refinement is , however , only worthwhile if the experimental data are of excellent quality ( e.g. collected using synchrotron radiation ) and the input structure is close enough to the experimental one . in the case of small particle size or poor crystallinity , like for many pigments , the low quality of the powder diffraction patterns can not be improved by using more powerful x - ray sources . moreover , rietveld refinement is not efficient if the candidate crystal structure is not close enough to the experimental one and too few peaks in the simulated and experimental powder patterns exactly match ( which happens very often ) . an example of the intrinsic ambiguity of powder data can be found in the structure of -qa that was deposited in the csd under the reference code qnacrd03 ( lincke & finzel , 1996 ) . this structure has a molecular packing very close to the criss - cross arrangement of molecules in -qa ( and far from -qa , as predicted by us ) ; only the angle between the molecules is different . however , this criss - cross -qa structure produces a powder pattern very close to the experimental pattern of -qa , which leads to confusion . the reason to have more confidence in our variant of -qa is that it represents a real local minimum in a reliable force field . in this case and in general , similarity of powder patterns of different polymorphs is not uncommon [ for example , two polymorphs of terephthalic acid ( bailey & brown , 1967 ; brown , 1984 ) ] . thus , the methods that use the powder patterns or other types of experimental information solely to distinguish the polymorphs contain by default some degree of uncertainty . for more accuracy , a combination of different physico - chemical characterization methods should be applied . another indication of the quality of the present prediction is the stability order of the polymorphs . according to the calculated lattice energy of the structures , the order of descending stability for the predicted polymorphs is or ( table 1 ) , depending on which structure to accept for -qa . as was mentioned before , experimental data about the stability of these polymorphs are not available . -qa is often mentioned in the literature as the most stable form and -qa as the least stable form ( jaffe , 2001 ) ( -qa is therefore not commercially important ) . , 2007 ) , we observed that -qa sublimed faster and at a lower temperature than -qa . this leads us to the conclusion that , at least at the temperature of sublimation ( 673 k ) , the polymorph is less stable than the polymorph . possibly , -qa is less stable than -qa for the whole range of temperatures ( monotropically related polymorphs ) , since no polymorphic transition could be observed till 773 k ( jones et al . assuming such a monotropic relation , our experimental data confirm the predicted stability order for - and -qa . experimentally observed metastable polymorphs , in general , are not necessarily next in this ranking , even in the case of perfect prediction results , as kinetics determine the circumstances under which they are formed and kinetics are not part of the prediction method . the structures of three polymorphs of quinacridone were predicted using polymorph predictor in combination with xrpd patterns of limited quality . the known structure was predicted correctly as first in the ranking . three different structures were proposed for the unknown -qa that closely match the experimental powder pattern . for -qa , the present work demonstrates a method to obtain crystal structures using polymorph predictor in combination with experimental xrpd patterns . ab initio prediction ( without any help of additional information , like xrpd ) is still not very reliable , even for simple molecules , mainly due to the limited quality of the force field . the use of x - ray powder data helps to improve the reliability of the prediction . the effectiveness of the latter step is limited by the flexibility of the molecule and/or the quality of the xrpd pattern . in the present prediction method , the xrpd pattern need not be indexable . we expect this method to be useful in the case of rigid organic molecules , if a corresponding non - indexable powder pattern of reasonable quality is available . pigments are a good example of the application of this method , since pigment molecules are often rigid and xrpd patterns of pigments are usually difficult to index because of the peak broadening as a result of small crystal sizes . while the paper was in press , another paper on the single - crystal x - ray structure determination of -quinacridone was published ( nishimura et al . the structure reported in this paper fits very nicely our 1-structure , which is the highest in ranking for the predicted -structures .

the structures of the , and polymorphs of quinacridone were predicted using polymorph predictor software in combination with x - ray powder diffraction patterns of limited quality . the present work demonstrates a method to obtain crystal structures of industrially important pigments when only a low - quality powder pattern is available .

Introduction Materials and methods Results Discussion Conclusions

PMC3508359

using exome sequencing and a variant prioritisation strategy that focuses on loss - of - function variants , we identified biallelic , loss - of - function cep57 mutations as a cause of constitutional mosaic aneuploidies . cep57 is a centrosomal protein and is involved in nucleating and stabilizing microtubules . our data indicate that these and/or additional functions of cep57 are crucial in maintaining correct chromosomal number during cell division .

Supplementary Material

PMC5147473

dacron arterial grafts are widely used in thoracic aortic replacement , but few cases of non - anastomotic aneurysmal dilatation or rupture have been reported . in most cases that have been reported , knitted , rather than woven , we report the clinically successful result of thoracic endovascular repair ( tevar ) of the late rupture of a woven dacron graft . the dacron graft had been implanted in the patient s descending thoracic and upper abdominal aorta 20 years previously . a 56-year - old man with a descending thoracoabdominal aortic aneurysm underwent descending thoracic and upper abdominal aorta replacement with a hemashield 20-mm woven dacron graft ( meadox medicals inc . , he was followed up as an outpatient , and no adverse events were noted until 2009 , when he presented with sudden - onset chest pain and severe dyspnea . at that time , chest computed tomography ( ct ) revealed a massive right hemothorax , a large pseudoaneurysm around the implanted graft just above the diaphragm level , and marked contrast medium extravasation from the graft ( fig . 1 ) . the patient underwent an emergency open thoracotomy , in which a disruption was found at the site of the anastomosis between the dacron graft and lumbar intercostal arteries . the disrupted site was repaired with 2 layers of running sutures , and the graft was wrapped with the calcified aneurysmal sac . in 2012 , the patient presented with back pain , abdominal pain , and indigestion . a follow - up chest ct revealed that the size of the aneurysm in the descending aorta had increased in comparison with the findings of the previous ct examination . dye collection in the space between the graft and native aneurysmal sac was also observed , and was suspected to be indicative of a late rupture of the dacron graft itself ( fig . the rupture was unrelated to the previous anastomosis or disrupted site . due to the high morbidity and mortality associated with reoperation , we decided to perform thoracic endovascular stent therapy as an alternative to open thoracotomy for the late graft failure . the primary goal of using tevar was to cover the entire length of the dacron graft in the descending aorta with a stent graft . the right common femoral artery was exposed under general anesthesia , and a guidewire and angiographic catheter were introduced through it . aortic angiography revealed multiple dye leakages from the graft that were caused by late failure of the dacron graft ( fig . 3 ) . after obtaining an aortogram , two stent grafts ( 3030150 mm and 3636100 mm ; s&g biotech inc . , seongnam , korea ) were also introduced through the right femoral artery and placed in the dacron graft . the proximal end of the stent graft was deployed distally to the left subclavian artery , and the distal end was placed just above the celiac trunk . the total length of the thoracoabdominal aorta covered by the stent graft was approximately 220 mm . a postoperative ct scan showed that the disruption of the dacron graft had been successfully treated ( fig . the patient was discharged with no procedure - related complications and , during the 1-year follow - up period , was free of any symptoms . dacron vascular grafts are known to be durable and effective as aortic vascular prostheses . however , a few cases of non - anastomotic aneurysmal dilatation or rupture of these grafts after implantation in the descending thoracic aorta have been reported . the probable cause was degeneration over time of the graft fibers or the round black yarn used as the guideline . intrinsic graft failure of dacron grafts is a rare complication ( 0.5%3% ) ; and in most reported cases , knitted , not woven , dacron grafts had been used . in our case , woven dacron graft rupture occurred 20 years after the initial operation , probably caused by the degeneration of graft fibers over a long time . although woven dacron graft rupture is extremely rare , reoperation is inevitable when it does occur . however , the hospital mortality after aortic reoperation remains high ( 8%15% in many reports ) , and is even higher ( 25%50% ) among patients receiving urgent or emergency care . tevar is increasingly being used for descending aortic diseases as a safe and feasible alternative to open graft repair . in light of the high morbidity and mortality associated with reoperation , we decided to perform endovascular therapy as an alternative management strategy for late graft failure in our patient . to the best of our knowledge , the endovascular repair of primary graft rupture in a woven dacron graft has not been reported . the case presented here demonstrates the effectiveness and feasibility of tevar implantation to treat the non - anastomotic rupture of a dacron graft after descending aortic replacement .

the intrinsic structural failure of a dacron graft resulting from the loss of structural integrity of the graft fabric can cause late graft complications . late non - anastomotic rupture has traditionally been treated surgically via open thoracotomy . we report a case of the successful use of thoracic endovascular repair to treat a dacron graft rupture in the descending aorta . the rupture occurred 20 years after the graft had been placed . two stent grafts were placed at the proximal portion of the surgical graft , covering almost its entire length .

Case report Discussion

PMC4142335

we describe here a method that can rapidly and accurately compute the potentials of mean force ( pmfs ) between spherical univalent ions in water . the pmf between an ion a and ion b represents the reversible work ( as a function of distance between the two ions , r ) , averaged over all the configurations of the surrounding solvent , needed to bring a and b from infinite separation to a distance r. pmfs are central to many solvation processes . for example , some pairs of ions form a stable contact pair or salt bridge ; others form a stable solvent - separated pair and have intervening bridging water molecules . the pmf is relevant to understanding the solubilities of salts in water , the equilibria and dynamics when a drug molecule approaches and binds to a protein binding site , and how some parts of a protein molecule approach other parts as the protein molecule folds . currently , the gold standard for computing the pmfs between large molecules such as are typical in biology is to use explicit - solvent simulations with semiempirical force fields . however , these simulations are often computationally too expensive for many of the calculations that would be of practical interest . even in the case of various sampling techniques have been introduced to mitigate the computational expense : constraints , umbrella sampling , and weighted histogram methods , for example . on the other hand , treating water explicitly in analytical theories is challenging , because of the need to properly account for the orientation ( angular ) effects of water . for simple salt solutions , integral - equation theories often predict qualitatively correct trends but not quantitative details . an alternative is to use implicit - solvent models ( such as poisson boltzmann ( pb ) or generalized born ( gb ) ) , in which the solvent is approximated as a dielectric continuum . implicit - solvent models have the advantage of computational speed : a pmf costs around 100 h of md simulation time in explicit water but only minutes on a personal computer for pb pmf or fractions of a second for gb . pmfs of a model nacl from explicit - solvent md simulation ( symbols ) and implicit - solvent theories : pb ( continuous blue line ) and gb ( dashed green line ) . pb calculations were done with apbs 1.3 software , and gb results were obtained according to the gb model of still et al . the permittivity of water was set to 78.54 in the pb and gb calculations . parameters from dang et al . were used for na and cl ions ( see table 1 ) . however , implicit - solvent models do not capture the subtle features of pmfs , which require some representation of particulate water ( see figure 1 ) . to correct for the energy errors in implicit modeling implicit - solvent models can allow interstitial high dielectrics ( e.g. , water ) , but they give incorrect pmfs between nonbonded atoms . salt bridges , for example , treated via gb models are typically too stable compared to explicit - solvent results , which leads to oversampling such conformations in md simulations . much work has been done to overcome these difficulties , such as the following . a method for including a single explicit bridging water between the pairs of the salt bridge charges was proposed by yu et al . , and it was shown that this significantly improves the predictions . geney et al . introduced an empirical correction to the dielectric radii of hydrogen atoms of charged protein groups , bringing the contact depth of gb pmfs in better agreement with explicit solvent for the tested salt bridges . recently , nguyen et al . did more systematic fits to improve the gb solvent model parameters for protein simulations . to account for the proper coupling between nonpolar and polar solvation energies in implicit - solvent models , dzubiella et al . proposed a variational formalism where the gibbs free energy of the system is expressed as a functional of the solvent volume exclusion function . the method captures the sensitivity of solvent expulsion in cases of simple spherical solutes . proposed an extension of the gb model to correctly describe the solvent - excluded volume of each pair of atoms , and that improved the nonbonded pmfs . an empirical model of chen and brooks captures the context dependence of the effective surface tension , and the authors showed that it can resolve deficiencies in pmfs of nonpolar peptide side chain analogues . we first perform md simulations in explicit solvent as our sort of gold standard for what we aim to capture in a fast computational method . we describe here a way which we call interpolation pmf ( i - pmf)to determine any ion ion pmf from this set of presimulated md explicit - water pmf simulations . i - pmf gets its speed because we simply capture the explicit results by simple interpolations of the precomputed results that we store in look - up tables . we show here that this simple approach results in fast and accurate pmf determinations . i - pmf is in the spirit of a recent solvation model called sea ( semi - explicit assembly ) in which solvation free energies of single spheres are computed through an explicit - simulation precompute step , and then assembled into the solute of interest at run - time by summing regional free energies . we describe below the two components of the i - pmf method : first , we performed extensive md simulations of small spherical ions of different charges and radii , and harvested their pmfs . second , we describe our interpolation algorithm , the i - pmf , that allows us to capture the presimulated md results with interpolation fits . next , we show that the i - pmf method gives good agreement with md and experimental results . we performed md simulations , mostly on the titan supercomputer at oak ridge national laboratory , using version 4.6.2 of gromacs . each simulation consisted of two charged solute particles and 635 water molecules in a rhombic dodecahedron box . hoover thermostat ( coupling constant of 2 ps ) and parrinello rahman barostat ( coupling constant of 10 ps ) . the equations of motion were integrated using the leapfrog algorithm with a time step of 0.2 ps . the smooth version of the particle mesh ewald ( pme ) was used to treat electrostatics ( grid spacing of 0.12 , pme order of 4 , real - space cutoff of 9 , and ewald screening parameter of 0.347 ) , and a 9 cutoff for lennard - jones ( lj ) interactions was used along with long - ranged dispersion corrections for energy and pressure . the lj size parameter ( lj ) of the solutes ranged from 2 to 5.5 in increments of 0.5 , while the lj energy parameter ( lj ) was set to 0.1 kcalmol for all solutes . the choice for the lj range and for the lj value was based on the values for the alkali metal ( na , k , rb , cs ) and halogenide ( cl , br , i ) ion lj parameters of the dang force field . the solute particles were each given a formal charge of 1 or + 1 . we simulated all possible combinations of solute pairs ( of different sizes and charges ) , a total of 136 combinations ( + 1:1 , + 1:+1 , and 1:1 ion pairs ) for a given water model . we obtained the pmfs from md simulations in which constraints were applied to hold the solute pairs at a series of fixed interparticle separations during the course of the simulation . the closest distance between the solute particles was 1.6 , and the largest interparticle distance was 12 . in the interval from 1.6 to 5 , the separation step was 0.1 , from 5 to 8 it was 0.2 , and 0.4 increments were used for the rest ( up to 12 ) . therefore , to construct the pmf for a given solute pair , 60 independent simulations were performed where , after energy minimization and 0.1 ns equilibration , the trajectory over 3 ns was collected ( in total , this amounted to 136 60 = 8160 independent md runs ) . the individual pmf was calculated by integrating the average mean force over the solute separation distance , while the entropic force due to the increase in phase space with solute separation was added to the average mean force prior to integration . uncertainties in the average mean force values were estimated from the limiting value of the block averages , and the errors in pmf were calculated by integrating the variances of the mean force . in short , in the presimulation step of the i - pmf method , we performed explicit - solvent ( tip3p water ) md simulations to obtain the pmfs for different combinations of solute charges ( q1 , q2 ) and lj size parameters ( lj,1 and lj,2 ) . we tabulated the values of the presimulated pmfs for given ( q1 , q2 ) and ( lj,1 , lj,2 ) at the end of the supporting information file . to validate that the i - pmf method is predictive , we performed additional test md runs for selected charge and size combinations of ions with the same protocol . the lj energy parameter was the same for all ions ( lj , ion = 0.1 kcal mol ) . a pmf is the free energy as a function of distance r between the two ions . in addition , here we aim to account for two other variables at the same time , lj,1 and lj,2 , the diameters of the two ions , each of which has a given fixed charge . below is the procedure for performing this interpolation from our md simulation data , and we use this process to determine pmfs for any given pair of charges on the ions . here is a short summary of how interpolations are performed on two variables at the same time . suppose that we are given a matrix of functional values , f(xi , yj ) , that correspond to points on a two - dimensional ( n m ) grid of points ( xi , yj ) . here , i goes from 1 to n and j goes from 1 to m. in a two - dimensional interpolation , we want to estimate , by interpolation , the function f at some point not tabulated ( xa , yb)we want to find f(xa , yb ) . the point ( xa , yb ) falls into a grid square ; i.e. , four tabulated points surround the desired interior point . say that xi xa xi+1 and xj yb xj+1 ; let us denote these four functional points in the following way1 in the simplest case of interpolation on the grid square t [ 0 , 1 ] and u [ 0 , 1 ] are3 in order to obtain a smoother interpolated surface , higher - order interpolation techniques are applied . for example , in bi - cubic spline interpolation , we enforce the smoothness of some of the derivatives as the interpolating point cross grid square boundaries . to do a bicubic interpolation within a grid square , one needs to know the function f and the derivatives fx = f/x , fy = f/y , fxy = f/xy at each of the four corners of the square . the values of the derivatives at the grid points are determined globally by one - dimensional splines . the interpolating function has the following form4and we need to find 16 coefficients cmn by first solving the equations for the square grid points . the function f was in our case the pmf , and xi and yj were the lj . besides bilinear and cubic spline interpolation , we tested the performance of two additional interpolation techniques ( nearest - neighbor and piecewise - cubic - hermite - polynomial interpolations ) . for a more detailed discussion regarding the two - dimensional interpolation algorithms , see refs ( 52 ) and ( 53 ) . now , we describe the i - pmf interpolation methodology for capturing the pmf for any particular pair of ion sizes and charges and water model . for a given pair of solutes with charges q1 and q2 ( qi = + 1 or 1 ) and for a given interparticle separation distance r , a matrix of pmf values corresponding to different combinations of the solutes lj size parameters ( lj,1 and lj,2 ) was built from the presimulated pmfs . the span of lj,1 and lj,2 was from 2 to 5.5 in increments of 0.5 , so the matrix dimension was 8 8 . in total , 60 such matrices were constructed , each belonging to a given interparticle separation distance r [ 1.6,12 ] ( see section above ) . for every matrix , a two - dimensional interpolation fit through the grid points was performed . for that purpose a matrix of interpolated values was constructed and used as a look - up table to determine the value of the pmf at a given r for an arbitrary pair of lj,1 , lj,2 [ 2 , 5.5 ] values . lj,1 and lj,2 are different from the size parameters lj,1 and lj,2 of the ions used in the grid . we stored the interpolated values of the pmf for every lj,1 in the range from 2 to 5.5 in increments of lj,1 = lj,2 = 0.01 ( this means that the matrix of interpolated values was of dimension 351 351 at each r ) . although , for example , lj values of ions in dang force field are given to five significant digits ( see table 1 ) , no significant differences in predicted pmfs were observed if smaller lj were used to store the interpolated data . we tested different interpolation algorithms . by comparing the pmf obtained via an interpolation scheme and a full explicit - solvent simulation , we concluded that the cubic spline interpolation method worked best in the majority of cases . details of this comparison are shown below . for our initial i - pmf implementation , it takes approximately 8 s on a personal computer ( pc ) to build tables of presimulated values , interpolating through the grid points , making the look - up tables of interpolated values , and extracting from them the pmf for a desired combination of charges and ion sizes . after the interpolation is performed a single time and the look - up tables are available , the pmf can be extracted in 23 s on a pc . our md pmfs required a cluster of computers with many processors ( the value of the pmf at each r is its own simulation ) , and in total , it takes more than 100 h of cpu time to get a single pmf . presimulating a systematic series of ion size and charge combinations on a supercomputer , oak ridge national lab s titan in this case , provides us with a data set of accurately calculated pmfs . we perform independent two - dimensional interpolations over these solute size combination grids to determine the interaction strength for two arbitrarily sized solutes across the full range of particle separation distances . the estimated pmf is finally assembled from this interpolation data for this specific solute pair . figure 3 shows the results of the i - pmf method utilizing four different interpolation algorithms . a size asymmetric + 1:1 system mimicking potassium iodide ( lj,+ = 3.3345 , lj, = 5.1705 ; see table 1 ) at infinite dilution was selected as a representative case . a similar analysis of i - pmf predictions in other cases shown in this paper is provided in the supporting information . the simulated pmf shows a deep first minimum , corresponding to the contact ion pair ( rcp 3.6 ) , a desolvation barrier at r 4.6 , and further peaks involving the solvent shared / separated pairs ( cf . table 2 ) . we see that all of the interpolation algorithms give a pmf that qualitatively agrees with the simulated one . nevertheless , the performance of the nearest - neighbor interpolation is the least accurate of the four interpolation algorithms over a broad range of interparticle separation distances . it overestimates the contact pair ( cp ) peak and underestimates the desolvation barrier ( numerical data are collected in table 2 ) . the bilinear interpolation underestimates the depth of the contact pair minimum , and the values at short r ( repulsive wall ) are shifted to somewhat larger r. the value of the cp which follows from piecewise cubic hermite polynomial interpolation lies on the border of the experimental error , but the rest of the pmf agrees within the error bars with the md results . the pmf obtained via the cubic spline interpolation through the grid points gives the best agreement with the simulated pmf . the positions of the characteristic peaks are within the error bars ( table 2 ) . here , we want to find the best interpolation method . the goodness of the interpolation schemes for the i - pmf method can be expressed in terms of the reduced parameter ( see the supporting information file for the definition ) . the values of red are 22.0 for bilinear interpolation , 8.2 for nearest - neighbor interpolation , 1.8 for piecewise cubic hermite polynomial interpolation , and 0.4 for cubic spline interpolation algorithm . the large value of red in the case of the bilinear interpolation algorithm originates in the differences between the predicted and simulated pmf at small interparticle separation distances ( the repulsive wall of the pmf ) . the values of the pmf on the steep repulsive wall are on the order of 10 kcalmol ( and larger ) , but the error in the case of + 1:1 ion pairs is small ( 10 kcalmol ) . even though the differences between the predicted and simulated pmfs are only around 1 kcalmol , the red becomes extremely large due to the small error in the simulated pmf . this region of the pmf is not very important when one discusses the structural aspects of ion ion and ion water interactions ( the red was indeed defined in such a way as to avoid points on the repulsive wall ; see the supporting information file ) . if two points on the steep repulsive wall , lying just before the cp minimum ( r < 3.6 ) , are not taken into consideration when calculating red , then the value of red for bilinear interpolation lowers to 6.1 . this smaller value compared to the red for the nearest - neighbor case agrees with the previous visual judgment of a better overall prediction of the bilinear interpolation compared to a nearest - neighbor fit . as a rule , a value of red = 1 suggests that the extent of the agreement between observations and estimates is in accord with the error variance . red > 1 indicates that the fit has not fully captured the data ( or that the error estimate has been underestimated ) , while red < 1 speaks in favor of an unexpectedly good fit . we have concluded that ( in the majority of cases ) the most satisfactory interpolation algorithm for predicting the pmf of opposite - charged as well as like - charged pmfs involves the cubic spline interpolation ( see tables s1s4 of the supporting information file ) . the method gives quantitative agreement within the error bars in most of the relevant cases displayed in the rest of the figures and in data presented in the supporting information . in the following subsections , we show a few characteristic cases that further demonstrate the quality of the proposed pmf prediction scheme . here we use only a single value of the lj energy parameter ( lj ) , regardless of the charge or size of the ions . this is true for the selected ions in the dang ion force field ( cf . table 1 ) , but in principle , each ion could require its own lj value . in that case , the presimulated pmfs would need to be collected for different combinations of lj . instead of performing a two - dimensional interpolation , a four - dimensional interpolation through the pmf values corresponding to ( lj,1 , lj,1 ; lj,2 , lj,2 ) would need to be conducted . comparison of i - pmf predicted pmfs using different interpolation algorithms for pmf prediction in the case of a model ki salt in tip3p water : nearest - neighbor interpolation ( blue line ) , bilinear interpolation ( orange line ) , piecewise cubic hermite polynomial interpolation ( green line ) , and cubic spline interpolation with smooth first and second derivatives ( red line ) . the cubic spline interpolation algorithm best captures the magnitude and shape of pmf features . here , we compare the results of the i - pmf method to the results of the precomputed md simulations . we find that i - pmf works quite well across the whole range of solutes , from small ions with high charge density to large ones having small charge density . it captures the position and the depth of the characteristic wells corresponding to the contact and solvent shared / separated pairs and the height of the peaks of the desolvation barriers . the method is even capable of capturing the complex behavior of the pmf in the case of small ions having a large charge density ( <3 ) , where the path from the solvent shared to solvent separated pair involves multiple transition barriers . i - pmf prediction ( lines ) and md simulation ( symbols ) pmfs for model size symmetric cations and anions . solute sizes are lj,+ = lj, = 2.75 ( blue ) , 3.25 ( violet ) , 3.75 ( purple ) , 4.25 ( brown ) , 4.75 ( green ) , and 5.25 ( yellow ) ; inset , 2.25 . in all cases , we find that the predictions are nearly indistinguishable from md simulations , and i - pmf even captures the structural features of extreme ion pairing cases where the ion sizes are unphysically small ( inset ) . up to a distance of the cp minimum ( i.e. , for the values on the repulsive wall ) , the cubic hermite polynomial interpolation performs slightly better than cubic spline interpolation ( not shown ) . in an extreme case of lj = 2.25 , the cubic hermite polynomial interpolation works better up to the distance of the desolvation barrier . however , we need to stress that in these cases the selected size parameters of ions are small ( 2.25 ) and do not correspond to practical model situations . cases ( inset in figure 4 ) . improved performance of the interpolation could be achieved by providing a finer grid at the size edges , i.e. , between 2 and 2.5 , and equivalently between 5 and 5.5 for larger solutes . this would , however , require an increase in the number of pmfs calculated in the presimulation step . figure s1 of the supporting information shows how the interpolated pmf values change with increasing size of the ions ( size symmetric case ) for few selected interparticle separation distances . comparison to the simulated values shows that the deviations between predicted and simulated values are the largest for solutes between 2 and 2.5 . the red values for the cases displayed in figure 4 are given in table s2 of the supporting information file . in the cases where red is smaller for the cubic hermite polynomial interpolation compared to cubic spline interpolation , this originates from the data on the repulsive wall . for the relevant domains of the pmf we also find that , in addition to working for ions having identical sizes , i - pmf works for ions with different sizes . figure 5 shows pmfs for combinations of small and large cations ( na and cs , respectively ) with small and large anions ( cl and i , respectively ) , using ion parameters listed in table 1 . results of predicted pmfs for nacl and nai ion pairs in tip3p water are given in panel a , and pmfs for cscl and csi , in panel b. displayed md simulation data ( symbols ) were obtained independently of the prediction scheme . in all cases , the predicted pmf ( cubic spline interpolation through a presimulated grid of pmf values ) agrees quantitatively with the reference md simulations . results for other combinations of ions given in table 1 are shown in figure s3 of the supporting information file . the method performs somewhat worse in the case of kcl , kbr , rbbr , and csbr where some parts of the pmf are either slightly over- or underestimated compared to corresponding md results . the differences are , however , small , and the agreement can still be considered very good . the quality of the prediction was estimated for all the shown cases in terms of the red parameter ( table s1 of the supporting information file ) . out of all four interpolation algorithms tested here , the cubic spline interpolation performs the best . we find that i - pmf gives more accurate pmfs ( figure 5a ) than the simple pb or gb models in figure 1 with similar computational performance . we also computed pmfs for like - sign pairs , such as na na and cl cl ion pairs . previously , such quantities have been studied by integral - equation theories and computer simulations . again , we find excellent agreement between the interpolation formulas and the md simulations ; see figure 6 . . examples of pmfs for other like - charged pairs of ions from table 1 are given in figure s4 of the supporting information file . i - pmf prediction ( lines ) and md simulation ( symbols ) pmfs for salts with dissimilarly sized cations and anions . displayed are sodium ( panel a ) and cesium ( panel b ) chlorides ( green ) and iodides ( purple ) . we find i - pmf gives quantitative agreement in all cases , indicating that more complex mixed - size solute pairings are no more challenging than the previous size - symmetric solute pairings . in this section , we explore the application of our pmf modeling to ion solubilities in water . a quantitative measure of the ion pair formation is given by the corresponding change in standard free energy . i - pmf predicted ( lines ) and md simulation ( symbols ) pmfs for na na ( yellow down - triangles and line ) , cl cl ( green up - triangles and line ) , and na cl ( brown circles and line ) . these results indicate that i - pmf can successfully predict all the potential pair interactions ( including like - charge interactions ) present in a given salt solution simulation . the i - pmf nacl curve also can be compared with the implicit results in figure 1 to highlight the method s ability to capture the detailed pairing features seen in explicit md simulations . the apparent association constant for the process of forming an ion pair is in the case of univalent ions ( a + b ab ) defined as ka = ( [ ab]/c)([a]/c)([b]/c ) , where [ x ] denotes the molar concentration of species x and c = 1 mol dm is the standard molar concentration . the change in standard free energy of ion pair formation is related to the equilibrium constant via a thermodynamic relation g = rt ln ka . by conducting equilibrium md simulations where the free energy change is obtained by counting the time that the ions spend in the associated and dissociated states this , however , requires running long explicit - solvent md simulations for a given ion pair in order to obtain reasonable statistics . both approaches imply that a definition of associated / dissociated state is given . here , we define an ion pair as a configuration of a cation and anion where the two are not separated more than a certain distance rcp . we take this distance to be the first minimum in the cation anion radial distribution function gab(r ) = exp[pmfab(r)/rt ] ( r denoting the gas constant ) , corresponding to the so - called contact pair distance . for all r > rcp concentrations of free ions and of the ion pair can be estimated by integrating the gab(r ) . the ratio of the concentration of the ion pair to the total concentration of the electrolyte ( ctot ) is equal to5and the concentration of the free ions is [ a ] = [ b ] = ctot [ ab ] . rmax is the largest interparticle separation distance ( 12 in our case ) . the total concentration of the electrolyte is ctot = 3/(na4rmax3 ) , where na is the avogadro constant . in table 3 , we compare predicted association constants for ion pair formation , obtained from integrating the appropriate parts of the predicted pmfs ( eq 5 ) , with the ka from reference md simulation pmfs . we report the values for all combinations of cation anion pairs given in table 1 . prior to integration , a cubic spline interpolation was used to smooth the original pmfs . it was demonstrated in previous studies that this does not significantly affect the value of ka . the error in the association constant was determined as ka = |ka ka|/2 , where ka and ka correspond to constants obtained from a pmf increased and decreased by its variance , respectively . from table 3 , we see that the agreement of the predicted association constants with the ones obtained by integration of the simulated reference pmfs falls in most cases within the limits of the estimated errors . the predicted ka is systematically smaller than the reference one in the cases of kcl , kbr , and rbbr , and larger in the case of rbi and csi . small differences in predicted and simulated pmfs accumulate in the integration process , resulting in larger ka differences than one might expect through visually comparing both pmfs . also , the rmax in eq 5 should in principle be , but we were restricted by the dimensions of the simulation box . differences in pmfs at larger interparticle separation distances amplify by integration and are reflected in estimated ka . in figure s6 of the supporting information , we show predicted association constants for size symmetric + 1:1 ion pairs as a function of ion sizes . we see that the predicted ka agree well with constants determined from reference md simulation pmfs . finally , we explore the law of matching water affinities of k. d. collins . in short , this is the idea that small small ion pairs are insoluble in water ( because of strong electrostatic attractions ) , that large large ion pairs are poorly soluble in water ( because they act largely like hydrophobic spheres ) , but that large small ion pairs are very soluble in water . here since we can consider the cp state as a precipitated form of the salt , the ka correlates with the solubility of the salt in question . figure 7 therefore shows inverse values of the experimentally determined solubilities in panel a and our predicted ka values in panel b. experiments show that nai and cscl ( one ion big and the other small ) have large solubilities ( s is small ) , whereas csi and nacl ( two big and two small ions ) have the smallest solubilities ( s is large ) . with respect to a given cation , the predicted association constants show a qualitatively similar pattern as experiments : for cs salts ka diminishes from csi to cscl , for rb and k there is not much difference with respect to the anion , while for na the constants increase going from nai to nacl . for potassium salts , experiments show a more significant increase in s for kcl than is reflected in the predicted ka values . for salts with a common anion , the predicted ka values increase with increasing size of the cation ( from na to cs ) . such a trend is expressed also in experimental data for i and br salts but not for cl . it seems that the predicted ka value for kcl is too small and the one for csi too large . in that respect , the predicted ka for cscl does not follow the law of matching water affinities . it should be noted that these ka results are only for the set of ion parameters listed in table 1 . many other sets of ion parameters have been proposed , and alternate sets may show different behavior . figure s7 of the supporting information shows a density map of predicted ka values for a systematic variation in the sizes of anion cation pairs ( from 3 to 5.5 ) . we see that ka is the largest in the case of two big ions and the smallest for the combination of one big and one small ion , in agreement with collins proposed behavior . inverse values of the experimentally determined solubilities , s ( panel a ) , and ion - association constants from predicted pmfs , ka ( panel b ) , for selected alkali metal halides . we performed extensive md simulations of the pmfs between ions of different sizes and charge in tip3p water . by applying an interpolation scheme ( i - pmf ) on this presimulated pmf data set , we can calculate pmfs accurately and quickly for an arbitrary combination of size and charge of the two ions . we tested various interpolation formulas and found that the cubic spline interpolation algorithm gives the most accurate representation of the majority of md results . the accuracy of the prediction is scalable based on the density of the presimulated pmf grid . improved agreement could come from a denser grid of pmfs or an expanded set of sizes . the advantage of the interpolations is about a 5 order of magnitude gain in speed , without loss of predictive accuracy ( within the error bars ) . our interpolated pmfs are consistent with experimentally observed ion - pair solubilities and with collins law of matching affinities . the i - pmf method may be useful where there is a need to calculate accurate pmfs rapidly .

we perform extensive molecular dynamics ( md ) simulations between pairs of ions of various diameters ( 25.5 in increments of 0.5 ) and charge ( + 1 or 1 ) interacting in explicit water ( tip3p ) under ambient conditions . we extract their potentials of mean force ( pmfs ) . we develop an interpolation scheme , called i - pmf , that is capable of capturing the full set of pmfs for arbitrary combinations of ion sizes ranging from 2 to 5.5 . the advantage of the interpolation process is computational cost . whereas it can take 100 h to simulate each pmf by md , we can compute an equivalently accurate i - pmf in seconds . this process may be useful for rapid and accurate calculation of the strengths of salt bridges and the effects of bridging waters in biomolecular simulations . we also find that our data is consistent with collins law of matching affinities of ion solubilities : small small or large large ion pairs are poorly soluble in water , whereas small large are highly soluble .

Introduction Theoretical Methods Results and Discussion Conclusions

PMC4208899

laparoscopic sleeve gastrectomy ( sg ) was introduced by gagner and colleagues as a first - step procedure to minimize surgical risk for super - super obese ( sso ) or high - risk patients , followed by either laparoscopic biliopancreatic diversion with duodenal switch or laparoscopic roux - en - y gastric bypass ( rygb ) . sg is now established as a primary bariatric procedure apart from being a first - stage procedure in a planned bariatric approach for high - risk patients . super obese ( so ) patients with a body mass index ( bmi ) 50 kg / m are a difficult - to - manage population because of the huge liver , leading to limited working space ; excessive torque on the instruments because of the thick abdominal wall ; comorbidities ; and high - risk anesthesia . the management of patients with super - super obesity ( bmi 60 kg / m ) also remains a challenge . robotic surgery may be a suitable alternative to the laparoscopic approach in so and sso patients . robotic surgery allows for more precise manipulations and increased dexterity by downscaling the surgeon 's movements and filtering out physiological tremor . it liberates the surgeon from torque on the instruments because of the thick abdominal wall and decreases the port - site trauma because of remote - center technology , thereby conferring improved ergonomic positioning . furthermore , a true 3-dimensional view of the intra - abdominal anatomy with robotic surgery facilitates fine tissue dissection . the main disadvantages associated with robotic surgery remain its high cost and longer setup time compared with the laparoscopic equivalent for many procedures . with increased experience , it may be assumed that this setup time will be reduced , and in the future , operative costs may decrease as material prices are reduced . this study evaluates our initial experience with and technique for robot - assisted sleeve gastrectomy ( rasg ) in morbidly obese ( mo ) and so patients . a robotic platform was used for bariatric surgery starting in march 2012 at our well - established center of laparoscopic bariatric surgery . the patients met the national institutes of health consensus criteria , as well as the institutional policies , for undergoing a bariatric procedure . after the required training in robotic surgery , the surgeons initially performed a few simple cases such as cholecystectomy , hernia repair , and hiatal surgery before proceeding with bariatric procedures . preoperative evaluation included bmi , anthropometry , routine blood parameters , nutritional markers ( vitamin b12 , folic acid , iron , ferritin , vitamin d3 ) , diabetes profile ( fasting blood glucose , hemoglobin a1c , fasting insulin , and c - peptide levels ) , lipid and thyroid profile , sleep study , upper gastrointestinal endoscopy , pulmonary function test , dobutamine stress echocardiogram , chest radiography , and ultrasonography of abdomen . the following parameters were evaluated : patient demographic characteristics , body mass index ( bmi ) , comorbidities , total operative time ( further split into docking time and imbrication time ) , mean blood loss , conversions , perioperative complications and deaths , length of hospital stay , and excess body weight loss at 6 months after the procedure . the operative time was defined as the time from skin incision to skin closure , including any associated procedures . the so ( bmi 50 kg / m ) subset of patients was compared with the mo patient group based on the previously mentioned parameters . statistical analysis was performed using spss software , version 19.0 ( ibm , armonk , new york ) . the patient is placed in the supine position with his or her legs by the side . a 38f gastric calibration tube is used after induction to empty out the gastric contents and is kept in place to guide the sleeve formation . pneumoperitoneum is created with a veress needle at palmer 's point up to a pressure of 15 mm hg . all distances for port placement are measured after insufflation because measurements obtained before pneumoperitoneum is achieved may be misleading . pre - emptive infiltration of 0.5% bupivacaine is performed before incision at all port sites . the camera port ( 12-mm - diameter , 150-mm - long endopath xcel trocar ; ethicon endo - surgery , cincinnati , ohio ) is placed 20 cm below the xiphisternum slightly to the left of the midline under vision using a 0 10-mm laparoscope . the ports for the robotic arms ( r1 , r2 , and r3 ) are established next . r1 ( 8-mm da vinci cannula ; intuitive surgical , sunnyvale , california ) is placed at the left midclavicular line approximately 20 cm from the xiphisternum . r2 ( 5-mm da vinci cannula ) is placed in the right hypochondrium at the midclavicular line , with care taken to ensure that the entry of the port is below the margin of the liver . r3 ( 5-mm da vinci cannula ) is placed in the left flank at the level of the camera port . the assistant port ( 12-mm - diameter , 100-mm - long endopath xcel trocar ) is placed between the camera port and r2 with a distance of at least 10 cm from both of them . a 5-mm epigastric port is made and used to place a nathanson liver retractor for retraction of the left lobe of the liver ( cook inc . , a1 = assistant port ; n = nathanson retractor ; r1/r2/r3 = ports for robotic arms ; mcl = mid clavicular line ; sul = spino - umbilical line . all 3 arms of the da vinci surgical system ( intuitive surgical ) are used . the patient cart is brought from the head end of the patient , and docking is performed . the instruments used for rasg are as follows : 8-mm da vinci harmonic ace curved shears ( ethicon endo - surgery , intuitive surgical , sunnyvale , ca , usa ) ( r1 , for gastrolysis ) , 5-mm bowel graspers ( r2 and r3 ) , and 5-mm needle driver ( r1 , for over - sewing staple line ) . a 5-cm umbilical tape is used to measure the distance from the pylorus , and gastrolysis is performed along the greater curvature of the stomach using a harmonic scalpel ( ethicon endo - surgery , intuitive surgical , sunnyvale , ca , usa ) . the short gastric vessels from the gastrosplenic ligament and posterior gastric adhesions with the pancreas are divided . one can appreciate a bilaminar parchment like membrane if one remains close to the stomach on the greater curvature . care is taken to ensure that the bougie is withdrawn in the esophagus during this step ; otherwise , it can lead to difficulty in dissection around the hiatus . the gastric calibration tube is pushed forward by the anesthetist and guided by the surgeon into the first part of the duodenum using bowel graspers . rotation of the bougie by the anesthetist and correct amount of traction by the surgeon help in negotiating the pylorus . this leads to the bougie nicely resting along the lesser curve of the stomach and helps in formation of an even sleeve . a sleeve of stomach is created using an articulating stapler from the assistant port while the console surgeon applies traction on the stomach so that it lies in the right orientation . sequential stapling first with a green load and subsequently with blue loads is performed while not being very snug to the bougie . after the sleeve is formed , the staple line is imbricated using no . 2 - 0 polydioxanone running suture with a sliding loop at one end . gastroscopy with saline solution immersion is performed in all cases to rule out any leak , bleed , or obstruction . the patient is placed in the supine position with his or her legs by the side . a 38f gastric calibration tube is used after induction to empty out the gastric contents and is kept in place to guide the sleeve formation . pneumoperitoneum is created with a veress needle at palmer 's point up to a pressure of 15 mm hg . all distances for port placement are measured after insufflation because measurements obtained before pneumoperitoneum is achieved may be misleading . pre - emptive infiltration of 0.5% bupivacaine is performed before incision at all port sites . the camera port ( 12-mm - diameter , 150-mm - long endopath xcel trocar ; ethicon endo - surgery , cincinnati , ohio ) is placed 20 cm below the xiphisternum slightly to the left of the midline under vision using a 0 10-mm laparoscope . the ports for the robotic arms ( r1 , r2 , and r3 ) are established next . r1 ( 8-mm da vinci cannula ; intuitive surgical , sunnyvale , california ) is placed at the left midclavicular line approximately 20 cm from the xiphisternum . r2 ( 5-mm da vinci cannula ) is placed in the right hypochondrium at the midclavicular line , with care taken to ensure that the entry of the port is below the margin of the liver . r3 ( 5-mm da vinci cannula ) is placed in the left flank at the level of the camera port . the assistant port ( 12-mm - diameter , 100-mm - long endopath xcel trocar ) is placed between the camera port and r2 with a distance of at least 10 cm from both of them . a 5-mm epigastric port is made and used to place a nathanson liver retractor for retraction of the left lobe of the liver ( cook inc . , a1 = assistant port ; n = nathanson retractor ; r1/r2/r3 = ports for robotic arms ; mcl = mid clavicular line ; sul = spino - umbilical line . all 3 arms of the da vinci surgical system ( intuitive surgical ) are used . the patient cart is brought from the head end of the patient , and docking is performed . the instruments used for rasg are as follows : 8-mm da vinci harmonic ace curved shears ( ethicon endo - surgery , intuitive surgical , sunnyvale , ca , usa ) ( r1 , for gastrolysis ) , 5-mm bowel graspers ( r2 and r3 ) , and 5-mm needle driver ( r1 , for over - sewing staple line ) . a 5-cm umbilical tape is used to measure the distance from the pylorus , and gastrolysis is performed along the greater curvature of the stomach using a harmonic scalpel ( ethicon endo - surgery , intuitive surgical , sunnyvale , ca , usa ) . the short gastric vessels from the gastrosplenic ligament and posterior gastric adhesions with the pancreas are divided . one can appreciate a bilaminar parchment like membrane if one remains close to the stomach on the greater curvature . care is taken to ensure that the bougie is withdrawn in the esophagus during this step ; otherwise , it can lead to difficulty in dissection around the hiatus . the gastric calibration tube is pushed forward by the anesthetist and guided by the surgeon into the first part of the duodenum using bowel graspers . rotation of the bougie by the anesthetist and correct amount of traction by the surgeon help in negotiating the pylorus . this leads to the bougie nicely resting along the lesser curve of the stomach and helps in formation of an even sleeve . a sleeve of stomach is created using an articulating stapler from the assistant port while the console surgeon applies traction on the stomach so that it lies in the right orientation . sequential stapling first with a green load and subsequently with blue loads is performed while not being very snug to the bougie . after the sleeve is formed , the staple line is imbricated using no . 2 - 0 polydioxanone running suture with a sliding loop at one end . gastroscopy with saline solution immersion is performed in all cases to rule out any leak , bleed , or obstruction . a total of 35 patients ( 15 female and 20 male patients ) with a mean bmi of 48.17 11.7 the bmi ranged from 33 to 82.6 kg / m , with 3 patients having a bmi > 80 kg / m . the mean operative time was 116.3 24.7 minutes , and the mean docking time was 8.9 5.4 minutes . the mean time taken to imbricate the staple line was 21.4 6.4 minutes . mean blood loss was 19.36 4.62 ml , and there were no complications , conversions , or perioperative deaths in the series . a steep decline in operative time and docking time was observed after the initial 10 cases . we believe that the operating room team surpassed the learning curve for robotic bariatric surgery by the 10th case . the team was already well - versed in laparoscopic bariatric procedures and could adjust to the new system fairly quickly . the so group was compared with the mo group regarding patient demographic characteristics , bmi , and comorbidities ( table 1 ) , as well as perioperative outcomes , complications , and weight loss ( table 2 ) . the comparison showed no significant difference in operative time ( p = .145 ) , blood loss ( p = .523 ) , and length of hospital stay ( p = .213 ) . the excess body weight loss at 6 months ' follow - up was also not significantly different between the 2 groups . perioperative outcomes , complications , and ebw loss in morbidly obese versus super obese patients ebw = excess body weight . laparoscopic bariatric surgery , especially in so patients , presents a complex situation in which the surgeon has to deliver the best results despite compromised technology . with the advent of robotic technology , its application to bariatric procedures may eliminate certain limitations such as torque on rigid instruments because of the thickness of the abdominal wall and difficulty in dissection or suturing in a limited working space because of a heavy liver and excess omental fat . it also provides for a stable and ergonomically comfortable platform with 3-dimensional vision and precise movements without any tremor . sg , on the other hand , is considered a relatively simple procedure compared with rygb . however , there are certain peculiarities in sg ; for example , it uses a long staple line with a potential to leak , and precise and safe dissection is required in the area of the left crus and hiatus to entirely mobilize the fundus . compared with laparoscopic surgery , robotic surgery offers the possibility of endowrist application ( intuitive surgical ) , and this action facilitates the over - sewing of the staple line . over - sewing of the staple line was accepted by most of the surgeons ( 95% ) participating in the international sleeve gastrectomy expert panel . any leak with sg is a high - pressure leak and is difficult to manage compared with an rygb leak . these difficulties become more pronounced when operating in an so patient because of limited working space . so and sso patients are high - risk candidates , and sg is considered the first step in a 2-stage procedure or , more and more often , is performed as a single - stage procedure . from a surgical point of view , so patients remain a difficult population to manage , usually with outcomes slightly less favorable than those for mo patients . there have been comparisons of robot assistance in rygb in so patients versus mo patients , and there are studies that state that rasg is a safe and feasible operation . however , there are no or very few studies comparing the perioperative outcomes of using a robot for sg in so patients versus mo patients . the results of rasg for the so group versus the mo group were compared to look for any advantages provided by the robotic system in the so group . in our experience , the outcomes in both the so and mo groups were the same , with no complications in either group . this occurred despite the fact that there were a few very high - risk sso patients in this series , including 3 patients with a bmi > 80 kg / m . in addition , a robotic system helps in precisely over - sewing the staple line ( as is the routine practice in our center ) even in sso patients with a severely limited working space . thus , using a robotic system may help overcome many difficulties in so patients and enables the surgeon to perform a similar procedure to that in mo patients with equal precision , similar time requirement , and little or no extra effort . we believed that during introduction of robotic surgery to a bariatric program , sg was a relatively simple and safer procedure to start with . it enables the whole team to become accustomed to the setup of the robotic system , docking , and troubleshooting . once the team is comfortable with rasg , it can proceed to robotic rygb and revision surgical procedures . in this series the learning curve for rasg was 10 procedures for a team well - versed in laparoscopic bariatric surgery and well - trained in handling the da vinci surgical system . after 10 cases , our docking time , setup time , and console time plateaued with a < 5% change in the operative time . first , the study had a small sample size , and patients were not randomized . yet , it described our initial experience with rasg and tried to address issues such as safety , outcomes , and the learning curve . second , the study included the learning curve of the team , which may have increased the mean operating room times . third , the study did not analyze the additional cost of using a robot , but we believe that clinical outcomes are much more important and that costs are bound to decrease with time . in addition , the study did not compare rasg with laparoscopic sg , and such a comparison is required to ascertain the real benefit of using a robotic system . in addition , the ease and precision with which the staple line is imbricated are definite advantages . rasg can act as a good procedure to introduce robotics in a bariatric surgery center before going on to perform rygb and revision procedures . the learning curve for rasg is about 10 cases for an established bariatric surgery center . the real advantages in comparison with the laparoscopic approach are still being debated and require further studies for the subpopulation of so patients .

background and objectives : this study evaluates our technique for robot - assisted sleeve gastrectomy for morbidly obese and super obese patients and our outcomes.methods:a retrospective analysis of patients who underwent robot - assisted sleeve gastrectomy at a single center was performed . the procedure was performed with the da vinci si hd surgical system ( intuitive surgical , sunnyvale , california ) . the staple line was imbricated with no . 2 - 0 polydioxanone in all cases . the super obese ( body mass index 50 kg / m2 ) subset of patients was compared with the morbidly obese group in terms of demographic characteristics , comorbidities , operative times , perioperative complications , and excess body weight loss.results:a total of 35 patients ( 15 female and 20 male patients ) with a mean body mass index of 48.17 11.7 kg / m2 underwent robot - assisted sleeve gastrectomy . of these patients , 11 were super obese and 24 were morbidly obese . the mean operative time was 116.3 24.7 minutes , and the mean docking time was 8.9 5.4 minutes . mean blood loss was 19.36 4.62 ml , and there were no complications , conversions , or perioperative deaths . when compared with the morbidly obese patients , the super obese patients showed no significant difference in operative time , blood loss , and length of hospital stay . there was a steep decline in operating room times after 10 cases of robot - assisted sleeve gastrectomy.conclusion:this study shows the feasibility and safety of robot - assisted sleeve gastrectomy . robotic assistance might help overcome the operative difficulties encountered in super obese patients . it shows a rapid reduction in operative times with the growing experience of the entire operative team . robot - assisted sleeve gastrectomy can be a good procedure by which to introduce robotics in a bariatric surgery center before going on to perform roux - en - y gastric bypass and revision procedures .

INTRODUCTION MATERIALS AND METHODS Operative Technique: RASG RESULTS DISCUSSION CONCLUSIONS

PMC4842443

the hormones dominating the phases are gonadotropin releasing hormone ( gnrh ) , follicle - stimulating hormone ( fsh ) , luteinizing hormone ( lh ) , estrogen and progesterone . these hormones can be changed by exercise estrogen and progesterone increase after acute exercise . in the menstrual period , progesterone and estrogen secretion is suppressed by chronic exercise1 , 8 , 13 , 26 . the performance of female athletes during their menstrual periods has attracted the attention of researchers for many years . , studies have not detected any decrease in the performance of female athletes during their menstrual periods4 , 6 , 10 , 24 . in this study , the effect of maximal aerobic exercise the luteal phase on some gonadal hormones and alpha - fetoprotein ( afp ) in female athletes was researched . it has a molecular weight of 70 kd is and it synthesized by the yolk sac , fetal gastrointestinal tract , and liver20 . it is an important parameter in pregnancy follow - up as the afp level increases in intrauterine fetal deaths , hydrocephalus , hemolytic diseases , placental separations , and neural - tube defects in pregnant women21 . the measurement of afp level is important for tracing prognosis and diagnosis hepatoma and germ cell tumors18 , 21 . serum afp is also elevated in 524% of malignancies of the pancreas , stomach , and colon20 . in a 12-week study performed in 2014 , ko et al . found no change in the afp level of women aged around seventy after 1 , 3 and 5 days of an exercise program . our study will contribute to the literature in terms of the relationship between afp and exercise . estrogen is an 18-carbon corticosteroid molecule released from the ovaries of women and from the testis of men . furthermore , it is also affects the cardiovascular system , muscle skeletal system , immune system , and central nervous system . it plays a role in the repair of the skin , nervous system , and hepatic tissues . while the estrogen level in women is not changed by acute exercise , it is suppressed by chronic exercise16 . kraemer et al . found an increase in estradiol with mild exercise . during the luteal phase , the progesterone level increases and exercise nakamura et al . detected an increase in both estrogen and progesterone after exercise in the mid - luteal phase . the menstrual disorders observed in female athletes result from differences in the release of oligomenore and amonore gonadotropin releasing hormones , and the release of fsh and lh is decreased by exercise11 . twelve healthy female soccer players with normal menstrual cycles ( regular for one year , 282 days ) were included in the study as subjects . all of the subjects were conforming to the luteal phase period ( 22nd and 25th day ) according to body temperatures ( table 1table 1.the subjects characteristics ( n=12)age ( years)19.61.3 ( 1822)sports age ( years)5.40.7 ( 47)height ( cm)1664.7weight ( kg)55.45.7 ) . the experimental protocol of this study was approved by the local ethics committee . all the subjects were informed about the purpose and risks of the study before signing a written consent . the test started with slow running ( 8 km / h ) , and the subjects ran 2 meters apart from each other between two lines . then , the same distance was run again and the second run was started . the running speed was adjusted by the signals and it was increased by 0.5 km / h per minute . the test was stopped when a subject failed to follow the set pace of the beeps for two successive shuttles or stopped voluntarily14 . all the subjects who completed the test were successful . the subjects were instructed not to eat anything three hours before the exercise and not to do any sports from two days before the exercise to control the test and these conditions . afp , estradiol , progesterone fsh , and lh values were measured using an enzyme - linked immunosorbent assay ( elisa ) and original kits in a laboratory . repeated measure anova was used to compare afp , estradiol , progesterone fsh , and lh serum levels at baseline and immediately and 1hour after the test . afp levels had significantly decreased after 1 h of recovery from exercise in the female soccer players ( p<0.05 ) . in the female soccer players , estradiol and lh levels had significantly increased immediately after the exercise but had returned to resting levels after 1 h of recovery ( p<0.05 ) . progesterone levels had significantly decreased immediately after the exercise but had returned to resting levels after 1 h of recovery ( p<0.05 ) . the value of fsh1 h after exercise was lower than its value before the exercise ( p<0.05 ) ( table 2table 2.repeated measure of anova comparison of pre and post ( immediately and 1-hour after ) shuttle run test effect on hormone level ( n=12)pepsep1hseafp ( ng / ml)1.50.51.50.381.40.5*fsh ( mu / ml)5.52.46.22.75.12.4*lh ( mu / ml)4.83.55.634.94*estradiol ( pg / ml)96.75.5104.259.19558.9*progesterone ( ng / ml)6.45.95.456.16.2*pe : before exercise , pse : after exercise , p1hse : one hour after exercise ) . our findings of heart rate before and after maximal aerobic exercise are shown in table 3table 3.before exercise ( pe ) and after exercise ( pse ) heart rate values ( n=12)pepse heart rate ( pulse / min)63.15159.110.3*. pe : before exercise , pse : after exercise , p1hse : one hour after exercise in the present study , the effects of acute maximal exercise on afp , estradiol , progesterone , fsh , and lh in female soccer players during the luteal phase of the menstrual cycle were researched . afp is an oncofetal glycoprotein with a molecular mass of approximately 70 kd17 , 21 . measurements of maternal serum and amniotic fluid afp during pregnancy are important and widely applied in screening methods for the detection of congenital anomalies of the fetus17 , 21 . measurement of serum afp plays an important role as a diagnostic or in screening tests for in certain malignancies . hepatocellular carcinoma is the most frequently occurring malignant disorder for which afp is used as a biomarker17 , 21 . other cancer types that increase serum afp levels are pancreatic and gastrointestinal carcinomas , and germ cell tumors of the testis or children s brains17 , 21 . it is our supposition that decreases in the afp values , a cancer parameter , taking place with exercise , could be used to determine the importance of exercise in cancer treatments . we found only one publication in the literature concerning the change in afp with exercise . in a study of women aged over seventy , it was found that the afp level did not change with exercise5 . in our study , estradiol and progesterone are gonadal hormones which decrease with aging , and this may lead to problems such as metabolic syndrome and sarcopenia16 . furthermore , estrogen also affects other endocrine responses during exercise9 . while the effect of exercise on anabolic hormones is maximal in the young women with normal menstruation , estrogen and progesterone levels decrease more in the women with the menarche disorder11 , 25 . consequently , female athletes may show menstrual disorders such as amenore ( no menstruation ) , delay in the age of menarche , oligomenore ( frequency of menstruation of more than 35 days ) and insufficiency of the luteal phase3 , 12 , 15 , 19 , 22 , 23 , 25 . in our study , it has been mentioned in the literature that chronic exercise suppresses estrogen and progesterone25 . in the present study , the progesterone level had decreased 1 hour after the exercise a result that is consistent with the literature . in female athletes , the pulsatile release of gonadotropin - releasing hormones decreases . this suppresses the release of lh and fsh from the ovaries ; therefore , the release of estrogen decreases . low luteinizing hormone levels explain menarche disorders and secondary amenore in female athletes3 , 15 . in our study , lh increased immediately after the exercise and returned to its former level after 1 hour . this may show there is a positive effect of acute exercise on lh . on the other hand , the decrease of the fsh level to under its original value 1 hour after the exercise shows that intense exercise suppresses fsh . this result is also consistent with the literature3 , 15 , 19 , 22 , 23 , 25 . in the present study the effect of maximal aerobic exercise in the luteal phase during which athletes show the most menstrual problems on fsh , lh , estradiol and progesterone hormones , and afp was studied . the observed of suppression progesterone and fsh values is the literature consistent with other reports while the increase of estradiol and lh levels immediately after exercise shows that acute exercise can be used as a method of training for female athletes . on the other hand , the significant decrease in the value of afp , which is a cancer parameter , 1 hour after the exercise , may be of valuable in the future studies of the relationship between exercise and cancer .

[ purpose ] the performance of female athletes during their menstrual period has attracted the attention of researchers for many years . it is known that the menstrual period changes with exercise . alpha - fetoprotein ( afp ) is an oncofetal protein . in this study , the effect of maximal aerobic exercise in the luteal phase on some hormones and afp in female athletes was researched . [ subjects and methods ] twelve volunteers and healthy female footballers with normal menstrual cycles volunteered for this study as subjects . all the participants performed a shuttle run test . blood samples were taken before , after , and one hour after exercise . serum afp , estrogen , progesterone , follicle - stimulating hormone ( fsh ) , and luteinizing hormone ( lh ) values were measured using an auto analyzer and original kits . heart rate measurements were performed before and after the exercise . [ results ] afp activity had significantly decreased after 1 h of recovery from the exercise in the female soccer players , and estrogen and lh activity had significantly increased immediately after the exercise . progesterone activity had significantly decreased immediately after the exercise . fsh values had significantly increased immediately after the exercise . [ conclusion ] the results of the present study show there were significant decreases in the values of afp , which is a cancer parameter , 1 hour after the exercise . this result may be valuable in future physiotherapy studies on the relationship between exercise and cancer .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC4264627

the use of sound information is a growing area of application of signal processing techniques in healthcare and biomedicine . in particular , multiple groups have reported on the development of systems that analyze audio recordings of patients and identify bouts of coughing [ 15 ] or other sounds indicative of health concerns [ 69 ] . in addition to their value as a monitoring or screening tool , the automated nature of these systems potentially enables new modes of disease management , especially in conjunction with advances in mobile technology . moreover , the type of approach presented here may be especially suited to improving outcomes and addressing chronic disease in populations that have been previously underserved . this paper studies cough as a symptom of asthma in adolescents . for a variety of reasons , including social and developmental pressures , adolescents with an asthma diagnosis typically have an inaccurate understanding of their disease condition primarily due to poor symptom perception or downplaying symptoms [ 11 , 12 ] . these issues can undermine effective symptom monitoring and medication adherence which can ultimately lead to poor condition management and high healthcare utilization . to address this problem a device is proposed that continuously monitors asthma symptoms , particularly coughing which is the most common symptom in pediatric asthma patients [ 13 , 14 ] . the identified symptoms can be stored and later retrieved by a patient or healthcare provider as an objective reference indicating the levels of asthma control . mobile technology has advanced sufficiently such that the hardware requirements of a personal asthma monitoring device are already met in existing consumer electronics platforms . the problem may , therefore , be approached within the context of mobile application development . owing to its broad install base and mature software development kit ( sdk ) apple 's ios devices were the target platform ( the 4 g ipod touch specifically ) . like other audio systems for cough monitoring reported in the literature the present approach is based upon proven technology and methods in the fields of speech recognition , keyword and key audio effect detection [ 1517 ] , machine learning , and content - based audio analysis [ 1823 ] . the symptom recognition algorithm consists of a frame - based feature extractor followed by temporal analysis with a hidden markov model ( hmm ) based viterbi decoder . mel - frequency cepstral coefficients ( mfcc ) plus a log - energy measure were employed as the audio features while both left - to - right and ergodic / connected hmm topologies were investigated . the mfcc / hmm recognition framework has also been reported for cough monitoring by , for example , matos et al . [ 3 , 25 ] . use a similar method however ; in their case , they use an original variant of cepstral analysis dubbed energy cepstral coefficients ( ecc ) and have inserted an artificial neural network discriminator prior to the hmm decoder . the cough detection algorithm presented here processed the audio data in two steps . in the initial preprocessing step the incoming stream of digital audio samples was rearranged into fixed - length frames from which a set of audio features ( feature vector ) were computed . the sequence of feature vectors was then passed to a hmm viterbi decoder ( using a token passing implementation as described in ) using hmms trained on the collected database of symptom and background sounds . as discussed in detail in the following sections the hmm was a composite consisting of separate models for coughing , background , and silence connected together with a simple parallel grammar . prior to development of the automated device for asthma monitoring ( adam ) application , a large amount of audio data was collected in trial recordings intended to closely approximate the circumstances in which the device would be expected to function . participants with asthma were 1317 years old , had active asthma symptoms within the previous 24 hours , and used english as a primary language . those with other health conditions producing respiratory symptoms ( e.g. , cold , cardiac disease , or cystic fibrosis ) were excluded . twenty - nine adolescents participated in the audio data collection trials ; details of the sample characteristics are provided elsewhere . participants with asthma were 1317 years old , had active asthma symptoms within the previous 24 hours , and used english as a primary language . those with other health conditions producing respiratory symptoms ( e.g. , cold , cardiac disease , or cystic fibrosis ) were excluded . the participants were instructed to continuously record the sounds of their respiration for 24 hours in vivo wearing a personal digital recorder ( olympus ws-331 m ) and lapel microphone while they went about their daily routines . the external microphone was attached to the shirt collar to optimally capture the respiratory sounds of the subject . the recorder was carried in a pocket or in a carrying case provided by the study . were instructed to place the recorder and microphone near the head of the bed to capture any symptoms that occurred during the night . for the purposes of developing the signal processing component of the adam device the large dataset was split into a series of files of more manageable size . representative selections of the audio containing cough sounds , silence , and background noise across all of the participants were aggregated into individual files . the classification and validation of symptomatic sounds were carried out by the principal investigator and three other clinicians trained in pediatric medicine and pulmonology . coughing sounds that were used to train the recognition algorithm were cropped to only include the sound itself . as a consequence the corresponding audio files had variable length ( i.e. , the duration of the cough ) . all other audio files ( including the training data for the silence and background hmms ) had a fixed duration of 6 seconds . the files were standard mono 16-bit pulse code modulation ( pcm ) audio format with a sample rate of 11025 hz . to keep training times manageable ( especially for the hmms with a large number of mixture components and states ) representative portions were selected from the audio database totaling approximately 2 hours for the training of background and silence hmms . the performance metrics that were of most practical interest , given the task , were the false positive rates rfp and true positive rates rtp ( sensitivity ) . to estimate rfp , approximately 6 hours of representative , cough - free , background noise was compiled . to estimate rtp the recognition algorithm was tested on 30 examples of cough that were withheld during training . the preprocessing step consisted of dividing the time - domain digital audio data into frames and then computing a sequence of per - frame descriptive feature vectors . these are implemented natively by the open source hidden markov toolkit ( htk ) which was used to train the symptom recognition algorithm . frame size was chosen to be 256 samples overlapping by one - half of a frame . the overall frame rate was thus 0.0116 seconds . to compute the mfccs features a 256-point hanning window multiplied the given frame of audio samples and the results were stored in an array a[k ] . a 32-point mel - filterbank ( figure 2 ) , setting array sizes to an appropriate power of 2 greatly simplifies programming when discrete fourier transforms are needed . importantly , the indices of a[k ] were reordered to introduce the symmetries needed to compute a discrete cosine transform ( dct ) with a dft . the result of the second dft was stored in an array c[k ] and the dct coefficients were found via the following formula : ( 1)mfcc[k]=r{q[k]}r{c[k]}i{q[k]}i{c[k]}2 . here , q[k ] is a fixed array arising from the definition of the discrete cosine transform . for k ( 1,31 ) q[k ] is equal to a complex exponential with an additional factor of 1/2 when k = 0 : ( 2)q[k]={22neik/(2n),k(1,31),22neik/(2n),k=0 . this value was not retained because a different log - energy measure was used instead to track sound intensity . the base of the logarithm is e , n is the frame size equal to 256 , and x[n ] are the values of the time - domain audio samples . although the computation of the mel - cepstrum is not an invertible operation it is reasonable to assume , based upon the performance of the classifier , that these mfccs contained enough discriminative acoustic information for the task . as stated above , the zeroth mfcc is an energy measure . in speech recognition , the higher mfccs , however , are not known to admit any simple perceptual interpretations [ 27 , 28 ] . the first twelve mfccs and the log - energy defined one - half of the feature vector for each frame . an example of these features for a given frame is shown ( figure 3 ) . the other half of the feature vector was defined to be the first differences of the mfccs and log - energy . if i is the index of the frames in time then the first difference of any quantity f at time i is defined as ( fi+1 fi1)/2 . at frame boundaries for simplicity in eventually transferring the recognition algorithm to a mobile application a preemphasis filter was not applied . hidden markov models are often used to describe a sequence of observations as the probabilistic evolution of a finite state machine . associated with each state is a probability distribution that describes the elements of the observation vector . the sequential nature of the relevant data or process is captured in the state - to - state transition probabilities . hmms are partially specified as directed graphs and two example topologies are shown ( figure 5 ) . the nodes in the graph represent states and the arrows represent allowed transitions between states . two topologies were examined : an ergodic or connected topology and a left - to - right topology ( 5 ) . following , connected models were used for background and silence hmms while both the connected and left - to - right topologies were tested for identifying coughs ( the key effect ) . clearly , for a fixed number of states , left - to - right models are much more constrained than connected models as quantified by the total number of possible paths through the model . comparing the two , the role of context partially determines the grammar for the recognition task ( equivalently , context determines how the background , silence , and cough models should fit together in a composite hmm ) . generally , for coarse segmentation of the audio stream , contextual information was not expected to greatly improve the performance of the classifier . this contrasts with speech where context can provide a significant assistance for recognition and even help to overcome poor phoneme level transcription ( i.e. , possible sequences of phonemes are constrained by the vocabulary of the language and , moreover , the sequence of words is constrained by grammatical rules ) . the gray boxes at the left and right indicate dummy states that route all of the output states to all of the input states so that the system can be run in a continuous fashion . ultimately , the structure of the composite hmm reflects in the transition matrix pij of the model . in addition to the topology , an hmm is also specified by an output probability distribution in the feature space for each state of the model . usually , for a given hmm , these probability distributions all have the same functional form . for the recognizer , the feature vector is denoted by f = [ f1 , , fm ] . the mixture model is a weighted sum of such terms : ( 5)bf(f)=i=1jibf,i,i . the total number of mixture components is denoted by l in this case . the silence model used in this paper was a 3-state connected model with 3 mixtures per state . for background , a variety of connected hmms were tested with the number of states ranging over the values ( 3,6 ) and the number of mixture components per state ranging over the values ( 10,30,60 ) . likewise , for cough , connected and left - to - right topologies were tested with 5 states and the number of mixtures ranging over the values ( 10,60 ) . after selecting the orders of the hmms the models were trained . as stated above , the hmms reported on in this paper were trained with tools provided in the hidden markov toolkit ( htk ) . specifically , each hmm was initialized using a single pass of viterbi training until convergence followed by a single pass of baum - welch training until convergence . in the nomenclature of htk this is accomplished with the command line programs hinit and hrest , respectively ( using the whole - word strategy ) . to recognize a new audio stream viterbi decoding the viterbi decoding is implemented with token passing [ 24 , 29 ] . in the recognition context the behavior of hidden markov models is best understood using a lattice where the horizontal axis is the discrete time index and the vertical axis an index of the states . a token can be visualized as an entity which moves through the state lattice and keeps a record of the path and the likelihood of the path it has taken . the likelihood is simply the sum of the appropriate values of the emission probabilities b(fn ) ( where n is a timing variable at the frame rate ) and the state - to - state transition probabilities pij . viterbi decoding finds the most likely path through the lattice given the data . using the token idea the decoding algorithm ( rule1 ) to update a new frame of data , or equivalently to move to the next time step in the lattice , compute all possible transitions for the current set of tokens ; ( rule2 ) for each state keep the incoming token that has the highest likelihood . in general , if there are n frames of data and l states then arrays of size n l must be kept in memory . in particular , the likelihoods and the states where the tokens originated are retained in memory . to recover the actual state alignment the algorithm begins at the end of these arrays and works backwards , following the path of the most likely token . the update rule can also be stated mathematically as a recursion : ( 6)sj[n]=maxi(si[n1]+logpij)+logbj(fn ) . an example of a decoding pass for some audio data containing coughs is presented ( figure 7 ) ( note that for continuity this is the same audio data shown above ( figure 3 ) ) . a time - domain waveform of the audio being decoded and the resultant state sequence are shown ( figure 7 ) . the horizontal axis represents time ( at the frame rate ) while the vertical axis is an integer of the state index . the black marks indicate states associated with the cough model ( in this case , a left - to - right topology ) . after the viterbi decoder , the rule for deciding whether a frame of audio data contains coughing is simply whether the state sequence ever passed through the cough model . this is equivalent to observing when the maximum likelihood path passed through the middle branch of the composite hmm ( figure 6 ) . note that when the cough model uses a left - to - right topology it forces any detected cough event to have visited each state of the cough hmm in a linear manner which is observed in the state sequence ( figure 7 ) . as discussed briefly above , this constraint partially explains why the rates of false positive are generally better for the left - to - right versus connected cough models . as stated in the introduction , the ultimate goal of this project was to create a symptom recognition application for asthmatics that could run , continuously , on a mobile device . consequently , apple developer credentials were obtained and the recognition algorithm described above was implemented in the xcode software development environment and ios software development kit . standard core audio apis were used for interfacing to the audio hardware of the mobile platform ( 4 g ipod touches ) . all of the required signal processing was facilitated by the fast fourier transform routines provided in the accelerate framework . moreover , custom cabling assemblies were constructed that allowed olympus me-15 tie - clip microphones to interface with the ipod . numerical underflow ( or overflow ) is a potential pitfall of working with hmms and the viterbi algorithm . equations ( 4 ) and ( 5 ) show that the probabilities involved can quickly accrue large exponents especially for a greater number of features and especially in conjunction with the viterbi algorithm . therefore , computations are carried out , where possible , with log - likelihoods . by taking care of computing the probability for each mixture component it was found that 64-bit floating point arithmetic could be used without underflowing . the exact algorithm for this computation again , the feature vector was 26-dimensional consisting of twelve mfccs , the log - energy measures , and the first differences of these quantities . here , f , , v , and are , respectively , the feature vector , the mean vector for the given component , a vector of the values along the main diagonal of , and the mixture weight . the likelihood for the entire state is found by running algorithm 1 for each associated mixture component and then taking the natural log . two example instances of the mobile adam application running in a demonstration mode that was used for debugging are shown ( figure 8) . the data presented in the screenshots is similar to the viterbi decoding examples shown earlier ( figure 7 ) with the exception that the audio is being obtained and processed live on a mobile device . the top trace is the time - domain audio waveform and the bottom trace is a consolidation of the corresponding maximum likelihood state sequence found by token passing . let aseq[m ] for 0 < m < 514 be maximum likelihood state sequence ( for the chosen frame rate , 6 second blocks of audio result in 515 feature vectors ) . from aseq another sequence is created that is defined as ( 7)acough[m]={1,aseq[m]coughstate,0,aseq[m]otherwise . thus acough[m ] indicates where coughs occur in the audio according to the recognition algorithm . the left screenshot shows the application identifying a sequence of coughs from the database while the right screenshot shows the device discounting some nonsymptom speech ( figure 8) . these tests were conducted in an anechoic environment with the test sounds played through high - quality active speakers . this is not to claim that such tests recreate the conditions of a patient wearing the device in a nonlaboratory setting . rather , the tests were meant to verify that the recognition framework and application could return positive results in circumstances where any concerns over the reproducibility of test signals were minimized . it should be stated here that the performance measures given in the next section were determined by cross - validation trials with the test database . the logical next step in evaluating the device is to trial it with a population of patients . such a trial will not only measure the accuracy of the recognition algorithm but will also measure the scope of the test database ( i.e. , how well the algorithm generalizes ) . the columns in table 1 are from left to right as follows : the number of states in the background model , the number of mixtures per state for the background model , the number of states in the cough model , the number of mixtures per state for the cough model , the topology of the cough model , the rate of false positive per 1 hour , and the sensitivity . it is also noted that the number of states listed in table 1 follows an htk convention of including the dummy states . therefore , the number of states that are actually active during decoding is the given number minus two . firstly , 6 seconds of pcm audio was collected and split into frames with 256 points and half a frame overlap . feature vectors for each frame were computed and the audio was segmented in time by a hidden markov model based viterbi decoder . the entire 6 seconds of audio was classified as either containing a cough or not containing a cough based upon the criterion explained above ( figure 7 ) . in the classification task the data is skewed since it was expected that cough would occur relatively infrequently when compared to the frequency of other background noises . here , a pair of metrics was used : the rate of false positive ( or false alarms ) per 1 hour fp/1 h and sensitivity or true positive rate . the rate of false positive is found by counting the number of false positives and dividing by the total length of time that was analyzed ( in units of hours ) . this can be estimated robustly since it is not a particular challenge to collect large volumes of audio data that are free of coughs ( i.e. , any hit is guaranteed to be a false alarm , and these are easy to count ) . let tp , tn , fp , and fn be , respectively , the total number of true positives , true negatives , false positives , and false negatives returned by a classifier . the sensitivity is defined as ( 8)sensitivity = tptp+fn . sensitivity measures how often the classifier misses symptoms . the composite hmm was tested with 30 examples of cough that were withheld during training . the results presented in the previous section show that certain model topologies and complexities provide a reliable detection and segmentation of cough and background sounds . clearly , the prototype used for the cough hmm has a powerful effect upon both performance measures . as expected , the connected cough models result in a higher false alarm rate ( keeping the other variables fixed ) . also , it is noted that the cough model requires a larger number of mixture components per state to achieve a decent rate of false positive . in trying to select among these alternative algorithms the goal is to provide data to the asthma patients that can give them a clearer picture of their disease state . a significant rate of false alarm will negatively impact the user 's trust in the feedback provided by the device . thus , aiming for low fp/1 h a large background hmm and a left - to - right cough hmm were used . describe the leicester cough monitor ( lcm ) [ 3 , 25 ] . like the system presented here , the lcm uses a recognition system consisting of preprocessing to calculate the cepstral features followed by hmm analysis . a very high sensitivity ( 85.7% ) and low false positive ( 0.8 per hour ) rate were achieved using this approach . however , in the lcm the audio analysis is performed offline and the ambulatory component of the study consisted strictly of an audio recorder . this differs critically from the present study where all of the processing is performed on the mobile device and the results are provided as feedback in real time . the inherent time and computational restrictions in the present study prevented the use of hmms of the same complexity ( in terms of the number of hidden states ) as the lcm . like adam and the lcm , shin et al . report on a cough detection system that uses hmm analysis of audio features . instead of using cepstral features directly shin et al . processed cepstral features with an artificial neural network before passing them to the hmm viterbi decoder . while their system successfully discriminated cough from environmental noise it was only validated using synthetic examples at most 10 minutes in length so it is not directly comparable to the in vivo situation that is of concern here . also , the present study is the first to examine the feasibility of ambulatory cough detection on a consumer mobile device in an adolescent asthma population . effective asthma management relies upon the accurate monitoring of symptoms by patients . however , current monitoring techniques have had limited success with adolescent groups . around their peers , adolescents with asthma may feel embarrassed about their condition and may also be reluctant to take medication when needed . adam provides reliable and objective symptom monitoring throughout the day that can be accessed privately through an attractive mobile interface . also , since adam is deployed on an already popular consumer platform , the device can be used inconspicuously and the self - management benefits it provides will not be hampered by social pressures . overall , it is expected that adolescents who have access to a technology such as adam will demonstrate greater adherence to treatment regimens , better awareness of symptom severity and , in the long term , require fewer emergency room visits . although cough is the most common symptom found in pediatric patients with uncontrolled asthma [ 13 , 14 ] there are other respiratory symptoms such as wheeze and the initial intention was to design a device to monitor both coughing and wheezing . however , wheeze was encountered infrequently during the data collection phase and the examples of wheeze that were recorded showed significant audio variations between each other . this frustrated attempts to design a reliable algorithm for identifying wheeze and distinguishing it from background noise . in principal , the methods described that were used for detecting cough could be applied to wheeze but it would require a more representative audio database than was available in the present study . firstly , because adam continuously records and processes sounds it is more resource intensive than typical current generation mobile applications . this impacts battery life negatively and may interfere with other applications that may be running on the device concurrently . future iterations of the application with better optimized code and power management will be needed to address these concerns . although mobile technology is becoming increasingly ubiquitous adam is still not appropriate for all situations . the device generally needs to be carried on the person and the lapel microphone must be positioned appropriately . certain activities , such as sports , may require the patient to set the device aside or the patient may be dressed in a manner that makes the adam device awkward to carry ( i.e. , clothing without a convenient pocket ) . moreover , in certain social settings , an adolescent may not want to be seen wearing a microphone . these practical limitations are expected however ; as long as patients make reasonable effort to use adam whenever possible the device will provide benefit in terms of asthma self - management . using a large audio database collected in a controlled study an algorithmic approach to the automated assessment of coughing in adolescent asthmatics was explored . this work represents the technical development of a device ( adam ) capable of providing objective round - the - clock feedback to adolescents with an asthma diagnosis . the eventual goal is to leverage the semantic information that can be gleaned from an audio stream to benefit the patient . in conjunction with the user interface capabilities and also the ubiquity of modern mobile phones this work enables new standards in 24-hour asthma symptom monitoring .

the development of an automated system for asthma monitoring ( adam ) is described . this consists of a consumer electronics mobile platform running a custom application . the application acquires an audio signal from an external user - worn microphone connected to the device analog - to - digital converter ( microphone input ) . this signal is processed to determine the presence or absence of cough sounds . symptom tallies and raw audio waveforms are recorded and made easily accessible for later review by a healthcare provider . the symptom detection algorithm is based upon standard speech recognition and machine learning paradigms and consists of an audio feature extraction step followed by a hidden markov model based viterbi decoder that has been trained on a large database of audio examples from a variety of subjects . multiple hidden markov model topologies and orders are studied . performance of the recognizer is presented in terms of the sensitivity and the rate of false alarm as determined in a cross - validation test .

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

PMC4759980

within clinical trials ( cts ) there are three dimensions where activities , their duration , and the conditions on which these time - bound activities unfold decide the fate of the study . these are : ( 1 ) assessment of safety prediction if a potential medicinal product will have any safety concerns through the evaluation of its potential adverse effects , ( 2 ) proof of concept and large studies efficacy , ( 3 ) postmarketing surveillance , pharmacovigilance , and periodic safety update reports . the coordination of an individual ct project and the general management of the projects running simultaneously have a strategic importance to ensure timely marketing authorization of the promising candidates in the portfolio . in all phases of cts , the common management items tend to remain the same , i.e. , the scope , resources , and the timelines . the productivity of the pharmaceutical industry has decreased over time , and the costs of producing new medicines have been rising sharply . critical path analysis ( cpa ) is a method which is intended to improve drug development and reduce uncertainty and cost by applying scientific tools to the clinical development process . when activity durations are deterministically or experientially known , the cpa can be applied to manage time and resources for a given trial . the motivation for cpa and overall project management for cts come from the fact that there are many dependent and independent ( parallel ) activities involved , and also there are many uncertainties . to meet the objective of systematic planning , cpa is one of the many network techniques which have been widely used for planning , scheduling and controlling the large and complex projects . cpa formally identifies tasks which must be completed on time for the timely completion of the whole project . it also identifies which tasks can be delayed if the resource needs to be reallocated to catch up on missed or overrunning tasks . a further benefit of cpa is that it helps us to identify the minimum length of time needed to complete a project . where we need to run an accelerated project , it helps us to identify which project steps we should accelerate to complete the project within the available time . the disadvantage of cpa , if we use it as the technique by which our project plans are communicated and managed against , is that the relation of tasks to time is not as immediately obvious as with gantt charts . it is not always possible to sort out completely identifiable activities and their start and finish times . , we tried to design and develop an innovative ct management approach for a project - specific operating plan during the conduct of the study . this ct management approach does not replace an understanding of or adherence to the requirements contained in all applicable regulations , guidelines , or standard operating procedures governing these studies but ensures optimization of ct design , analysis , trial management , and cost . initially , the activity list of a model ct was listed in different 78 activities , which was further merged into the major 35 activities . after further consideration and dependence analysis , it was finalized in the 25 activities which were taken in activity predecessor table for the purpose of network diagram and cpa considering patients , conduct and outcome [ table 1 ] . activities are inclusive ; describe the trial entirely with accuracy , in chronological and logical sequences . dependency nature is both qualitative and quantitative [ table 2 ] and also validated by experts . primary and secondary outcomes were appropriate , ethically approved , approved by the concerned regulatory agency , scientifically standardized as well as patients were well cared [ figure 1 ] . these 25 activities were taken to create a network diagram and perform cpa considering patients , conduct , and outcome activity - predecessor relationship to describe how each activity is dependent or independent from another activity which precedes this given activity network diagram based on the activity - predecessor table including activities from start to finish . in this figure , all 25 activities mentioned in tables 1 and 2 were taken to design the network a y . the activities which were linked to another activity ( ies ) are connected with the direct line to show inter - dependent relationships to determine the flow of these activities , we need to create a precedence diagram . activities which must be completed before a particular activity starts are called predecessor activities , and those which must follow a particular activity are called successor activities . after deciding the precedence order , the activities are put in a logical sequence using the graphical notations . while constructing the network , to ensure that the activities fall in a logical sequence , the following questions need to be answered : ( 1 ) what activities must be completed before a particular activity starts ? ( 2 ) what activities follow this ? ( 3 ) what activities must be performed concurrently with this ? a delay in any of the critical path activities will delay the entire project , regardless of whether the other project activities are completed on or before time . the precedence diagram shown in figure 2 has multiple paths highlighted as the green line , red line . the red line of this diagram describes the critical path of the activities from the starting point to completion of a clinical trial y slack and float both refer to the amount of time by which a particular event or activity can be delayed without affecting the time schedule of the network . the total float of an activity is the difference of its latest start time and its earliest start time . the activities which have zero floats are critical activities and are on the path of critical activities , the critical path . total float represents the time by which an activity can be delayed without affecting the project completion time . in this network diagram , critical activities such as development of protocol and investigator brochure , informed consent form translations , review , approval , translation certificate , study insurance certificate , site initiation visit ( siv ) preparation , dispatch of investigational product and other site requirements , siv , data entry , data validation , data clarification form generation and tracking , reconcile final records , and close study plays vital role . some of the noncritical activities of this project include development of drug supply management plan , clinical trial registry of india registration , preparation of ethics committee submission packets , and allocation of sites to the clinical research associate [ figure 3 ] . the diagram shown above describes the multiple paths highlighted with different colors with multiple activities and longest path that is , red lined path is the critical path of the project the longest path without any slack is the critical path . from the above diagram , we can see that path red lined is the longest as compared to green and blue lined path . therefore , if activity on this path is delayed , then the project will be delayed . after identifying the critical path , we can deduce the activities that when delayed will not impact the project . calculating the float of each activity using the cpa to determine the amount of time an activity may be late without delaying the project . a delay in any of the critical path activities will delay the entire project , regardless of whether the other project activities are completed on or before time . the precedence diagram shown in figure 2 has multiple paths highlighted as the green line , red line . the red line of this diagram describes the critical path of the activities from the starting point to completion of a clinical trial y slack and float both refer to the amount of time by which a particular event or activity can be delayed without affecting the time schedule of the network . the total float of an activity is the difference of its latest start time and its earliest start time . the activities which have zero floats are critical activities and are on the path of critical activities , the critical path . total float represents the time by which an activity can be delayed without affecting the project completion time . in this network diagram , critical activities such as development of protocol and investigator brochure , informed consent form translations , review , approval , translation certificate , study insurance certificate , site initiation visit ( siv ) preparation , dispatch of investigational product and other site requirements , siv , data entry , data validation , data clarification form generation and tracking , reconcile final records , and close study plays vital role . some of the noncritical activities of this project include development of drug supply management plan , clinical trial registry of india registration , preparation of ethics committee submission packets , and allocation of sites to the clinical research associate [ figure 3 ] . the diagram shown above describes the multiple paths highlighted with different colors with multiple activities and longest path that is , red lined path is the critical path of the project the longest path without any slack is the critical path . from the above diagram , we can see that path red lined is the longest as compared to green and blue lined path . therefore , if activity on this path is delayed , then the project will be delayed . after identifying the critical path , we can deduce the activities that when delayed will not impact the project . calculating the float of each activity using the cpa to determine the amount of time an activity may be late without delaying the project . by executing this approach repeatedly , an arbitrarily large number of realizations of project completion time may be generated allowing inference of its uncertainty . in addition , cost uncertainty with time - dependent effects can also be quantified by secondary calculations in the project network . some believe that a project plan is just a list of specific tasks and deliverables , organized across time and into larger groupings that begin and end with major project milestones . however , a project plan is not just a timeline management but also achieving the objectives clearly and being answerable to stakeholders by managing well all sorts of resources . major factors in determining the success or failure of clinical development projects are how well the planning process is carried out , whether the clinical project managers are involved early enough to understand the product development goals , executive sponsorship , and stakeholder involvement . senior level leadership in the planning phase is common in established pharmaceutical companies , but we recommend that the attention is given to developing strategic development plans also extend to individual clinical study plans . in smaller biopharmaceutical companies without dedicated project management offices , strategic product development planning conducted to satisfy investors usually lacks a rigorous operational component . in the absence of mid to senior management leadership , this planning is almost nonexistent , increasing the risks for each individual clinical study . in the clinical research the project team works within a matrix reporting environment to ensure that the tasks required for completing the project are done within the project timeline and with the quality that meets or exceeds the client 's expectations . within a clinical research organization , project management can include satisfying the client , supporting the project team , and senior management . most of the time , the project manager ends up sitting on a fence between what the client wants and what senior management wants . at the end of each day , the project manager must ensure that both sides are happy with the project 's performance . because the planning gap for clinical projects is persistent and widespread , managing each section of a typical project plan during the definition and planning stages is key to the success of the study . as the need to meet deadlines becomes more important , producing good and stable project plans become imperative . we can see that the critical path method or cpa is very useful for determining activities that can lead to project delay . based on actual results , resource reallocation can be considered for further better management of complex cts . the management of ct resources can only be carried out effectively if a series of accurate resource models can be produced that can provide a range of project execution alternatives . in this way

clinical research operates in a strictly regulated environment under various management models , but a distinct management model of clinical trial ( ct ) still needs exploration and research . critical path analysis ( cpa ) is a management approach can be used for monitoring , analysis , and prediction of success of its time - bound operational activities . a model ct was compiled with 78 activities , which were further merged into 35 major activities . after performing dependence analysis , the list was finalized with 25 activities which were taken in activity predecessor to create a network diagram and perform cpa considering patients , conduct , and outcome . activities were inclusive , described the trial entirely with accuracy , and were in chronological and logical sequences . this approach does not replace an understanding of or adherence to the requirements contained in all applicable regulations , guidelines or standard operating procedures governing clinical studies but ensures the proper use of operational and decisional approaches including optimal resource management . as the need to meet deadlines becomes more important and the need to produce good , stable project plans , cpa is very useful for determining activities that can lead to project delay . with this approach , project may be effectively monitored , and realistic schedules can be maintained .

INTRODUCTION RATIONAL METHODOLOGY, APPROACH, AND RESULTS Critical path analysis DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest

PMC3783794

aerial parts are used in the treatment of kidney stones , hypertension , fever and cough ( http://www.forestry.gov.my/ ) . the earlier studies have shown that endophytic microorganisms isolated from medicinal plants produce the same metabolites as their hosts . therefore , there is a great potential in exploring endophytes as a source of therapeutic natural products . however , despite several traditional medicinal applications of nepenthes spp . the objective of this study was to isolate and to identify the endophytic bacteria ( eb ) from nepenthes spp . namely , nepenthes ampullaria , n. gracilis , n. macfarlanei , n. mirabilis , n. rafflesiana and n. sanguinea were collected from frim , selangor , malaysia . however , leaves and twigs from one to three individual plants of nepenthes spp . , namely , n. alba , n. albomarginata , n. gracillima and n. sanguinea were collected from the gunung jerai ( gj ) , gurun , kedah , malaysia . leaves with petioles were thoroughly washed under running tap - water and the surface - sterilization of plant material samples was carried out as reported elsewhere . the stem pieces were soaked in 70% ethanol and flamed to make their surface sterile . aseptically , the leaf and stem tissue pieces were inoculated in the petri plates containing luria - bertani ( lb ) agar medium . the plates were incubated in an incubator at 37c ( 3c ) for 18 - 20 h in the dark . the isolation , cultivation of endophytes , amplification of 16s rdna , sequencing of 16s rdna , identification of endophytes and rooted phylogenetic tree construction was carried out as reported by bhore et al . incubation of the inoculated leaf discs and stems pieces on lb agar medium enabled cultivable eb to grow , and the colonies of grown eb were visible on the margins of the leaf and stem tissues . all 96 isolates were identified based on 16s rdna sequence blast ( megablast ) hits analysis . the annotated 16s rrna gene fragment ( 16s rdna ) nucleotide sequences of all isolates have been submitted to the international dna database ( genbank / ddbj / embl ) under accession numbers : jf819686-jf819713 and jf938974-jf939041 . were from the bacilli ( 59.4% ) class , followed by gammaproteobacteria ( 35.4% ) and betaproteobacteria ( 5.2% ) [ table 1 ] . the genera of endophytic bacterial isolates ( ebis ) isolated from 9 nepenthes plant species the 16s rdna multiple sequence alignment output from clustalw was used in the construction of a rooted dendrogram . rooted dendrogram showing clustering of 44 diverse types of endophytic bacteria ( eb ) isolated from nine nepenthes spp . the accession number of 16s rdna ( rrna gene ) sequence of the respective isolate is given in front of the species name in this short and snappy study , we isolated and identified 96 isolates from nine nepenthes spp . eb have been reported from several medicinal plants , for instance glycyrrhiza spp . however , to the best of our knowledge , our study is the first to illustrate diversity and types of eb in nine stated nepenthes spp . comparison between the annotated 16s rdna fragment sequences from isolates and the sequences from genbank / ddbj / embl database using the blastn program revealed the identity of the respective isolates . the rooted dendrogram clearly showed the clustering of the species from the bacilli , betaproteobacteria and gammaproteobacteria groups . endophytes are found abundantly in various plant species studied to date , and soil bacteria such as bacillus spp . , pseudomonas spp . and azospirillum spp . however , we did not find any pseudomonas spp . and azospirillum spp . in our isolates . during isolation of eb , the growth medium used might be directly affecting the number and type of endophytic microorganisms that can be isolated from the plant tissues . the tissue samples used in the isolation of eb were from a single or few plants of each nepenthes spp . the location and the conditions in which plant species are grown also determine the types of endophytes in it . we have used plant samples from nepenthes spp . that were collected from their wild habitat in gj and diverse collection available at frim . this could be the reason for the wide diversity of eb in the studied nepenthes spp . it is important to note that from 96 isolates , 22 isolates ( representing 15 species ) belonged to the enterobacteriaceae family , which contains human enteric pathogens . a number of species from the enterobacteriaceae family have been reported as endophytes , viz entrobacter cloacae and klebsiella pneumonia in maize , entrobacter asburiae in cotton , and klebsiella spp . and entrobacter cloacae in banana . of the 44 species of eb isolated from nepenthes spp . however , we have not found any published record that reported acinetobacter soli , bacillus cibi , b. horneckiae , b. indicus , b. koreensis , b. stratosphericus , citrobacter gillenii , c. youngae , kluyvera ascorbata , providencia alcalifaciens and serratia liquefaciens as endophytes . perhaps , this is the first study that reports these bacterial species as endophytes . however , the benefits derived by nepenthes spp . from these bacterial endophytes and its quantum are not clearly understood yet . from this study contains diverse types of cultivable eb , and that the majority of bacterial endophytes ( 59.4% ) were from the bacilli class . nonetheless , these research findings could serve as a foundation in further research on the therapeutic properties of nepenthes spp . in correlation with their bacterial endophytes . we hypothesize that in nepenthes spp . , these eb might be involved in producing bioactive compounds of pharmaceutical importance , and further research is required to ascertain the same .

background : nepenthes species are used in traditional medicines to treat various health ailments . however , we do not know which types of endophytic bacteria ( eb ) are associated with nepenthes spp.objective:the objective of this study was to isolate and to identify eb associated with nepenthes spp.materials and methods : surface - sterilized leaf and stem tissues from nine nepenthes spp . collected from peninsular malaysia were used to isolate eb . isolates were identified using the polymerase chain reaction - amplified 16s ribosomal dna ( rdna ) sequence similarity based method.results:cultivable , 96 isolates were analyzed ; and the 16s rdna sequences analysis suggest that diverse bacterial species are associated with nepenthes spp . majority ( 55.2% ) of the isolates were from bacillus genus , and bacillus cereus was the most dominant ( 14.6% ) among isolates.conclusion:nepenthes spp . do harbor a wide array of cultivable endophytic bacteria .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION

PMC5060847

asked how they make a decision , health professionals , either individually or as part of a multidisciplinary medical team , will often say something liketogether with the patient we look at the available options to see how well each performs on the main effect benefit , then take into account the side effect and adverse event harms , the burdens of the treatment and so on , finally weighing the benefits and harms and any other considerations to arrive at a conclusion as to the best option . we naturally bear in mind what the most recent relevant high quality guidelines have to say . together with the patient we look at the available options to see how well each performs on the main effect benefit , then take into account the side effect and adverse event harms , the burdens of the treatment and so on , finally weighing the benefits and harms and any other considerations to arrive at a conclusion as to the best option . we naturally bear in mind what the most recent relevant high quality guidelines have to say . a patient responding to the same question will probably come up with something similar , albeit expressed in different words such as taking the pros and cons into account ' and giving all the considerations due weight. clearly , these are not accurate characterizations of all clinical decisionmaking processes , but would seem to be reasonably descriptive of many . more importantly , they would certainly be common responses when the prescriptive question is asked : how should a clinical decision be made? these sorts of statements indicate that we operate in a healthcare system where some form of shared decision making is accepted as the aim . the majority of health professionals routinely talk the talk of informed choice and patientcentred care , increasingly emphasizing patientimportant outcomes as promoted by the grading of recommendations assessment , development , and evaluation ( grade ) collaboration1 and the newly established patientcentered outcomes research institute ( pcori ) ( http://www.pcori.org ) among many other individuals and groups . they do so with genuine conviction and intent , but find it more difficult to walk the walk2 , 3 and even to agree on what the key steps should be in terms of pace , direction and support . the presence of cultural and socioeconomic variations , together with great individual heterogeneity within cultures and classes , is at the heart of the challenge posed in pursuing shared decision making ( and informed choice ) within an overall philosophy of person and patientcentred care . the challenge to the professionals is mirrored by that of the individuals with whom they engage . all parties lack a simple and generic way to picture and communicate about the decisions that need to be made in health care . we seek to address this major handicap to progress towards all three goals . for convenience , the discussion is focused on the encounter between individual clinician and patient , but we regard our proposal as applying beyond the microclinical setting , to the meso and macrolevels of healthcare decision and policymaking . as it takes some form of argumentation conducted in words , even if it refers to numbers as inputs , we feel an appropriate shorthand term for it is verbal multicriteria decision deliberation ( mcdd ) . it embraces all forms of decision making that occur through deliberative processes , including those which are based on decision aids and support grounded in descriptive theories of human decision behaviour , usually involving descriptive theories of expert decision making.4 it dominates recent work in relation to shared decision making and patientcentred decision support.5 , 6 , 7 mcdd is a useful term because it highlights the key similarities and differences with the alternative decision ( and decision support ) technology that we argue should be included in the portfolio of clinical decisionmaking competencies of both health professionals and patients . this alternative is based on the wellestablished , theoretically grounded , prescriptive technique of multicriteria decision analysis ( mcda).8 to make the comparison with verbal mcdd even clearer , we can imagine the adjective numerical preceding it . in short , we are suggesting , along with dolan,9 van hummel and ijzerman10 and liberatore and nydick11 that numerical mcda ( hereafter simply mcda ) be added to the competency portfolio of all those involved in clinical decision making . we regard their studies as establishing that mcda based clinical decision support systems can be successfully developed and deployed . however , despite the high quality of the efforts of these researchers , the implementation of mcdabased decision support in health care has been fairly limited not that the success of mcddbased aids in routine practice has been spectacular to date.12 , 13 the reasons for this undoubtedly include the usability and communicability of the current software implementations of mcda technique , computerization being a necessary condition for its application , in contrast to mcdd . in this respect , we believe it reasonable to infer that an implementation which is superior in these respects will be more successful and can be regarded a priori as a workable clinical decision support system . but the reasons also trace back to the fundamentally different theoretical paradigm from which mcda itself emanates , compared with that underlying current clinical practice and the majority of decision aids built for use within it ( a comprehensive inventory of patient decision aids is available at http://decisionaid.ohri.ca/index.html ) . there are two key similarities between the two broad modes of multicriteria decision making , the umbrella term . first , both imply that in every clinical decision , two sorts of judgement are needed : ( i ) on the performance of each of the available options on each of the multiple relevant considerations and ( ii ) on the relative importance of those multiple considerations . second , that these conceptually different types of input must be integrated / synthesized / combined in some way to arrive at a decision . the key differences are reflected in the final words of the labels deliberation and analysis and in the preceding , implied adjectives verbal and numerical . ( in this paper , we include a graphical representation of data within the scope of the latter term . ) it might be asked why we characterize the distinction as a verbal / numerical contrast , rather than a qualitative / quantitative one . we do so because it is crucial to accept that mcdd is replete with the quantification of magnitudes . this quantification is simply done in predominantly verbal ways during the decisionmaking parts of the discourse . this applies in relation to the performance magnitude judgements , for example , of different medications reducing the chance of pain , where terms such as low probability , good chance and very likely are used to characterize the chances of the criterion being met for this patient . it also applies to the relative importance judgements , for example , of the importance of pain reduction relative to medication side effects , where again a variety of terms such as paramount , trivial and major or simply very important and not very important are deployed . the word analysis is used because the explicit aim in mcda ( and in fact of any version of decision analysis , including its costeffectiveness and costutility forms ) is to arrive at a result an opinion is our preferred term by a process of analytical calculation on the basis of numerical judgments . of course , the process of arriving at those numerical judgements almost certainly involves extensive verbal , nonnumerical elements and hence deliberation , in the same way that the deliberative discourse of mcdd may contain many judgments of magnitudes , including some expressed numerically . deliberation on the other hand is an interpersonal process where the provenance of the emerging conclusion inheres in the social process adopted and the participants involved in it . unless the deliberation is structured as an mcda,14 the conclusion can not be detached from them in the form of a graphic summary , or equation , or set of numerical option scores . a process that would benefit from the perceived strengths of each approach is an attractive prospect , and such a hybrid form has been implemented by proctor and drechsler in the context of environmental policy formation.14 a stakeholder jury was used to structure an mcda through a deliberative process and populate it with the help of experts . mcdd then followed as the final stage . the extensive time and resources involved , as well as the environmental policy context , make the empirical conclusions of limited relevance , but the hybrid case is well made in general . however , such a hybrid involves compromise from both sides , and this is not easy once it becomes clear that paradigmatic principles are involved , not merely syntactic or semantic differences that can be addressed by this means establishing whether improvements can be made , identifying where improvements could be made and providing support that will lead to improvements . purely descriptive approaches , which focus on describing how decisions are made by individuals or by organizations , communities and other groups provide no basis for change as they have no basis for identifying what would be an improvement.15 purely normative approaches , which focus on establishing , without reference to how decisions are made , the fundamental principles and processes that an ideal decision maker would implement , are simply impractical . on what basis can such desirable , potentially decisionimproving prescriptions for decisionmaking processes be identified ? decision analysis is essentially the ideal processes of decision theory converted into processes that are practical , given the time , resource and cognitive constraints of the real world . lipshitz and cohen4 call prescription arrived at on this basis analysisbased prescription , and this is exactly what we mean when we say mcdabased decision aids are ' prescriptive ' . they produce an opinion which reflects , as closely as practicable for many reasons this may not be very close at all the logical processes of an idealized decision maker . interestingly these principles and processes have been endorsed by many people if they are asked how a decision should be made , even when they do not follow the principles and processes themselves.4 the other possible basis of prescriptions for improvement in decision making is description of the decision processes of expert decision makers , on the one hand , in contrast to nonexpert decision makers on the other . identifying the differences between them what makes the experts expert can lead to what lipshitz and cohen call expertisebased prescription . despite the accuracy of this term , in the world of decision support the term those who favour the expertisebased approach prefer to characterize themselves as operating within an descriptive approach , which in many ways is true , even though , by definition , prescription is necessary to distinguish experts from nonexperts and good from notgood results.16 expertisebased description / prescription has been virtually the only route to improvement in decisions considered professionally acceptable in clinical medicine , and this is reflected in the curricula of medical schools and in clinical practice . decision analysis is rare to nonexistent in both the curricula of medical schools and in clinical practice . it is also the basis for the regular attacks on the expected utility / value principle which underlies analysisbased prescription . these critiques , most recently that of russell and schwartz,17 are always derived from the descriptive inadequacies of the expected value principle . but such inadequacies are ultimately irrelevant within a prescriptive paradigm because it is not derived from actual behaviour . as it is hard to conceive of unbiased crossparadigm evaluation , it is not surprising that this is never proposed in such critiques , which ultimately reflect the intuitive appeal of descriptive approaches that seek to take into account the complex characteristics , history and contexts of the individual . the issue is not whether these inadequacies and complexities exist are descriptively true but whether a user would prefer to be supported by analysisbased prescription or expertisebased description . the ethical responsibility is to make clear the paradigmatic origins of the type of support offered . the analysisbased prescriptive approach has one compelling advantage in the provision of patient / personcentred care and genuinely shared decision making . in its multicriteria form , decision analysis provides a generic approach to all decisions , that is , it is not condition specific and does not mandate the reasoning expertise and knowledge acquisition in the particular area ( e.g. a disease ) required to follow and share expertisebased prescriptions . as long as expertisebased prescription is the sole basis of the clinical encounter , patient empowerment will be a very difficult and demanding task . an mcdabased prescriptive approach allows the person / patient to input their preferences as importance weights for criteria in a straightforward manner and to have them transparently combined with the published evidence and the clinician 's expertise . this paper focuses on mcda as an appropriate technique for facilitating personcentred health care in relation to the adoption decision deciding what to do given the available options . it is important to distinguish this decision from two other decisions where we also regard mcda as an appropriate support technique . one is the quality decision deciding how good the decision just taken was , given the decision technology used . an mcdabased instrument for measuring decision quality has been developed and is presented in j. dowie , m. kjer kaltoft , g. salkeld and m. cunich ( submitted ) . the other is the decision decision deciding how to decide , given the available decision technologies . in our case , the decision decision is whether the adoption decision is to be made by the exercise of the health professional 's clinical judgment , by some form of mcddbased decision making , or in conjunction with some type of mcdabased decision support ? meta question later . a great number of software implementations of mcda exist , reflecting both widely varying versions of the technique and particular judgements about the extent and type of complexity to be catered for and the time and cognitive resources required.18 , 19 these range from implementations of a smart ( simple multi attribute rating system ) in a simple spreadsheet , implementations using the analytic hierarchy process ( ahp ) as executed either in a spreadsheet template or a dedicated software package , notably expert choice , http://expertchoice.com/products-services/expert-choice-desktop/ , to specific mcda implementations such as v.i.s.a http://www.visadecisions.com , hiview http://www.catalyze.co.uk/index.php/software/hiview3/ , webhipre http://hipre.aalto.fi/ and logical decisions http://www.logicaldecisions.com/. the latter two packages also contain an ahp option . the prime motivation for developing an mcda decision support system in the form of annalisa was that none of the existing implementations of mcda had , despite proving themselves as clinical decision support systems , made significant progress in health care . that was the situation when annalisa was first conceived and we feel it remains true now , despite the growing research in this area ( see dolan,9 maarten ijzerman and colleagues,20 and other examples cited in the liberatore review).11 most of the increasing use of mcda in health care is at the policy and health technology assessment level , with the recent developments within the evidem framework and software in the forefront21 , 22 confined to this setting . in deliberately implementing the simplest , compensatory however , annalisa seeks to provide enhanced interactive online usability by way of the numerous customizing and personalizing functionalities provided in the survey program elicia , into which the annalisa file is normally embedded . for example , annalisa enables personalization of the performance ratings of options on criteria on the basis of patient characteristics and personalization of the weightings of the criteria by the patient at the point of decision . thus , the focus of this paper is not to reintroduce mcda or confirm its value as the basis for clinical decision support systems , but to introduce a particular software template , annalisa , as a practical and personcentred implementation for use at the individual level , not only in shared decision making , but also in the community , especially in relation to cancer and other disease screening decisions and policies . the ahp has been the mcda implementation used most widely in the clinical healthcare context and warrants special mention . in an extensive series of papers , james dolan has expounded and investigated the ways it can contribute to both shared decision making and the wider issues involved in clinical decision support.8 , 9 , 23 however , in its standard form , ahp involves a level of complexity that imposes high demands on both the developers and implementers / users of ahpbased support systems . primarily responsible for this increased complexity is the hierarchical attribute structure which ahp permits and indeed encourages ( hence its name ) and the unique pairwise comparison method used to establish criteria importance weights and performance ratings , devised by its founder , thomas saaty.24 while this increased complexity can be seen as leading to a high level of performance by some standards of normative rigour and comprehensiveness,25 it creates the difficulties in the development and delivery of ahpbased decision support26 that have hindered its wider dissemination . we use them , as summarized by riabacke et al.,28 to highlight the basis of the claims of annalisa in each respect . all the implementations of mcda mentioned in this paper embody some version of the weightedsum principle which is at the centre of decision theory . annalisa implements this basic principle in a very simple way while still retaining its key principles . feasibility . the template was explicitly designed to reduce the complexity and resulting cognitive demands that is possible and tends to be facilitated by increasingly sophisticated implementations within the other available packages . no provision for sensitivity analysis is built into annalisa , but both the weightings and ratings can be directly varied and the effect on scores instantly observed . the weightedsum principle is not only explained and illustrated prior to the user 's interaction with annalisa , but also during interaction where appropriate , for example when and whether the individual panels ( weightings , ratings , scores ) are opened or closed at different stages during engagement . this criterion relates to the model developed in the software rather than to its basic functionalities . it is up to the user of annalisa to develop a model relevant to their context and setting . the embedding of annalisa in the online survey program elicia makes interactivity and cyclical iteration simple and efficient and leaves the amount of each entirely in the user 's control . all the implementations of mcda mentioned in this paper embody some version of the weightedsum principle which is at the centre of decision theory . annalisa implements this basic principle in a very simple way while still retaining its key principles . feasibility . the template was explicitly designed to reduce the complexity and resulting cognitive demands that is possible and tends to be facilitated by increasingly sophisticated implementations within the other available packages . no provision for sensitivity analysis is built into annalisa , but both the weightings and ratings can be directly varied and the effect on scores instantly observed . the weightedsum principle is not only explained and illustrated prior to the user 's interaction with annalisa , but also during interaction where appropriate , for example when and whether the individual panels ( weightings , ratings , scores ) are opened or closed at different stages during engagement . this criterion relates to the model developed in the software rather than to its basic functionalities . it is up to the user of annalisa to develop a model relevant to their context and setting . the embedding of annalisa in the online survey program elicia makes interactivity and cyclical iteration simple and efficient and leaves the amount of each entirely in the user 's control . the final point requires further development in the light of the increasing interest in the comparison and evaluation of decision aids on the basis of multiple criteria relating to development , performance , accessibility and impact . within its essential prioritization of simplicity over complexity , the functionalities provided by the annalisainelicia software create the possibility of building decision aids that should perform well on most criteria of adaptability and personalization . according to the eiring et al . coding scheme for the personalization of decision aids,29 the basic components of personalization are media content , user features , user model construction and representation and adaptive system behaviour . user features can broadly be classified into the users knowledge level , interests , preferences , goals / tasks , background , individual traits and context . adaptive system behaviours include adaptive navigation support , adaptive selection , organisation and presentation of content , adaptive search , adaptive collaboration and personalized recommendations . used in conjunction with elicia , any implementation of annalisa should provide a medium to high degree of personalization in all these respects and hence compare favourably with the 10 of the initial 259 decision aids that were subject to detailed classification in the eiringled study . it is up to the developer of a decision support tool within this software to determine what degree and type of adaptational flexibility and personalization to individuals or groups is to be offered in terms of attributes ( such as content , language , connectivity and presentation ) . it is also up to the developer to determine whether these are to be provided on an optin or optout basis . while compared with some ideal decision aid there are limitations in all these respects , a tool built in the annalisa software is capable of technically matching , or possibly surpassing , any of the actual decision aids subjected to intensive analysis in the eiringled project . most differences that arise will not be for reasons of functionality , but be traceable to their mcda basis , because this is what influences what is offered and required and how it is offered and required by way of adaptation and personalization . this point is worth emphasizing because it is important that a particular decision aid is assessed as an implementation of the underlying technique and philosophy within which it is built . a tool using annalisa is based on the paradigm of analysisbased prescription , as opposed to that of expertisebased description within which all the other aids examined by eiring et al . what is paramount in the development and use of a particular decision support tool or system is to make the potential user very clear about its underlying paradigm , so that questions concerning lower levels of functionality are relevant to it . the appropriate evaluation of annalisabased aids is therefore in comparison with other mcdabased ones and such a comparison , involving annalisa and ahp ( using expert choice ) , carried out within hiview , is that has been undertaken by pozomartin ( personal communication ) . this makes the 2005 french and xu30 survey of the five mcda packages cited earlier hiview , v.i.s.a , webhipre , expert choice and logical decisions a survey of current decision analysis software , including full technical and operational details , is provided biennially by informs . fifteen packages offer some form of multicriteria da , but this is a purely descriptive listing of information provided by 24 vendors , with no comments or assessments added . french and xu compared the five programs in terms of the aspects in which they differed , notably ( using annalisa terminology ) decision structuring , weighting elicitation , rating elicitation , data presentation and sensitivity analysis . when annalisa , in conjunction with elicia , is added to the comparison , two things stand out . first , the package fails to provide the vast majority of the functionalities and features that these five offer , considerably augmented in the 8 years since the survey . these functionalities and features are entirely appropriate where complex analysis is of benefit , such as in major projects with numerous stakeholders involved and large amounts of resources used , but even in such contexts the limited use of these packages either in practice or as the basis of decision support templates is noteworthy . and even where used , the complexity of the analysis is rarely matched in , or warranted by , the extensive deliberation that follows , as exemplified in a swedish exercise in participatory democracy.31 somewhat paradoxically it is the failure of annalisa to provide alternative and/or more sophisticated and complex methods for key tasks ( including determining the criteria and eliciting weights ) that we regard as its positive virtue , because it will provide the potential for much wider use . the growth of product comparison websites and recommender systems within ecommerce32 , 33 , 34 , 35 is a clear sign that multicriteria analysis is eminently accessible to large sections of the population , but only at an appropriate level of complexity . william buxton has pointed out that the speed of technological progress captured in moore 's law ( a technology generation is 18 months and decreasing ) is in complete contrast to his god 's law , which states that the capacity of human beings is limited and does not increase over time our neurons do not fire any faster , our memory does n't increase in capacity , and we do not learn or think faster as time progresses.36 the problem this creates for the evaluation of innovative and disruptive systems of all kinds is succinctly captured in martin buxton 's law it is always too early [ for rigorous evaluation ] , until suddenly it 's too late.37 the second difference is closely associated with the pace of change in both the hardware and connectivity within which any mcda software will operate . both french and xu and an earlier study by belton and hodgkin in 199938 saw three main settings for use of an mcda package : doityourself use by a single individual , an analystfacilitated group meeting , and offline analysis by a consultant sandwiched between facetoface meetings with decision makers . subsequent developments in communication technology and connectivity means that there are now many more possibilities , including one in which a prestructured ( options , criteria ) and evidencepopulated mcdabased decision aid is made available online . decision makers then need only to have their preferences ( utilities , importance weights ) elicited to obtain an opinion on the merits of the alternative options . in terms of data presentation and display , as well as interactivity , annalisa benefits from being developed specifically for online use within the latest technology . now written in html5 , with tablet use prominently in mind , it has mobile presence on touchoperated devices using ios , android and other operating systems . such interactive mobile accessibility is likely to trump most other functional considerations in the coming years , making a level of complexity compatible with such operation a paramount consideration . annalisa was designed to embody the following practical principles : it should be possible to undertake an analysis within a very short time , such as the 510 min often available in time / resource pressured situations , to ensure that the possible benefits of even a modicum of slow thinking should not be lost.39 this was in no way , of course , intended to prevent weeks or months being devoted to generating the detailed structure and inputs if the time and other resources are available.irrespective of the time available at the point of decision ( and therefore including 510 min ) , the decision owner should not be asked to make the necessary tradeoffs among more than 7 2 criteria.40 , 41 ( annalisa actually has a maximum of 10.)all the elements of the decision ( preferences and evidence ) and the outcome ( best option ) should be simultaneously visible on the screen , providing a complete picture of all elements of the decision and with the effects of changing any weighting or rating dynamically visible in real time.popups on the screen should provide access to additional information , especially the provenance of the option performance ratings ( including external links where appropriate ) . it should be possible to undertake an analysis within a very short time , such as the 510 min often available in time / resource pressured situations , to ensure that the possible benefits of even a modicum of slow thinking should not be lost.39 this was in no way , of course , intended to prevent weeks or months being devoted to generating the detailed structure and inputs if the time and other resources are available . irrespective of the time available at the point of decision ( and therefore including 510 min ) , the decision owner should not be asked to make the necessary tradeoffs among more than 7 2 criteria.40 , 41 ( annalisa actually has a maximum of 10 . ) all the elements of the decision ( preferences and evidence ) and the outcome ( best option ) should be simultaneously visible on the screen , providing a complete picture of all elements of the decision and with the effects of changing any weighting or rating dynamically visible in real time . popups on the screen should provide access to additional information , especially the provenance of the option performance ratings ( including external links where appropriate ) . giving higher weight to practical considerations , annalisa adopts the simplest and most colloquially familiar form of mcda . in the decision matrix weightedsum approach , all attributes exist at the same level ( there is no hierarchy of criteria and subcriteria ) ; the performance of each option is directly rated on each attribute ; the importance of each attribute is directly weighted in relation to that of all the other attributes ; and the option scores are calculated by summing an option 's ratings on the attributes multiplied by the attribute weightings . an illustrative example of a completed annalisa screen is provided in figures 1 and 2 . these might be seen as either those for two different patients or those of the same patient at two points of time ( where fig . 2 is produced at the next encounter i.e. time 1 ) . in the ratings panel of both instances , we can see that new treatment is better at maximizing the main effect benefit than current treatment ( 0.70 vs. 0.50 ) , is better at minimizing the treatment burden than the current treatment ( 0.80 vs. 0.70 ) , but is worse at minimizing side effects ( 0.20 vs. 0.50 ) . the two are equally good in relation to minimizing adverse events ( both 0.90 ) . given the relative weightings of the four attributes in fig . 1 , new treatment emerges with the highest score in a simple expected value calculation.score for current treatment=(0.500.50)+(0.500.30)+(0.900.10)+(0.700.10)=0.56 score for new treatment=(0.700.50)+(0.200.30)+(0.900.10)+(0.800.10)=0.58 figure 2 presents the scores when the weight assigned to minimizing side effects harm is increased , with correspondingly reduced weight to maximizing main effect benefit . current treatment now has the highest score , which means we interpret this option as the opinion emerging from the annalisa . from wherever and however they are derived , both the ratings and the weightings entered into annalisa are treated as measures on a ratio scale running from a ( true ) zero to 1 or 100% . zero on the ratings scale means either zero probability ( literally , and in many case logically , no chance ) or zero fulfilment of the attribute concerned ; 1 means 100% probability or complete fulfilment . similarly , zero on the weightings scale means of no importance whatsoever , and 1 means all important to the exclusion of all other attributes . the choice of the simple weightedsum approach , among many other decisions in the design and development of the annalisa template , was made in the light of our value judgments as to the weight to be assigned to particular considerations . preeminently , we have assigned high weights to relevant practical considerations in both development and delivery , in full recognition and awareness that these may lead to poorer ratings on other criteria , preeminently ones concerned with normative rigour . we do not see rigour / relevance and practicality / normativity as dichotomies , where one must make binary choices , but as matters of weighting and hence preference sensitive . annalisa , as with any implementation of mcdm including mcdd , embodies a particular view as to the criteria and weights to be used in deciding how to decide. this point continues to apply even when choosing among the candidates within the field of mcda . given that a patient faces multiple options and regards multiple criteria as relevant to choosing among them , should they stick with mcdd , the currently dominant decision technology , or move to mcda , at least as a decision support technology ? as discussed earlier , the two basic forms of mcdm and their many internal variations differ in important ways , as well as having key similarities . but from the clinical decisionmaking standpoint , we should not be thinking of making a choice between them at some general and abstract level . it hardly makes any sense to ask whether mcdd or mcda is better in general as a technique . neither is it particularly useful to ask whether ahp / expert choice or weightedsum / annalisa is a we need rather to focus on particular instantiations of each technique template in a tool for a particular clinical decision setting , where setting embraces such things as the organization , the professional , the patient and the condition involved . these criteria will probably include such higherlevel considerations as evidential strength and coverage , theoretical grounding , explicitness , precision , transparency , communicability and potential for social or institutional biasing . but given that this is clinical decision support , they should also include the basic resource requirements , such as the time and cognitive effort and commitment required from all parties , as well as any financial implications for them . it can be taken for granted that the performance of particular implementations of mcdd and mcda will vary on these criteria , not least because of conscious value judgementbased tradeoffs regarding the selection and weighting of the criteria made by individual parties in the case of mcdd and by the developers and implementers in the case of mcdabased decision support . for example , an mcdabased aid or mcddbased appointment designed to take no more than 20 min will ( should ) perform less well on a criterion such as coverage of the evidence than one assumed to have 40 min at its disposal . the various interactive decision support systems we are developing all allow customization of the support process to the time and other resources available , as well as personalization of the weightings by , and ratings for , the specific patient on the selected criteria . they explicitly assume , indeed emphasize , that such customization choices will impact on which aspects of the decision support will be accessed and that the personalization of weightings will affect the outcome ( opinion ) emerging from the analysis . thus , given that the decision on what decision procedure or decision support system to adopt involves multiple criteria and is therefore preference sensitive , it does not make sense to ask whether annalisa has , or ever can be , shown to work in some overall or average sense as the basis of a clinical decision support system . the answer will vary as a function of the particular decision maker 's preferences in the particular context as well as the quality of the instantiation . empirically , we can note that in a study with a small number of australian gps , 80% agreed that the demonstrated annalisabased tool for prostate cancer screening would be useful in discussions with their patients and half thought it would be useful and could be recommended for use in decisions on any health matter.42 pozomartin has recently established the preference sensitivity of decision support evaluation in a comparison of annalisa and the analytic hierarchy process for developing and delivering decision support for patients with advanced lung cancer in some spanish hospitals.43 finally , while it is not appropriate to report the full set of results of a rct involving annalisabased decision aids for psa testing here , table 1 provides information on the age of participants and their ratings on various criteria , such as difficulty in responding to the key items on criteria weighting , that confirm its accessibility . age of participants and individual ratings on criteria relating to usability of annalisa decision aids for psa testing given the resource requirements of decision making and decision supporting and hence their opportunity costs we suggest there is a strong case for deciding how to decide being approached analytically as an exercise in decision resource decision effectiveness analysis. this simply parallels in relation to the decision decision ( should we adopt this or that way of deciding whether , for example , to adopt this new drug or device technology ? ) the use of conventional costeffectiveness analysis in relation to the adoption decision ( should we , for example , adopt this new drug or device technology or not ? ) . as implied above , both numerator and denominator in decision resourcedecision effectiveness analysis are appropriately conceptualized as multicriterial indexes . a template designed to facilitate generic online multicriteria decision support in personcentred health care is presented in this paper as a valid addition to the portfolio of decision support systems available to clinicians and their patients . it is essential that any comparative evaluation of decision support systems makes the theoretical basis of each aid and process very clear to all respondents and decision stakeholders . in the context of personcentred care , this comparison will involve multiple criteria , of which the paradigmatic basis of the aid or process is a crucial one . the choice will be preference sensitive , with the weighting sometimes leading to an instantiation of multicriteria decision deliberation emerging as the best way of deciding and at other times to an implementation of multicriteria decision analysis . ultimately whether annalisa and similar templates have a role to play in personcentred care the empirical question , which will need to be iteratively asked and reasked as technology and attitudes change , concerns the precise roles it can play in the increasingly complex world of translational health . the contribution of professor salkeld and dr cunich was supported by the screening and diagnostic test evaluation program ( step ) funded by the national health and medical research council of australia under program grant number 633003 . jack dowie has a financial interest in the annalisa software , but annalisa and elicia are maldaba ltd .

abstractobjectiveto introduce a new online generic decision support system based on multicriteria decision analysis ( mcda ) , implemented in practical and userfriendly software ( annalisa).backgroundall parties in health care lack a simple and generic way to picture and process the decisions to be made in pursuit of improved decision making and more informed choice within an overall philosophy of person and patientcentred care.methodsthe mcdabased system generates patientspecific clinical guidance in the form of an opinion as to the merits of the alternative options in a decision , which are all scored and ranked . the scores for each option combine , in a simple expected value calculation , the best estimates available now for the performance of those options on patientdetermined criteria , with the individual patient 's preferences , expressed as importance weightings for those criteria . the survey software within which the annalisa file is embedded ( elicia ) customizes and personalizes the presentation and inputs . principles relevant to the development of such decisionspecific mcdabased aids are noted and comparisons with alternative implementations presented . the necessity to tradeoff practicality ( including resource constraints ) with normative rigour and empirical complexity , in both their development and delivery , is emphasized.conclusionthe mcda/annalisabased decision support system represents a prescriptive addition to the portfolio of decisionaiding tools available online to individuals and clinicians interested in pursuing shared decision making and informed choice within a commitment to transparency in relation to both the evidence and preference bases of decisions . some empirical data establishing its usability are provided .

Introduction: multicriteria decision making MCDD and MCDA: similarities and differences Developing an MCDAbased decision support system Deciding how to decide: the decision decision Conclusion Sources of Support Conflict of Interest

PMC4641944

autoimmune diseases ( aids ) are chronic disorders originated by the loss of immunological tolerance to self - antigens . this heterogeneous group of conditions present common genetic risk factors and share several pathophysiological mechanisms leading to overlapping clinical manifestations targeting specific organs or multiple organ systems . there is evidence that they share similar immunogenetic mechanisms , even though they exhibit varying epidemiological features and clinical manifestations [ 2 , 3 ] . underlying these diverse clinical phenotypes is a deregulated immune system with an enriched ability to respond against self - tissues . the fact that aids share several clinical signs and symptoms ( i.e. , subphenotypes ) and also share physiopathological mechanisms and genetic factors has been called autoimmune tautology and indicates that they may have a common origin . the immune system is in charge of the defence against external pathogens . for this purpose , t and b lymphocytes are responsible for the immune response through regulated cell - cell interactions and secretion of cytokines , chemokines , and other inflammatory mediators . this defence against external pathogens must occur without causing unnecessary harm to self . to achieve this delicate balance , the majority of self - reactive t and b lymphocytes are destroyed in the thymus and bone marrow through negative selection . nevertheless , this process is far from perfect , and self - reactive lymphocytes escape into the periphery . consequently , peripheral tolerance mechanisms are necessary to keep these self - reactive cells in check . activated self - reactive t and b cells promote autoimmunity when the effector and regulatory balance of the immune response is disturbed . major histocompatibility complex ( mhc ) molecules are widely distributed surface membrane glycoproteins that present antigenic peptides to t cell receptors ( tcrs ) . developing thymocytes encounter a highly heterogeneous repertoire of self ( endogenous ) peptide - mhc ( pmhc ) complexes on thymic epithelial cells , the main thymus antigen presenting cells . the affinity / avidity with which these thymocyte tcrs bind self pmhc determines if it is destined to perish or if it will survive . in this way , a repertoire of peripheral t cells that is essentially self - tolerant several hypotheses have been put forward to explain how mhc polymorphisms influence autoimmunity risk or protection . they must do so , somehow , by shaping the central or peripheral t cell repertoires toward autoimmune resistance or proclivity . a protective mhc profile could achieve this by the selection of a t cell repertoire with diminished pathogenicity . on the other hand , protective mhc molecules may keep autoimmunity in check by favouring the negative selection of particular self - reactive t cells [ 1214 ] . the functional basis of the association between specific hla alleles and development of aids can be classically explained by two possible etiopathogenic models . the molecular mimicry hypothesis proposes that certain hla alleles are more efficient in presenting pathogen epitopes that share structural features with self - peptides to mature t cells . once the response to the pathogen is initiated the self - antigen is also recognized and disease ensues . central selection failure proposes that certain hla alleles are less efficient at presenting self - peptides to developing t cells in the thymus , so negative selection fails . a different hypothesis proposes that different alleles can modulate the immunologic profile of an individual , through antigen - independent mechanisms , resulting in either promoting a higher autoimmune predisposition or , in opposition , a more efficient immune regulation . given the consistent association of hla - drb1 alleles with different autoimmune diseases ( table 1 ) , we explored the idea that the same hla - drb1 alleles could be influencing several different autoimmune diseases . to this end this study includes four autoimmune systemic diseases , namely , systemic lupus erythematosus ( sle ) , rheumatoid arthritis ( ra ) , psoriasis or psoriatic arthritis ( ps + psa ) , and systemic sclerosis ( ssc ) . patients with multiple sclerosis ( ms ) and myasthenia gravis ( mg ) were also included . a total of 1228 patients with aids , 213 patients with sle and 153 patients with ra diagnosed according to the american college of rheumatology ( acr ) criteria , 166 patients with ps + psa , 67 with ssc , 536 with definitive diagnosis of ms according to the revised mcdonald criteria , and 93 with mg , were recruited from the neurology and medicine outpatient clinic of centro hospitalar do porto - hospital de santo antnio ( chp - hsa ) . the hla - drb1 frequencies of patients were compared with the ones of a control group consisting of 282 unrelated individuals without disease and from the same geographic origin ( north of portugal ) . genomic dna was obtained from proteinase - k treated peripheral blood leukocytes by using a salting - out procedure . drb1 locus ( i.e. , 2-digit hla nomenclature ) was performed using polymerase chain reaction and sequence - specific primers ( pcr - ssp ) , based on methods previously described . in order to produce pcr - ssp reactions able to detect and discriminate each of the known hla - drb1 genes , primers were designed using sequence alignments comprising all hla - drb1 variants and were validated by the twelfth international histocompatibility workshop . pcr products were visualized under ultraviolet light after running in a 1.5% agarose gel containing ethidium bromide . to identify the hla - drb1 genes contributing to the six different aids , we applied stepwise logistic regression on an allelic level , using forward selection which involves starting with no variables in the model , testing the addition of each variable using a chosen model comparison criterion , adding the variable ( if any ) that improves the model the most , and repeating this process until none improves the model . it should be noted that odds ratios ( ors ) obtained in a multivariable logistic regression analysis are adjusted for all the other genes included in the model and therefore differ from those obtained when a given gene is compared with all other genes . a total of 1228 cases and 282 controls were analysed and different types of association between alleles and aids were found ( table 2 ) . these included three risk alleles for two or more aids , two protective alleles for two or more aids , and three risk alleles for a particular aid . hla - drb113 was a protective allele for four aids : sle ( 18.3% versus 29.8% , p = 0.016 , or = 0.58 , and 95% ci = 0.370.90 ) , ps + psa ( 19.3% versus 29.8% , p = 0.050 , or = 0.621 , and 95% ci = 0.391.00 ) , ra ( 16.3% versus 29.8% , p = 0.044 , or = 0.58 , and 95% ci = 0.340.98 ) , and ssc ( 11.9% versus 29.8% , p = 0.035 , or = 0.42 , and 95% ci = 0.190.94 ) . hla - drb103 was found to be a risk factor for sle ( 34.3% versus 15.6% , p = 4.2 10 , or = 2.49 , and 95% ci = 1.613.86 ) , ms ( 22.9% versus 15.6% , p = 0.003 , or = 1.81 , and 95% ci = 1.232.67 ) , and mg ( 35.5% versus 15.6% , p = 6.1 10 , or = 2.98 , and 95% ci = 1.755.07 ) . there were two risk alleles associated with two aids : hla - drb108 was positively associated with ms ( 12.1% versus 8.5% , p = 0.033 , or = 1.73 , and 95% ci = 1.052.87 ) and ssc ( 22.4% versus 8.5% , p = 0.004 , or = 3.01 , and 95% ci = 1.436.31 ) and hla - drb101 was found to be a risk factor for ra ( 32.7% versus 23.4% , p = 0.017 , or = 1.79 , and 95% ci = 1.112.88 ) and ssc ( 41.8% versus 23.4% , p = 0.006 , or = 2.28 , and 95% ci = 1.274.09 ) . hla - drb109 was negatively associated with sle ( 1.0% versus 5.0% , p = 0.027 , or = 0.18 , and 95% ci = 0.040.83 ) , ms ( 1.0% versus 5.0% , p = 0.004 , or = 0.22 , and 95% ci = 0.080.63 ) , and ra ( 0.0% versus 1.0% , p = 0.003 , or = 0.95 , and 95% ci = 0.930.98 ) . three risk disease - specific alleles were found : hla - drb104 for ra ( 47.7% versus 24.5% , p = 6 10 , or = 2.81 , and 95% ci = 1.794.39 ) , hla - drb107 for ps + psa ( 39.8% versus 25.5% , p = 0.006 , or = 1.79 , and 95% ci = 1.182.72 ) , and hla - drb115 for ms ( 32.7% versus 19.9% , p = 2 10 , or = 2.17 , and 95% ci = 1.533.10 ) . considering aids as a group , hla - drb103 frequency was significantly higher ( 23.9% versus 15.6% , p = 0.022 , or = 1.51 , and 95% ci = 1.02.15 ) compared with controls ; conversely hla - drb113 ( 20.0% versus 29.8% , p = 0.004 , or = 0.58 , and 95% ci = 0.430.79 ) and hla - drb109 ( 1.4% versus 5.0% , p = 1 10 , or = 0.23 , and 95% ci = 0.110.49 ) frequencies were significantly lower . through a systematic review of published works , cruz - tapias and collaborators , in 2012 , identified some common hla class ii alleles that contribute to susceptibility to aids in latin americans . the present study is , to date and to the best of our knowledge , the only one that addresses the hypothesis that a hla - drb1 allele could influence different autoimmune diseases , using a new cohort , encompassing six different autoimmune diseases . in this study we confirmed positive and negative associations in ms [ 24 , 25 ] , sle [ 1618 ] , ps + psa [ 19 , 20 ] , ra , ssc [ 22 , 23 ] , and mg , previously reported in our or other populations . when aids studied were considered as a group , hla - drb103 allele was significantly overrepresented , as already described . it has been shown that this allele has low affinity for clip ( class ii - associated invariant chain peptide ) and may not require hla - dm to ensure peptide presentation , preventing efficient peptide selection and allowing the binding of low stability peptides . concerning the observed negative association with hla - drb109 , we think that this is likely a spurious association , as this is a rare allele and the frequency found in controls is , for some reason , double the one reported for the portuguese population . our observations suggest that the presence of hla - drb113 allele may confer protection for aids . hla - drb113 is a high frequency allele in the general population both in portugal and worldwide . our results confirm that the lower frequency of hla - drb113 in every individual aids group is not secondary to the deviations granted by the concurrent positive associations . when the data obtained from previous studies are taken into consideration , the hla - drb113 allele seems to be a universal protective allele for ra . it was reported as protective against ra in asian [ 32 , 33 ] , turkish , and several european populations [ 3537 ] . recently this allele was also described to be protective in sle in the japanese population and for anca - associated vasculitis in the dutch population . specific amino acid patterns at the peptide binding cleft are involved in disease susceptibility , such as the well - known shared epitope first described in the ra susceptibility alleles hla - drb101 and hla - drb104 [ 37 , 39 ] . similar to the shared epitope classification of susceptibility alleles , protective hla - drb1 alleles have been categorized according to several models . one of the most accepted classifications proposes that protection against ra is conferred by the deraa sequence at positions 7074 of the hla - drb1 allele . other models suggest that protection is conferred by an aspartic acid at position 70 ( d70 allele ) or an isoleucine at position 67 ( i67 allele ) of the hla - drb1 molecule . because it was unclear which hla - drb1 alleles were protective a meta - analysis was performed involving four european populations with > 2,700 patients and > 3,000 control subjects . the objective was to investigate exhaustively which hla - drb1 alleles were associated with protection against ra . interestingly , this study showed that the protective effect attributed to deraa and d70 was no longer present after the exclusion of hla - drb113 . the authors concluded that this evidence indicates that hla - drb113 rather than deraa , d70 , or i67 is associated with protection . in a recent study van heemst and collaborators identify citrullinated vinculin , present in the joints of acpa ra patients , as an autoantigen targeted by acpa and cd4 t cells . these t cells recognize an epitope with the core sequence deraa , which is also found in many microbes and in protective hla - drb113 molecules , presented by predisposing hla - dq molecules . intriguingly , deraa - directed t cells were not detected in hla - drb113 donors , indicating that the deraa epitope from hla - drb113 could mediate thymic tolerance in these donors and explain the protective effects associated with hla - drb113 . they suggest that subjects born with hla - drb113 will present the hla - drb113-derived deraa - peptide in the thymus , leading to tolerization of the deraa - reactive t cell response . the negative association we describe here supports the idea that the hla - drb113 allele , possibly by its specific structural features , may as well confer resistance to several other aids . the protective effect of hla - drb113 could be explained by a more proficient antigen presentation by these molecules [ 43 , 44 ] , favouring an efficient thymic selection . as a result , negative selection and development of dr - driven autoreactive regulatory t cells a different model would relate hla molecules with the presence of specific endophenotypes indirectly associated with autoimmunity . other studies of our group suggest that the hla genotype may primarily influence the general activation state of cd4 t cells . the protective effect of hla - drb113 curiously , several reports have suggested an association of hla - drb113 and/or hla - dqb106 with slow disease progression in human immunodeficiency virus ( hiv ) infected individuals , meaning that among hiv controllers there is an association between the presence of certain class ii hla alleles and strong cd4 t cell responses [ 46 , 47 ] . although different alleles are associated with particular aids , the same allele , hla - drb113 , was underrepresented in all six diseases . this observation suggests that this allele confers protection to aids in general and particularly to rheumatic diseases .

autoimmune diseases ( aids ) are characterized by a multifactorial aetiology and a complex genetic background , with the mhc region playing a major role . we genotyped for hla - drb1 locus 1228 patients with aids-213 with systemic lupus erythematosus ( sle ) , 166 with psoriasis or psoriatic arthritis ( ps + psa ) , 153 with rheumatoid arthritis ( ra ) , 67 with systemic sclerosis ( ssc ) , 536 with multiple sclerosis ( ms ) , and 93 with myasthenia gravis ( mg ) and 282 unrelated controls . we confirmed previously established associations of hla - drb115 ( or = 2.17 ) and hla - drb103 ( or = 1.81 ) alleles with ms , hla - drb103 with sle ( or = 2.49 ) , hla - drb101 ( or = 1.79 ) and hla - drb104 ( or = 2.81 ) with ra , hla - drb107 with ps + psa ( or = 1.79 ) , hla - drb101 ( or = 2.28 ) and hla - drb108 ( or = 3.01 ) with ssc , and hla - drb103 with mg ( or = 2.98 ) . we further observed a consistent negative association of hla - drb113 allele with sle , ps + psa , ra , and ssc ( 18.3% , 19.3% , 16.3% , and 11.9% , resp . , versus 29.8% in controls ) . hla - drb113 frequency in the aids group was 20.0% ( or = 0.58 ) . although different alleles were associated with particular aids , the same allele , hla - drb113 , was underrepresented in all of the six diseases analysed . this observation suggests that this allele may confer protection for aids , particularly for systemic and rheumatic disease . the protective effect of hla - drb113 could be explained by a more proficient antigen presentation by these molecules , favouring efficient clonal deletion during thymic selection .

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

PMC3082169

altman and das ( 1965 ) proposed in 1965 the revolutionary idea that new neurons are born in the adult brain . for decades , the concept has been ignored until it was rediscovered in the early 1990s . it is now well accepted that new neurons are born throughout life , even in humans ( eriksson et al . , 1998 ; jin et al . , 2004 ; d'alessio et al . , 2010 ) and in aged animals ( kuhn et al . , 1996 ; kempermann et al . , adult neurogenesis has been clearly demonstrated and confirmed in two brain regions : the subventricular zone ( svz ) of the lateral ventricles and the subgranular zone ( sgz ) of the dentate gyrus ( dg ) of the hippocampal formation ( kempermann and gage , 1999 ) . cells born in the adult svz migrate through the rostral migratory stream and become granule neurons and periglomerular neurons in the olfactory bulb . cells born in the adult sgz migrate into the granule cell layer of the dg and become dentate granule cells ( dgc ) . it is only in the last decade that hippocampal adult neurogenesis has become a hot topic . many studies have been designed to characterize these newly generated neuronal cells ( kempermann et al . , 2004 ; zhao et al . , 2006 ) , to unravel the regulation of the different maturation stages ( zhao et al . , 2008 ) and to unveil the potential meaning of this phenomenon ( zhao et al . , 2008 ; the newly generated cells undergo different maturation stages to become functional mature neurons . as a consequence of neurons being born in a continuous manner , the dg is composed of a heterogeneous population of dividing and non - dividing , immature and mature , neuronal and non - neuronal cells . ( 2004 ) have developed a model of six developmental milestones of hippocampal adult neurogenesis based on their basic morphology and the combinatorial expression of six fairly stage - specific markers . they are characterized by their radial process extending to the inner molecular layer where they ramify , and by the expression of both nestin and glial fibrillary acidic protein ( gfap ) . nestin - positive cells that do not express gfap nor doublecortin are classified as type-2a putative progenitor cells . doublecortin is indeed expressed in type-2b , type-3 cells , both putative progenitor cells , and by immature granule cells . postmitotic immature neurons are further characterized by the emergence of dendrites and the coexpression of calretinin or neun . finally , mature dgc have developed a dendritic arborization and are calbindin- and neun - positive . recent studies have suggested that the electrophysiological and morphological properties of the newborn cells gradually emerge during neuronal maturation . these properties are needed by the young neurons for successful integration into the existing synaptic circuits ( zhao et al . , 2006 , 2008 ; the idea that new neurons are needed to build new memories has been quick to emerge . the concept implies hippocampal adult neurogenesis as being a new form of structural plasticity correlated to the processes of synaptic learning and memory formation . should this be the case , animals with impaired neurogenesis should , compared to wildtype animals , show alterations in acquisition , retention , recall or extinction of some kinds of information like spatial or contextual information . 2010 ) have used the synras transgenic mouse model in order to better understand the role of adult neurogenesis in learning and memory . the mouse has a synapsin1 promoter - driven overexpression of the constitutively activated g - protein p21ras in neurons . intriguingly , this mouse has significantly depressed rates of hippocampal adult neurogenesis , and this is associated with impaired performance in an object recognition task ( lafenetre et al . , 2010 ) . both , impaired neurogenesis and impaired object recognition could however be rescued by exposing synras mice to free access to a running wheel ( lafenetre et al . , 2010 ; proliferation , assessed by the number of bromodeoxyuridine ( brdu)-labeled cells , brain - derived neurotrophic factor levels , dendritic arborization of doublecortin - labeled cells and object recognition performance have been studied in wildtype ( wt ) and transgenic ( synras ) mice under basal conditions and with free access to a running wheel . ( 2010 ) , starting by presenting the regulation of adult neurogenesis by physical activity as an epigenetic factor and the possible implication of neurotrophins as molecular mediators . adult neurogenesis has been shown to be a dynamic process that can be regulated both positively and negatively by many factors , including epigenetic factors . indeed , in the last decades , five of them have clearly been identified and studied . whereas stress and aging downregulate adult neurogenesis , physical activity , environmental enrichment , and learning and memory have beneficial effects ( figure 1 ) . regulation of adult neurogenesis can modulate learning and memory . whereas aging and stress downregulate adult neurogenesis ( pink arrows ) , experience , enriched environment and physical activity ( blue arrows ) stimulate the generation of new neurons . ( 2010 ) to rescue the reduced adult neurogenesis of the synras mice due to reduced proliferation . voluntary physical activity is indeed the most potent enhancer of adult proliferation ( van praag et al . , 1999a ; olson et al . , 2006 ; fabel and kempermann , 2008 ) . the increase in neurogenesis is however region - specific and occurs only in the hippocampus and does not stimulate the olfactory bulb neurogenesis ( brown et al . , however , the modalities could vary from one experiment to another and could induce differential effects . first , the access to the wheel could be free or restricted to some hours per day or night ( holmes et al . , 2004 ) . second , running is limited to physical activity when rodents are housed individually but could also be considered as a social activity when the animals are housed per group . indeed , when rodents are isolated , the effects may be delayed or prevented ( stranahan et al . , 2006 ; leasure and decker , 2009 ) but this is still under debate ( kannangara et al . , 2009 , 2010 ) . thirdly , grouped animals could use the running wheel according to two modes : some are very active and seem to become addicted to running whereas other individuals are passive users . in the study by lafenetre et al . ( 2010 ) , grouped mice had free access to the running wheel : this paradigm gave the greatest opportunity to enhance adult neurogenesis . 2010 ) have thus confirmed the positive regulation of adult neurogenesis by physical activity that had been described earlier in wild type rodents ( van praag et al . this paradigm is also efficient in stimulating the neurogenesis of synras mice characterized by a reduced basal adult neurogenesis in the hippocampus ( figure 2 ; lafenetre et al . , 2010 ; manns et al . , the ways by which physical activity promotes proliferation must thus differ from the regulation of proliferation in basal conditions ( lafenetre et al . , 2010 ) . it has earlier been shown that neurogenesis could be partially rescued in aged animals by voluntary physical activity . this also suggests the regulatory mechanisms could be modified during aging but that the cells could still respond to external stimuli ( van praag et al . doublecortin - labeled cells in the dentate gyrus of wildtype controls ( upper left ) , wildtype runners ( upper right ) , synras controls ( bottom left ) , and synras runners ( bottom right ) . wheel running has also been shown to improve hippocampal - dependent spatial learning in rodents in the morris water maze ( fordyce and farrar , 1991 ; van praag et al . , 1999a ; vaynman et al . , 2004 ) and in the radial maze ( anderson et al . , 2000 ) . other tasks like contextual fear conditioning have also been used to show the running - induced improvement in cognitive performance ( baruch et al . , the beneficial effect of physical activity could be mediated by increased synaptic plasticity ( van praag et al . , 1999b ; farmer et al . , 2004 ) , neurotransmission and growth factor expression ( cotman and berchtold , 2002 ) that are observed both in running mice and rats . in their study , lafenetre et al . , ( 2010 ) have focused on the involvement of brain - derived neurotrophic factor ( bdnf ) as one of the potential mediators of these effects . brain - derived neurotrophic factor is a neurotrophin that is highly expressed in the hippocampus and has been implicated in synaptic plasticity and neuronal development ( binder and scharfman , 2004 ) . bdnf is known for its survival - promoting effects on new neuroblasts through the trkb receptor ( bath et al . , 2008 ) . environmental factors like environmental enrichment and physical activity induce an increase in bdnf expression level even after a short exposure to a running wheel ( cotman and berchtold , 2002 ) . moreover , running wheel activity increases the levels of the phosphorylated form of the bdnf receptor trkb ( vaynman et al . , 2003 ) . the functional role of bdnf in the regulation of the hippocampal adult neurogenesis is quite controversial . 2010 ) showed a running - induced increase in bdnf protein level that was associated with increased level of bromodeoxyuridine ( brdu)-labeled cells and doublecortin - labeled cells in both wildtype and synras mice . bdnf acts through the trkb receptor which is present on doublecortin - expressing cells ( donovan et al . , 2008 ; lafenetre et al . , 2010 the authors thus suggested that bdnf could not only promote the proliferation of doublecortin - expressing neuronal precursor cells but also stimulate the dendritic arborization of the immature neurons and facilitate their survival ( figure 3 ) . free and social access to the running wheel would induce an increase in bdnf release that could act on ( 1 ) the proliferation of doublecortin - immunoreactive precursor cells , ( 2 ) on the dendritic arborization of doublecortin - immunoreactive immature neurons , stimulating their maturation and their differentiation , ( 3 ) on the selective recruitment of immature cells . the infusion of bdnf for 2 weeks directly into the hippocampus increases neurogenesis of granule cells ( scharfman et al . , 2005 ) . however , classical genetic studies manipulating directly the expression of bdnf do not lead to such clear results . indeed , enhanced and reduced cell proliferation in heterogeneous bdnf knockout ( bdnf+/ ) mice have been reported ( lee et al . , 2002 ; sairanen et al . , the survival rate of the neurons must however be dependent on bdnf expression as impaired levels of cell survival were observed in both studies ( lee et al . , 2002 ; sairanen et al . , 2005 ) . moreover , enriched environment does not enhance the survival of newborn cells in bdnf+/ heterogenous knockout mice ( rossi et al . , 2006 ) . in accordance with the role of bdnf in promoting the dendritic differentiation of mature neurons ( mcallister et al . , 1996 ; wirth et al . , 2003 ) , the dendritic arborization of the dgc is more developed in bdnf - overexpressing transgenic mice ( tolwani et al . , 2002 ) . in a recent study , the role of bdnf in different stages of adult neurogenesis has been assessed in conditional knockout mice that lack the expression of bdnf in mature neurons of the adult hippocampus , resulting in 50% of the bdnf levels . bdnf has been suggested to play a critical role in the regulation of the survival and of the dendritic development of neuronal precursor cells but seems less important for exercise - induced proliferation of the cells ( choi et al . , 2009 ) . the conditional deletion of the trkb receptor in progenitor cells leads to impaired basic organization of synaptic connections and compromises the survival and the integration of the newborn neurons ( bergami et al . , thus , whereas the role of bdnf in the proliferation of neuronal progenitor cells is still unclear , the survival and the integration of these newborn neurons rely on the good functioning of bdnf / trkb signaling . due to the cellular heterogeneity of the dg , the understanding of the role of bdnf and other growth and neurotrophic factors , like vegf , igf1 , erythropoietin , remains difficult . presumably , they have to be tightly regulated and can act differentially on different developmental stages of the various cellular subsets . the functionality of the newly generated neurons has remained incompletely understood . with regard to the well - characterized role of the hippocampus in learning and memory , hippocampal adult neurogenesis has been proposed to be a new form of plasticity that underlies these processes . three technical approaches have been used to study the involvement of the newly generated neurons in learning and memory processes : the neural progenitor cells are ablated by pharmacological treatment , by irradiation or by genetic tools . . injections of the dna methylating agents , methylazoxymethanol ( mam ) , or more recently of temozolomide ( tmz ) , significantly reduce the rate of adult neurogenesis . x - ray irradiation has also been a very powerful , even stronger , tool . however , these techniques may induce side - effects that could cause other detrimental effects on brain physiology and function ( bruel - jungerman et al . , 2007 ) . with the generation of new genetic tools , it has been possible to specifically target the neural progenitor cells in the hippocampus and better assess the role of the new neurons in learning and memory processes . besides , the use of other transgenic mice has helped in the better characterization of some molecules that could be involved in the regulation of adult neurogenesis . as in lafenetre et al . , ( 2010 ) , many studies have used environmental stimuli that could affect both , the animals behavior and adult neurogenesis ( figure 3 ) . ( 2010 ) have recently reviewed the different behavioral tasks that have been performed with animals subjected to an ablation of progenitor cells . for instance , the involvement of adult neurogenesis in the acquisition of the morris water maze test , the most commonly used paradigm to test hippocampal functions , has been reported by dupret et al . by contrast , other studies have reported that adult neurogenesis is not required for the acquisition of the morris water maze test ( shors et al . , 2002 ; madsen et al . similarly , is adult neurogenesis necessary for long - term retention of the location of the hidden platform ? when progenitor cells are ablated by irradiation ( rat ; snyder et al . , 2005 ) , viral disruption of the wnt signaling ( rat ; jessberger et al . , 2009 ) and genetic disruption ( mouse ; dupret et al . , 2008 ; however , the retention is only affected 1 week after the viral injection in the genetic model of deng et al . , ( 2009 ) and only the retention of the reversal is affected by tmz treatment ( garthe et al . , 2009 ) . other studies have not found any positive correlation between reduced adult neurogenesis and impaired cognitive performance in the morris water maze ( shors et al . , 2002 ; meshi et al . , 2006 ; saxe et al . , 2006 ) and it has even been revealed that inhibiting adult neurogenesis could lead to better learning and memory performances ( kerr et al . , 2010 ) . similar contradictory results have been obtained in the contextual fear conditioning paradigm which is also a key test to assess the role of the hippocampus . some studies have found deficits in freezing behavior ( saxe et al . , 2006 ; winocur et al . , 2008 ; hernandez - rabaza et al . , 2009 ; snyder et al . , 2009 ) but no effect on freezing were reporting by others ( dupret et al . , 2008 ; zhang et al . , 2008 ; snyder et al . , while specific effects on the formation but not the extinction of contextual fear memory have been found in strongly irradiated mice , mice that have received mam injections ( ko et al . , 2009 ) or mice that have been irradiated to a lesser extent ( kitamura et al . , 2009 ; ko et al . , 2009 ) are not impaired . by contrast , in nestin - tk mice with ablation of actively dividing progenitor cells by ganciclovir , impairment in freezing or fear conditioning is not observed , yet they are not able to extinguish their fear response to the context as efficiently as the wildtype mice ( deng et al . , 2009 ) . the synras mouse has a reduced adult neurogenesis and is impaired in exploring and discriminating a novel object compared to a familiar object . however , stimulating adult neurogenesis by physical activity rescues these deficits ( table 1 ) . similar deficits have been observed in rats that have been injected with a lentiviral virus targeting the wnt signaling ( jessberger et al . , 2009 ) . several other studies employing hippocampus - dependent or independent tasks have also led to inconclusive results ( see deng et al . , 2010 for review ) . recent studies have shown how dramatic hippocampal activity varies between standard laboratory mouse lines including those used for genetic engineering . electrical activity of neuronal networks is a driving force , e.g. , for the production , release and action of bdnf , the structural differentiation of neurons and the ability to form meaningful circuits . in particular , mouse lines differ in the patterns of spontaneous and evoked gamma oscillations , which are considered to be central to cognitive performance ( jansen et al . , 2009 ) . strains also differ in the rates of proliferation and/or survival of newly formed cells ( kempermann and gage , 2002 ) and this correlates with deficits in learning tasks ( kempermann et al . , 1998b ) . even substrains of the frequently used c57 strain differ dramatically in many behavioral aspects ( siegmund et al . , 2005 ; matsuo et al . , the different ablation protocols may also affect different populations of cells that would be more vulnerable according to their maturation stage ( deng et al . , 2010 ) . equally important are the collateral side - effects , the parameters used to assess the different phenotypes , the cognitive demand of the test , age , sex , and presumably the life history of the individual animals . it is thus difficult to integrate the results of different studies with so many variables . it would thus be needed to standardize protocols to target stage - specific cells in specific conditions . an often overlooked aspect is that most experimental paradigms are primarily designed to assess the role of the hippocampus with all its input , intrinsic , and output networks . are the tests adequate for understanding the role of the newly generated granule cells in the dg ? of course , the dg is considered as the information gateway to the hippocampus and has been specifically shown to be involved in pattern separation ( gilbert et al . , 2001 ; computational models based on this function of the dg have been proposed for explaining either the addition of the new neurons in the network or the replacement of old dgc by these new neurons ( aimone et al . , 2009 , 2010 ; new born cells would thus help avoiding interferences between new and already established memories and promoting the individuals behavioral adaptation . studies directly assessing this role are thus needed to better understand the real involvement of adult neurogenesis in learning and memory processes . according to a recent study , the newly generated cells in the dg must have a functional implication in pattern separation only when stimuli are presented with little separation in space , but not when they are widely separated ( clelland et al . , 2009 ) . similarly , voluntary running improves the performance of the subtle discrimination of the location of two adjacent identical stimuli , which is tightly correlated to adult neurogenesis ( creer et al . adult new neurons may thus be important for pattern separation and for encoding fine spatial distinctions ( clelland et al . lafenetre et al . , ( 2010 ) have been able to show that voluntary physical activity could rescue two main phenotypes , the reduced adult neurogenesis and the impaired performance in an object recognition test , of a genetically modified mouse via an increase of bdnf . these results thus support the idea that adult neurogenesis is a dynamic process that is under the influence of environmental changes and that this form of plasticity is regulated by neurotrophic factors like bdnf . epigenetic factors , physical activity , enriched environment , aging , stress , and learning induce changes in both , the rate of adult neurogenesis and the behavior of the individual . however , the causal relationships are still not clearly determined and the role of adult neurogenesis in learning and memory processes remain obscure due to the many discrepant results . more studies are needed to better determine the effects of the various extrinsic factors on the progenitors and the subsequent stages of differentiation of the newly generated neurons , and their ability to integrate into the adult dg . furthermore , the functional involvement of the newly generated granule cells must be further studied in the restricted context of the direct role of the dg . computational models will thus help in elaborating theories that could be tested with animal models . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

neurogenesis occurs in two neurogenic zones in the adult brain : new neurons are born at the subventricular zone of the lateral ventricles and then migrate to the olfactory bulb , and at the subgranular zone to integrate the granular cell layer of the dentate gyrus . the hippocampus is involved in learning and memory and the generation of new hippocampal neurons has been suggested to be a new form of plasticity implicated in these processes . in the last decades , diverse intrinsic and epigenetic factors have been identified to influence adult neurogenesis but the underlying mechanisms remain unclear . in a recent study , lafenetre et al . ( 2010 ) showed the beneficial influence of physical voluntary activity on adult neurogenesis and cognitive performance in a transgenic mouse , the synras mouse via brain - derived neurotrophic factor . here we review how hippocampal neurogenesis can be regulated by environmental factors and the possible role of the newly generated cells in learning and memory .

Introduction Epigenetic Regulation of Adult Neurogenesis Neurotrophins and Adult Neurogenesis Adult Neurogenesis and Learning and Memory Conclusion Conflict of Interest Statement

PMC4272996

carpal tunnel syndrome ( cts ) represents the most common compressive peripheral neuropathy , predominantly presenting as a tingling sensation , pain and numbness in the distribution of the median nerve and it is estimated to affect about 1% of the population33,37 ) . cts is usually diagnosed on the basis of the patient 's history and clinical findings , and confirmed by electrophysiological studies . recently , ultrasonography and/or magnetic resonance imaging can also be used to evaluate the median nerve and surrounding structures to complement electrophysiological study in diagnosing cts12,35 ) . when conservative treatment including medications , splinting or local injection of steroid into carpal tunnel fails , simple decompression of the median nerve by division of the transverse carpal ligament is indicated as an effective surgical treatment . as surgical technique , open carpal tunnel release ( octr ) has been the gold standard method . since the introduction of endoscopic technique by okutsu et al.32 ) in 1987 for treatment of cts , endoscopic carpal tunnel release ( ectr ) has been widely used as a minimally - invasive carpal tunnel surgery22 ) . ectr techniques are divided into two categories depending on the number of skin incision used to access carpal tunnel : single - portal vs. dual - portal techniques . ectr has advantage over octr such as less postoperative pain and more rapid postoperative recovery but there is still major controversy between the two procedures regarding the outcome and the complication rates5,22,31,41 ) . successful results after endoscopic carpal tunnel decompression have been reported with range from 92 to 98%10,22,40 ) . in most cts patients with successful postoperative outcome , preoperative tingling sensation and pain decreased significantly within 1 - 7 days after ectr and completely resolved by one - postoperative month31,41 ) . however , a small number of patients showed delayed improvement after 1 month following ectr . although various studies have reported preoperative prognostic indicators of the outcome of carpal tunnel release8,23,30,43,44 ) , to the knowledge of the authors , there have been no studies that have analyzed the delayed improvement patients after endoscopic carpal tunnel surgery . we analyzed patient subpopulation with the delayed improvement after ectr to investigate the characteristics of those patients and to determine the predictable factors of delayed improvement . we retrospectively reviewed databases of cts patients who underwent ectr at our hospital from 2002 to 2011 . the preoperative assessment included a detailed history , symptoms and physical examination and all patients undergoing ectr had electrodiagnostic studies confirming compression of the median nerve at the level of the transverse carpal ligament . however , small number of patients with severe symptoms underwent ectr even though electrodiagnostic studies did not reveal any abnormalities , compatable with cts . patients with postoperative period more than 12 months were analyzed and patients with prior carpal tunnel surgery or patients with diabetes or an underlying disease , related to cts were excluded from the study . the clinical findings in the patient group were classified into 3 grades according to the consistency and severity of symptoms as follows17 ) . grade i : intermittent symptoms with paresthesia at night grade ii : persistent symptoms throughout the day with hypesthesia in the finger of the median nerve distribution grade iii : constant symptoms with weakness or thenar muscle atrophy the electrophysiological study was performed using a cadwell sierra wave ( cadwell laboratories , kennewick , wa , usa ) . the study consisted of motor and sensory median nerve conduction tests using standard techniques according to the practice parameters for the electrodiagnosis of cts of the american association of electrodiagnostic medicine , the american academy of neurology , and the american academy of physical medicine and rehabilitation3 ) . the electrophysiological abnormalities were classified into three grades according to stevens ' classification as follows39 ) . mild : prolonged median sensory distal latency moderate : prolonged median sensory and motor distal latency severe : abnormal needle electromyography other than the above two abnormalities , or no response in sensory and motor distal latency the modified menon 's single - portal method25 ) was applied to all of the 793 patients as a means of ectr by the same neurosurgeon . ectr was carried out under monitored anesthetic care by intravenous injection of propofol without endotracheal intubation . after exsanguination of the arm and inflation of a tourniquet up to 250 mm hg , a 1.0-cm transverse incision is made along the proximal wrist crease in the ulnar side of the palmaris longus tendon . subligamentous dissection toward the fourth finger is carried out to reflect the synovial tissue from the undersurface of the transverse carpal ligament ( tcl ) and to create a space for the endoscopic device with a synovial elevator . a washboard sensation could be felt in the undersurface of the tcl with the elevator and its tip could be felt on the palm while it passes the distal part of the tcl . a slotted cannula is introduced into the carpal tunnel and a rigid 30-angled 2.7-mm endoscope is inserted through the cannula . manifestation of reduced transillumination could be observed in the palm while the endoscope is under the tcl . white transversely running fibers of tcl and a fat pad distal to the distal edge of the tcl can be seen on the endoscopic view . after ensuring that there are no intervening structures in the operative field after the tcl is completely cut out , both an escape of palmar fat into the carpal tunnel and retraction of cut edges of the tcl out of the endoscopic view could be observed as well as increased transillumination through the palm . a right - angled probe is used to palpate the divided tcl . the tourniquet is released . wound closure is accomplished in layers following hemostasis and a bulky compressive dressing is applied . clinical severity and electrodiagnostic abnormality were compared between groups using chi - square test and demographic data including age and duration of symptoms was compared using pearson 's correlation analysis . all analyses were performed using spss , version 19.0 , commercially available software package ( spss inc . , chicago , il , usa ) . we retrospectively reviewed databases of cts patients who underwent ectr at our hospital from 2002 to 2011 . the preoperative assessment included a detailed history , symptoms and physical examination and all patients undergoing ectr had electrodiagnostic studies confirming compression of the median nerve at the level of the transverse carpal ligament . however , small number of patients with severe symptoms underwent ectr even though electrodiagnostic studies did not reveal any abnormalities , compatable with cts . patients with postoperative period more than 12 months were analyzed and patients with prior carpal tunnel surgery or patients with diabetes or an underlying disease , related to cts were excluded from the study . the clinical findings in the patient group were classified into 3 grades according to the consistency and severity of symptoms as follows17 ) . grade i : intermittent symptoms with paresthesia at night grade ii : persistent symptoms throughout the day with hypesthesia in the finger of the median nerve distribution grade iii : constant symptoms with weakness or thenar muscle atrophy the electrophysiological study was performed using a cadwell sierra wave ( cadwell laboratories , kennewick , wa , usa ) . the study consisted of motor and sensory median nerve conduction tests using standard techniques according to the practice parameters for the electrodiagnosis of cts of the american association of electrodiagnostic medicine , the american academy of neurology , and the american academy of physical medicine and rehabilitation3 ) . the electrophysiological abnormalities were classified into three grades according to stevens ' classification as follows39 ) . mild : prolonged median sensory distal latency moderate : prolonged median sensory and motor distal latency severe : abnormal needle electromyography other than the above two abnormalities , or no response in sensory and motor distal latency the modified menon 's single - portal method25 ) was applied to all of the 793 patients as a means of ectr by the same neurosurgeon . ectr was carried out under monitored anesthetic care by intravenous injection of propofol without endotracheal intubation . after exsanguination of the arm and inflation of a tourniquet up to 250 mm hg , a 1.0-cm transverse incision is made along the proximal wrist crease in the ulnar side of the palmaris longus tendon . subligamentous dissection toward the fourth finger is carried out to reflect the synovial tissue from the undersurface of the transverse carpal ligament ( tcl ) and to create a space for the endoscopic device with a synovial elevator . a washboard sensation could be felt in the undersurface of the tcl with the elevator and its tip could be felt on the palm while it passes the distal part of the tcl . a slotted cannula is introduced into the carpal tunnel and a rigid 30-angled 2.7-mm endoscope is inserted through the cannula . manifestation of reduced transillumination could be observed in the palm while the endoscope is under the tcl . white transversely running fibers of tcl and a fat pad distal to the distal edge of the tcl can be seen on the endoscopic view . after ensuring that there are no intervening structures in the operative field , the tcl is incised proximally to distally with a forward - facing knife . after the tcl is completely cut out , both an escape of palmar fat into the carpal tunnel and retraction of cut edges of the tcl out of the endoscopic view could be observed as well as increased transillumination through the palm . wound closure is accomplished in layers following hemostasis and a bulky compressive dressing is applied . clinical severity and electrodiagnostic abnormality were compared between groups using chi - square test and demographic data including age and duration of symptoms was compared using pearson 's correlation analysis . all analyses were performed using spss , version 19.0 , commercially available software package ( spss inc . , chicago , il , usa ) . single - portal ectrs were performed in 1194 hands of 793 patients with electrodiagnostically - proven idiopathic cts from 2002 to 2011 . among 1194 hands , 570 hands of 388 patients with minimal 1-year postoperative follow - up mean age of the patients was 53 years ( range , 24 - 82 years ) and mean follow - up period was 23.6 months ( range , 12 - 160 months ) . patients ' clinical outcomes were assessed with postoperative improvement of tingling sensation , pain and numbness at 1 , 3 , 6 , and 12 months postoperatively . among 570 operated hands , 545 hands ( 95.6% ) we divided the 545 satisfied hands into early ( group a ) and delayed ( group b ) improvement group according to improvement period of 1 month . the mean age of group a was 53 years ( range , 24 - 82 years ) . the mean age of group b was 52 years ( range , 32 - 71 years ) . mean symptom duration was 42.4 months in group a and 33.4 months in group b. table 1 shows the baseline characteristics of the study groups . in group b , 11 hands improved in postoperative 2-months , 15 hands in postoperative 3-months and 9 hands in postoperative 6-months , respectively . in group a / b , according to clinical severity , 60/1 hands were graded to i , 345/24 hands to ii , 105/10 hands to iii ( table 2 ) . in group a / b , based on electrodiagnostic abnormality , 57/3 hands were divided to mild group , 221/11 hands to moderate group and 222/21 hands to severe group with normal findings in 10 hands of group a ( table 3 ) . statistical analysis of these results between groups did not reach significance ( clinical severity : p=0.191 , electrodiagnostic abnormality : p=0.294 ) but severity of electrodiagnostic abnormalities or clinical severity had a tendency to affect on delayed response . in addition , age and duration of symptoms did not affect the postectr improvement period ( age : p=0.478 , duration of symptoms : p=0.1 ) . classic symptoms of cts include tingling sensation , pain and numbness in the median nerve distribution of the hand comprising the thumb , index finger , middle finger and radial half of the ring finger . however , sensory symptoms do not always involve the entire median nerve distribution of the hand . in some patients the primary pathophysiologic mechanism of cts development is known as an increase in interstitial pressure in the carpal tunnel , leading to median nerve compression . the normal interstitial pressure in the carpal tunnel is known to be 2.5 mm hg with the wrist in neutral position and it increases to approximately 30 mm hg with the wrist in either maximum flexion or maximal extension26 ) . symptoms of the median nerve compression including tingling sensation develop when carpal tunnel interstitial pressure increases above 30 mm hg , leading to a reduction of epineural blood flow , development of epineural edema and block in axonal transport26 ) . with prolonged compression of the median nerve at higher carpal tunnel interstitial pressure , manifestations of the median nerve compression generally progress over a period of months to years to develop more severe sensory symptoms and even thenar muscle weakness . there are numerous causes of this increase in carpal tunnel pressure but most cases of cts do not have identifiable cause42 ) . however , idiopathic cts is believed to be caused by combination of an anatomically small carpal tunnel and changes in the tenosynovium of the flexor tendons in the carpal tunnel such as edema , vascular sclerosis and fibrous hypertrophy26 ) . secondary causes of cts include neuropathic factor with diabetes , metabolic and physiologic factor such as hypothyroidism , rheumatoid arthritis and pregnancy , and space - occupying lesion with tumor or cyst42 ) . in addition , risk factors in the general population that contribute to cts include repetitive movement of wrist extension and flexion , obesity , rapid dieting , short height , hysterectomy without oophorectomy and recent menopause11 ) . we excluded the cases with secondary cts from the study because the predisposing conditions of an identifiable cause that contribute to cts may affect the surgical outcome , other than known preoperative prognostic indicators8,23,30,43,44 ) . decompression of the carpal tunnel is recommended when symptomatic discomfort interferes with work or activity of daily living despite conservative treatment or advanced cts with thenar muscle weakness even when sensory symptoms are not disabling . in these cases , decompression of the carpal tunnel can be used to improve clinical symptoms and physical findings of cts , to prevent progression and loss of hand function and to eventually minimize loss of time from work . two different types of surgical procedures are in use for the treatment of cts : open and endoscopic release of the transverse carpal ligament . octr is the traditional option and is easy to perform with good symptomatic improvement and a low complication rate . since the introduction of a less invasive carpal tunnel surgery using surgical endoscope by okutsu et al.32 ) in 1987 , endoscopic procedures have been widely used as an alternative treatment to octr , and single - portal agee technique1 ) and two - portal chow9 ) technique are the two most commonly used procedures . we used modification of single - portal menon technique25 ) using a forward - facing knife to divide the transverse carpal ligament . it is beyond the scope of this study to describe details of each technique so we do not discuss it in this article . since the introduction of endoscopic carpal tunnel surgery in the late 1980s , thereis still controversy regarding the efficacy , safety and complication rate of open and endoscopic procedures . jimenez et al.22 ) reported that ectr surgery offer similar success and complication rate compared to open surgery in an extensive review of published articles on ectr surgery covering six different types of techniques . in addition , he described patients undergoing ectr experienced less postoperative pain and earlier return to work or activity of daily living but endoscopic technique need a learning curve and surgical experience for a successful outcome . ashwoth5 ) also described in a critical review of 57 review articles regarding ectr that ectr surgery , compared to open surgery , seemed equally effective at improving symptoms and hand function in short and long outcome , and it may be more effective at reducing time to return to work . endoscopic surgery was reported to have more transient nerve injuries such as neuropraxia , numbness and paresthesia but open surgery to have more wound problems including scar tenderness , hypertrophic scar and infection . he also reported endoscopic surgery is more resource - intensive and demands a higher surgical skill than open technique . several studies have reported preoperative prognostic factors , affecting the outcome after surgical release of transverse carpal ligament and preoperative predictors included duration and severity of symptoms , grade of electrodiagnostic abnormalities , worker 's compensation and work activity8,23,30,43,44 ) . good outcome has been associated with preoperative tingling other than anesthesia or weakness as well as good response to corticosteroid injection into carpal tunnel and poor result has been correlated with preoperative weakness or atrophy , associated diseases such as diabetis and thyroid disease , worker 's compensation and heavy or repetitive work activity13,18,23,24,29,36,43,44 ) . the finding of delayed improvement in our series demonstrates late postoperative improvement group had more proportion of patient with severe clinical grade , but clinical severity did not differ significantly between early and late improvement groups . even though we can expect poor outcome in patients with severe clinical grade of cts , it is recommended that postoperative follow - up up to 6 months is necessary in idiopathic cts patient without improvement . the duration of cts has been shown to have no significant correlation with clinical outcome in patients undergoing carpal tunnel surgery19,43 ) , whereas , other studies found worse outcome with longer duration2,13,24,27,29,30,44 ) . pauda et al.34 ) reported that the main positive prognostic factors of idiopathic cts without treatment are short duration of symptoms and young age . our study has indicated that the chronicity of cts did not affect the time of improvement following ectr . the role of ncs in predicting the outcome of carpal tunnel surgery is not clearly determined2,4,8,21,27,44 ) . schrijver et al.38 ) noted that ncs improved significantly at postoperative 12 months but only a modest correlation existed between relief of preoperative symptoms after surgery and the improvement of ncs . however , other studies showed that ncss improve after surgery28 ) . in severe cts cases , nerve conduction studies results may not correlate with the clinical findings due to the varying nature of the impairment in different nerve fibers . in addition , nerve conduction studies will not accurately predict the recovery following release of the carpal tunnel , though neither do any of the other investigations predict this with any certainty6 ) . our study has shown that grade of electrophysiologic abnormalities does not correlate with the time course of improvement after carpal tunnel surgery , which means ncss are not helpful to predict the improvement period . several studies demonstrate that improvements after carpal tunnel release decreased in older patients who were at risk of severe cts , compared to a younger population14,15,16,20 ) . therefore , we can expect older patients to show a more delayed improvement than younger patients but we found no significant differences in the time of improvement following ectr between early and delayed improvement group . in addition , worker 's compensation can affect postoperative outcome and worker 's compensation group showed diminished outcome , compared to patients without worker 's compensation19 ) . in our study , worker 's compensation did not affect postoperative improvement period but we think it may delay postoperative improvement period . therefore , worker 's compensation status can be considered as a possible factor of delayed postoperative improvement in patients with worker 's compensation . endoscopic decompression of carpal tunnel is effective as a primary or alternative surgical technique for the treatment of cts , demonstrating symptomatic improvement within postoperative one - month in most patients . therefore , it is advised that postoperative observation for at least 6 months is necessary in the patients without symptomatic improvement to expect the delayed response .

objectivein most patients with carpal tunnel syndrome ( cts ) , pain and/or paresthesia disappeared or decreased in a month after endoscopic carpal tunnel release ( ectr ) . however , subpopulation of patients showed delayed improvement following ectr . we analyzed the delayed improvement hands to investigate the characteristics of those patients and to determine the predictable factors of delayed improvement.methodssingle-portal ectrs were performed in 1194 hands of 793 cts patients from 2002 to 2011 . five - hundred seventy hands with minimal 1-year postoperative follow - up were included . we divided the 545 satisfied hands into early ( group a ) and delayed ( group b ) groups according to improvement period of 1 month . demographic data , clinical severity and electrodiagnostic abnormality were compared between groups.resultsgroup a included 510 hands and group b included 35 hands . in group b , 11 hands improved in 2 months , 15 hands in 3 months and 9 hands in 6 months , respectively . in group a / b , according to clinical severity , 60/1 hands were graded to i , 345/24 hands to ii , 105/10 hands to iii . in group a / b , based on electrodiagnostic abnormality , 57/3 hands were classified to mild , 221/11 hands to moderate and 222/21 hands to severe group . statistical analysis between groups did not reach significance but electrodiagnostic or clinical severity had a tendency to affect the delayed response.conclusionit is difficult to predict the factors contributing to postoperatively - delayed response in subpopulation of cts patients . however , we recommend that postoperative observation for at least 6 months is necessary in patients without symptomatic improvement .

INTRODUCTION MATERIALS AND METHODS Patient population Clinical grade Electrodiagnostic grade Surgical technique Statistical analysis RESULTS DISCUSSION CONCLUSION

PMC4836244

psoriasis is a chronic , recurrent , inflammatory disorder of the skin affecting approximately 2% of the world 's population . psoriasis vulgaris ( psv ) is the most common type of psoriasis , which manifests as well - demarcated , scaly patches on the skin . psoriasis is considered severe if more than 10% of a patient 's body surface is affected or if a patient scores more than 10 on the psoriasis area severity index ( pasi ) . topical therapies are used for mild or localized disease while systemic therapies and phototherapy are used for patients with moderate to severe disease . extracts of the chinese herb tripterygium wilfordii hook f ( twhf ) , also known as lei gong teng , are used as one of the most common systemic treatments for inflammatory and immune disorders including psoriasis , rheumatoid arthritis ( ra ) , crohn 's disease , nephritis , and systemic lupus erythematosus . recently , extracts of twhf have been tested in western countries , with good efficacy . however , acitretin is a teratogen , causes dyslipidemia , the therapeutic effect is slow to onset , and it has other side effects that limit widespread use . although both therapies are effective , twhf has never been directly compared with systemic acitretin in a randomized clinical study . we compared the efficacy and safety of twhf and acitretin for the treatment of moderate to severe psv in a randomized , double - blind , double - dummy , parallel - group clinical trial . adults ( age > 18 years and < 75 years ) with moderate to severe psv were eligible to enroll in the study . eligible patients had a pasi score greater than or equal to 10 or a psoriasis - affected body surface area 10% or higher . exclusion criteria included : participation in other clinical studies during the past 30 days ; patients with psoriatic erythroderma , psoriasis pustulosa , psoriasis arthritis , or guttate psoriasis ; patients taking systemic corticoid , immunosuppressive agents , or biologicals therapy during the past 4 weeks ; patients taking local corticoid therapy or phototherapy during the past 2 weeks ; patients that are pregnant , breastfeeding , planning to become pregnant within 2 years ; patients with acute or chronic systemic infections , hepatitis b virus , hepatitis c virus , hiv , a history of malignancy , severe systemic disease , or elevation of aspartate transaminase , alanine transaminase , blood - lipoids reaching 1.5 times the normal range ; and patients with allergies to twhf or acitretin . this randomized , double - blind , double - dummy , parallel - group clinical study was conducted at peking union medical college hospital in china . the first patient was enrolled on september 27 , 2011 , and the last patient completed the study on june 14 , 2014 . patients were randomly assigned in a 1:1 ratio to receive either a chloroform methanol extract of twhf 20 mg 3 times a day plus a placebo matching acitretin 30 mg once a day or acitretin 30 mg once a day plus a placebo matching a chloroform methanol extract of twhf 20 mg three times daily , both for 8 weeks . a permuted block randomization design using a block size of four the random sequence was generated by an independent randomization group , principal investigators enrolled the study participants , and a third - party vendor assigned the interventions using the web response system . patients were assessed at baseline and at weeks 2 , 4 , and 8 . at the screening visit , the laboratory tests consisted of urinalysis , hematology , and blood biochemistry ( urea nitrogen , creatinine , cholesterol , triglycerides , high - density lipoprotein , aspartate transaminase , alanine transaminase ) at baseline , week 4 , and week 8 . differences from baseline to weeks 2 , 4 , and 8 were tested using paired samples t - test . differences in the primary outcomes between the two groups at weeks 2 , 4 and 8 were performed by analysis of variance ( anova ) test . all analyses were performed using spss software program version 15.0 for windows ( spss inc . , safety was evaluated by all adverse events ( aes ) , laboratory tests , and physical examinations . at each visit , patients were queried for any aes , and an assessment was made as to whether there were any possible , probable or definite relationships between study drug and aes . an ae was defined as any undesirable symptom or sign that occurred after the initiation of the treatment , regardless of its relation to the study drug . a serious ae was defined as any untoward medical occurrence that , at any dose , resulted in death , was life - threatening , required hospitalization , or resulted in persistent or significant disability , incapacity , or a congenital malformation , or required medical intervention . the endpoint of the study was the response of 75% improvement in pasi score ( pasi 75 ) at weeks 2 , 4 , and 8 . efficacy assessments ( pasi ) were performed by doctors who were blinded to the treatment group assignments . adults ( age > 18 years and < 75 years ) with moderate to severe psv were eligible to enroll in the study . eligible patients had a pasi score greater than or equal to 10 or a psoriasis - affected body surface area 10% or higher . exclusion criteria included : participation in other clinical studies during the past 30 days ; patients with psoriatic erythroderma , psoriasis pustulosa , psoriasis arthritis , or guttate psoriasis ; patients taking systemic corticoid , immunosuppressive agents , or biologicals therapy during the past 4 weeks ; patients taking local corticoid therapy or phototherapy during the past 2 weeks ; patients that are pregnant , breastfeeding , planning to become pregnant within 2 years ; patients with acute or chronic systemic infections , hepatitis b virus , hepatitis c virus , hiv , a history of malignancy , severe systemic disease , or elevation of aspartate transaminase , alanine transaminase , blood - lipoids reaching 1.5 times the normal range ; and patients with allergies to twhf or acitretin . this randomized , double - blind , double - dummy , parallel - group clinical study was conducted at peking union medical college hospital in china . the first patient was enrolled on september 27 , 2011 , and the last patient completed the study on june 14 , 2014 . patients were randomly assigned in a 1:1 ratio to receive either a chloroform methanol extract of twhf 20 mg 3 times a day plus a placebo matching acitretin 30 mg once a day or acitretin 30 mg once a day plus a placebo matching a chloroform methanol extract of twhf 20 mg three times daily , both for 8 weeks . a permuted block randomization design using a block size of four the random sequence was generated by an independent randomization group , principal investigators enrolled the study participants , and a third - party vendor assigned the interventions using the web response system . patients were assessed at baseline and at weeks 2 , 4 , and 8 . at the screening visit , the laboratory tests consisted of urinalysis , hematology , and blood biochemistry ( urea nitrogen , creatinine , cholesterol , triglycerides , high - density lipoprotein , aspartate transaminase , alanine transaminase ) at baseline , week 4 , and week 8 . differences from baseline to weeks 2 , 4 , and 8 were tested using paired samples t - test . differences in the primary outcomes between the two groups at weeks 2 , 4 and 8 were performed by analysis of variance ( anova ) test . all analyses were performed using spss software program version 15.0 for windows ( spss inc . , safety was evaluated by all adverse events ( aes ) , laboratory tests , and physical examinations . at each visit , patients were queried for any aes , and an assessment was made as to whether there were any possible , probable or definite relationships between study drug and aes . an ae was defined as any undesirable symptom or sign that occurred after the initiation of the treatment , regardless of its relation to the study drug . a serious ae was defined as any untoward medical occurrence that , at any dose , resulted in death , was life - threatening , required hospitalization , or resulted in persistent or significant disability , incapacity , or a congenital malformation , or required medical intervention . the endpoint of the study was the response of 75% improvement in pasi score ( pasi 75 ) at weeks 2 , 4 , and 8 . efficacy assessments ( pasi ) were performed by doctors who were blinded to the treatment group assignments . of the 115 patients enrolled , 58 were allocated to the twhf group and 57 were allocated to the acitretin group most patients were men ( 70.4% ) , and the mean age sd was 41.1 12.2 years . there was a higher attrition rate in the acitretin group ( 5.2% in the twhf group and 7.0% in the acitretin group by week 2 , and 8.6% in the twhf group and 12.3% in the acitretin group by week 8) . discontinuation occurred because of withdrawn consent ( n = 1 ) , loss to follow - up ( n = 8) , ae ( n = 1 ) , and disease progression ( n = 2 ) . reasons for discontinuing the study were similar between groups . baseline demographics and clinical characteristics of the patients sd : standard deviation ; twhf : tripterygium wilfordii hook f ; bmi : body mass index ; pasi : psoriasis area severity index . the pasi score decreased from a median of 23.8 ( range 7.559.5 ) to 11.1 ( range 0.346.9 ) in the twhf group ( n = 58 ; p < 0.0001 ) and from 23.2 ( range 7.256.7 ) to 13.0 ( range 0.446.4 ) ( n = 57 ; p < 0.0001 ) in the acitretin group within 8 weeks [ table 2 ] . the median pasi score improved in the twhf group by 50.4 31.0% and in the acitretin group by 42.7 45.7% . there was no significant difference in median pasi improvement between the two groups ( p = 0.317 ) . there was also no statistically significant difference between two groups at week 8 for pasi 25 ( 70.7% vs. 64.9% in the twhf and acitretin groups , respectively ; adjusted p = 0.553 ) [ figure 1a ] , pasi 50 ( 51.7% vs. 43.9% ; nominal p = 0.457 ) [ figure 1b ] , pasi 75 ( 19.0% vs. 17.5% ; nominal p = 1 ) [ figure 1c ] , and pasi 90 ( 5.2% vs. 5.3% ; nominal p = 0.204 ) [ figure 2 , 3 ] . at week 2 , there was also no significant difference in median pasi score improvement ( 18.7 41.1% vs. 18.3 16.5% in the twhf and acitretin groups , respectively ; adjusted p = 0.985 ) [ figure 1d ] , pasi 25 ( 50.0% vs. 31.6% ; nominal p = 0.058 ) [ figure 1a ] , pasi 50 ( 12.1% vs. 3.5% ; nominal p = 0.162 ) [ figure 1b ] , and pasi 75 ( 1.7% vs. 0% ; nominal p = 1 ) [ figure 1c ] . similarly , at week 4 , there was no significant difference in median pasi score improvement ( 34.4 34.9% vs. 31.6 24.7% in the twhf and acitretin groups , respectively ; adjusted p = 0.635 ) [ figure 1d ] , pasi 25 ( 55.2% vs. 59.6% ; nominal p = 0.707 ) [ figure 1a ] , pasi 50 ( 37.9% vs. 24.6% ; nominal p = 0.160 ) [ figure 1b ] , and pasi 75 ( 1.7% vs. 1.8% ; nominal p = 1 ) [ figure 1c ] . treatment efficacy of twhf and acitretin sd : standard deviation ; twhf : tripterygium wilfordii hook f ; pasi : psoriasis area severity index . ( a ) pasi 25 response ; ( b ) pasi 50 response ; ( c ) pasi 75 response ; ( d ) percentage pasi improvement . clinical appearance of a patient from tripterygium wilfordii hook f group at baseline , weeks 2 , 4 , and 8 . clinical appearance of a patient from acitretin group at baseline , weeks 2 , 4 , and 8 . at week 2 , the median pasi score improvement rate of the head , upper limbs , trunk , and lower limbs in the twhf group was 14.9 33.7% , 10.6 49.2% , 25.5 22.9% , and 9.7 61.0% , respectively . there was no significant difference in median pasi improvement between different parts of the body ( p = 0.222 ) [ tables 3 and 4 ] . there was also no significant difference in median pasi improvement between different parts of the body at weeks 4 ( p = 0.462 ) and 8 ( p = 0.882 ) in the twhf group . similarly , in the acitretin group , there was no significant difference in median pasi improvement between different parts of the body at weeks 2 , 4 , and 8 ( p = 0.339 , p = 0.144 , and p = 0.764 , respectively ) . treatment efficacy of different parts of the body ( meansd ) twhf : tripterygium wilfordii hook f ; pasi : psoriasis area severity index . median pasi improvement of different parts of the body ( % , meansd ) twhf : tripterygium wilfordii hook f ; pasi : psoriasis area severity index . there was no statistically significant difference between two groups at week 2 for median pasi improvement rate of the head ( 14.9 33.7% vs. 22.9 33.3% in the twhf and acitretin groups , respectively ; adjusted p = 0.221 ) , upper limbs ( 10.6 49.2% vs. 7.2 78.9% ; nominal p = 0.787 ) , trunk ( 25.5 22.9% vs. 18.0 21.4% ; nominal p = 0.084 ) , and lower limbs ( 9.7 61.0% vs. 14.9 19.8% ; nominal p = 0.550 ) . at week 8 , there was also no statistically significant difference between the two groups for median pasi improvement rate of the head ( 49.9 40.3% vs. 49.6 45.0% in the twhf and acitretin groups , respectively ; adjusted p = 0.979 ) , upper limbs ( 42.6 71.3% vs. 33.9 115.2% ; nominal p = 0.649 ) , trunk ( 48.8 42.0% vs. 36.9 86.9% ; nominal p = 0.381 ) , or lower limbs ( 45.5 39.7% vs. 38.9 38.2% ; nominal p = 0.394 ) . at week 4 , there was a statistically significant difference between two groups for median pasi improvement rate of the trunk ( 39.5 30.8% vs. 23.8 40.3% in the twhf and acitretin groups , respectively ; adjusted p = 0.026 ) . however , there was no statistically significant difference between the two groups at week 4 for median pasi improvement rate of the head ( 35.1 41.4% vs. 39.2 37.9% in the twhf and acitretin groups , respectively ; adjusted p = 0.597 ) , upper limbs ( 35.7 44.0% vs. 31.3 36.9% ; nominal p = 0.575 ) , and lower limbs ( 27.1 45.3% vs. 27.1 28.0% ; nominal p = 0.991 ) . more aes were reported in the acitretin group ( 78.1% ) compared with the twhf group ( 43.6% ) . the most frequent complaints in twhf group were menstrual disorders in females , dry mouth , gastrointestinal complaints , and swelling of the lower limbs . the results of blood biochemistry ( alanine transaminase , aspartate transaminase , urea nitrogen , creatinine , cholesterol , triglyceride , and high - density lipoprotein ) are shown in table 5 . compared with baseline , there was a significant increase in the level of aspartate transaminase and triglycerides at week 8 in the twhf group ( p = 0.026 and p = 0.011 , respectively ) . in the acitretin group , there was a significant increase in the level of alanine transaminase , cholesterol , and high - density lipoprotein at week 8 ( p = 0.03 , p < 0.01 , and p < 0.01 , respectively ) . laboratory tests of twhf and acitretin ( meansd ) twhf : tripterygium wilfordii hook f ; alt : alanine transaminase ; ast : aspartate transaminase ; hdl : high - density lipoprotein . of the 115 patients enrolled , 58 were allocated to the twhf group and 57 were allocated to the acitretin group most patients were men ( 70.4% ) , and the mean age sd was 41.1 12.2 years . there was a higher attrition rate in the acitretin group ( 5.2% in the twhf group and 7.0% in the acitretin group by week 2 , and 8.6% in the twhf group and 12.3% in the acitretin group by week 8) . discontinuation occurred because of withdrawn consent ( n = 1 ) , loss to follow - up ( n = 8) , ae ( n = 1 ) , and disease progression ( n = 2 ) . reasons for discontinuing the study were similar between groups . baseline demographics and clinical characteristics of the patients sd : standard deviation ; twhf : tripterygium wilfordii hook f ; bmi : body mass index ; pasi : psoriasis area severity index . the pasi score decreased from a median of 23.8 ( range 7.559.5 ) to 11.1 ( range 0.346.9 ) in the twhf group ( n = 58 ; p < 0.0001 ) and from 23.2 ( range 7.256.7 ) to 13.0 ( range 0.446.4 ) ( n = 57 ; p < 0.0001 ) in the acitretin group within 8 weeks [ table 2 ] . the median pasi score improved in the twhf group by 50.4 31.0% and in the acitretin group by 42.7 45.7% . there was no significant difference in median pasi improvement between the two groups ( p = 0.317 ) . there was also no statistically significant difference between two groups at week 8 for pasi 25 ( 70.7% vs. 64.9% in the twhf and acitretin groups , respectively ; adjusted p = 0.553 ) [ figure 1a ] , pasi 50 ( 51.7% vs. 43.9% ; nominal p = 0.457 ) [ figure 1b ] , pasi 75 ( 19.0% vs. 17.5% ; nominal p = 1 ) [ figure 1c ] , and pasi 90 ( 5.2% vs. 5.3% ; nominal p = 0.204 ) [ figure 2 , 3 ] . at week 2 , there was also no significant difference in median pasi score improvement ( 18.7 41.1% vs. 18.3 16.5% in the twhf and acitretin groups , respectively ; adjusted p = 0.985 ) [ figure 1d ] , pasi 25 ( 50.0% vs. 31.6% ; nominal p = 0.058 ) [ figure 1a ] , pasi 50 ( 12.1% vs. 3.5% ; nominal p = 0.162 ) [ figure 1b ] , and pasi 75 ( 1.7% vs. 0% ; nominal p = 1 ) [ figure 1c ] . similarly , at week 4 , there was no significant difference in median pasi score improvement ( 34.4 34.9% vs. 31.6 24.7% in the twhf and acitretin groups , respectively ; adjusted p = 0.635 ) [ figure 1d ] , pasi 25 ( 55.2% vs. 59.6% ; nominal p = 0.707 ) [ figure 1a ] , pasi 50 ( 37.9% vs. 24.6% ; nominal p = 0.160 ) [ figure 1b ] , and pasi 75 ( 1.7% vs. 1.8% ; nominal p = 1 ) [ figure 1c ] . treatment efficacy of twhf and acitretin sd : standard deviation ; twhf : tripterygium wilfordii hook f ; pasi : psoriasis area severity index . ( a ) pasi 25 response ; ( b ) pasi 50 response ; ( c ) pasi 75 response ; ( d ) percentage pasi improvement . clinical appearance of a patient from tripterygium wilfordii hook f group at baseline , weeks 2 , 4 , and 8 . clinical appearance of a patient from acitretin group at baseline , weeks 2 , 4 , and 8 . at week 2 , the median pasi score improvement rate of the head , upper limbs , trunk , and lower limbs in the twhf group was 14.9 33.7% , 10.6 49.2% , 25.5 22.9% , and 9.7 61.0% , respectively . there was no significant difference in median pasi improvement between different parts of the body ( p = 0.222 ) [ tables 3 and 4 ] . there was also no significant difference in median pasi improvement between different parts of the body at weeks 4 ( p = 0.462 ) and 8 ( p = 0.882 ) in the twhf group . similarly , in the acitretin group , there was no significant difference in median pasi improvement between different parts of the body at weeks 2 , 4 , and 8 ( p = 0.339 , p = 0.144 , and p = 0.764 , respectively ) . treatment efficacy of different parts of the body ( meansd ) twhf : tripterygium wilfordii hook f ; pasi : psoriasis area severity index . median pasi improvement of different parts of the body ( % , meansd ) twhf : tripterygium wilfordii hook f ; pasi : psoriasis area severity index . there was no statistically significant difference between two groups at week 2 for median pasi improvement rate of the head ( 14.9 33.7% vs. 22.9 33.3% in the twhf and acitretin groups , respectively ; adjusted p = 0.221 ) , upper limbs ( 10.6 49.2% vs. 7.2 78.9% ; nominal p = 0.787 ) , trunk ( 25.5 22.9% vs. 18.0 21.4% ; nominal p = 0.084 ) , and lower limbs ( 9.7 61.0% vs. 14.9 19.8% ; nominal p = 0.550 ) . at week 8 , there was also no statistically significant difference between the two groups for median pasi improvement rate of the head ( 49.9 40.3% vs. 49.6 45.0% in the twhf and acitretin groups , respectively ; adjusted p = 0.979 ) , upper limbs ( 42.6 71.3% vs. 33.9 115.2% ; nominal p = 0.649 ) , trunk ( 48.8 42.0% vs. 36.9 86.9% ; nominal p = 0.381 ) , or lower limbs ( 45.5 39.7% vs. 38.9 38.2% ; nominal p = 0.394 ) . at week 4 , there was a statistically significant difference between two groups for median pasi improvement rate of the trunk ( 39.5 30.8% vs. 23.8 40.3% in the twhf and acitretin groups , respectively ; adjusted p = 0.026 ) . however , there was no statistically significant difference between the two groups at week 4 for median pasi improvement rate of the head ( 35.1 41.4% vs. 39.2 37.9% in the twhf and acitretin groups , respectively ; adjusted p = 0.597 ) , upper limbs ( 35.7 44.0% vs. 31.3 36.9% ; nominal p = 0.575 ) , and lower limbs ( 27.1 45.3% vs. 27.1 28.0% ; nominal p = 0.991 ) . more aes were reported in the acitretin group ( 78.1% ) compared with the twhf group ( 43.6% ) . the most frequent complaints in twhf group were menstrual disorders in females , dry mouth , gastrointestinal complaints , and swelling of the lower limbs . the results of blood biochemistry ( alanine transaminase , aspartate transaminase , urea nitrogen , creatinine , cholesterol , triglyceride , and high - density lipoprotein ) are shown in table 5 . compared with baseline , there was a significant increase in the level of aspartate transaminase and triglycerides at week 8 in the twhf group ( p = 0.026 and p = 0.011 , respectively ) . in the acitretin group , there was a significant increase in the level of alanine transaminase , cholesterol , and high - density lipoprotein at week 8 ( p = 0.03 , p < 0.01 , and p < 0.01 , respectively ) . laboratory tests of twhf and acitretin ( meansd ) twhf : tripterygium wilfordii hook f ; alt : alanine transaminase ; ast : aspartate transaminase ; hdl : high - density lipoprotein . to our knowledge , this trial is the first randomized , double - blind , double - dummy , parallel - group clinical comparison of twhf with acitretin in moderate to severe psv . because twhf has several important advantages over acitretin ( e.g. , low risk for teratogenicity , dyslipidemia , or photosensitivity ) , it is important to analyze whether or not the two therapies have similar efficacy . the data from this trial clearly indicate that twhf and acitretin significantly improved psv within 8 weeks , indicating that both medicines are highly efficient for the treatment for moderate to severe psv . our results demonstrated no statistically significant difference in treatment efficacy between the two groups in patients with moderate to severe psv in 8 weeks . a decrease in safety signals psoriasis vulgaris is a common chronic relapsing inflammatory cutaneous disease . for patients with moderate - to - severe psv , currently , four conventional systemic agents : cyclosporin a , methotrexate , fumaric acid esters ( germany only ) , and acitretin , and five biological agents : infliximab , etanercept , adalimumab , alefacept ( united states and switzerland only ) , and ustekinumab are approved for chronic plaque psoriasis . twhf , is approved in china and often used to treat a variety of immune and inflammation - related diseases such as psoriasis and ra . acitretin is currently approved by the us food and drug administration for the treatment of severe psoriasis in adults . because it is teratogenic and should not be used in women who are pregnant , breast - feeding , or may become pregnant within 3 years of discontinuing acitretin , its use is substantially limited in female patients of childbearing potential . dyslipidemia may also occur and require dose reduction or treatment with lipid - lowering agents . the extracts of twhf contain more than 70 ingredients , but triptolide is the most potent bioactive substance . it has been shown to possess potent anti - inflammatory and immunosuppressive properties in vitro as well as in different animal models in numerous preclinical studies . inflammatory myeloid dendritic cells release interleukin-23 ( il-23 ) and il-12 to activate il-17-producing t cells , th1 cells , and th22 cells to produce abundant psoriatic cytokines il-17 , interferon- , tumor necrosis factor ( tnf ) , and il-22 . these inhibit the immunological response and maintain cutaneous immunological homeostasis , which prevents autoimmunity against autoantigens . triptolide can suppress the activation of t - lymphocytes , promote the differentiation of cd4 + t cells to foxp3 + tregs , and downregulate the level of tnf-. in addition , a recent study showed that the triptolide inhibits il-2 expression in t cells , and can inhibit the maturation and allogenicity of dendritic cells . these mechanisms might explain the pharmacological effects of twhf in psv treatment . in a literature search including the databases : medline , the cochrane library , china biomedical literature on disc , national science and technology library , vip , and all chinese medical journals , only ten randomized controlled trials ( rcts ) using twhf extracts to treat psoriasis patients were found . nine of these studies were performed using a group of patients who received a comparator drug , and only one was designed with a placebo group . the comparator drugs included erythromycin , azithromycin , glycyrrhizin , di yin pian , or ampepitidium et elementum , indigo naturalis ( a dark blue plant used to treat psoriasis in traditional chinese medicine ) , and bimolane . all the rcts demonstrated a statistically significant improvement of the disease after treatment with twhf extracts . however , most of these claims were derived from uncontrolled clinical trials or retrospective reports . the design of the above studies does not meet the criteria required for rigorous clinical trials . in addition , significant improvement of pasi in patients with only mild forms of psoriasis may bias a favorable efficacy outcome . therefore , rcts in a defined patient population with moderate to severe psv are needed to gain further insight into the potential benefits and risks of the use of twhf extracts . the major side effects of twhf extracts documented in the literature are gastrointestinal complaints , skin rash and pigmentation , decreases in white and red blood cells and platelets , and dysfunction of the male and female reproductive system such as dysmenorrhea , irregular menstruation , or reversible sterility . an adverse effect on renal function such as a decrease in creatinine clearance in elderly patients was also documented . the side effect - related withdrawal rate for the chloroform methanol extract of twhf in a 3-month trial was only 2.9% . dry mouth , gastrointestinal complaints , and swelling of the lower limbs were also common complaints . no significant abnormalities in urinalysis or blood panels were seen during the 8 weeks . compared with baseline , there was a significant increase in the level of aspartate transaminase and triglycerides within 8 weeks in the twhf group . we prescribed oral lipid - lowering drugs and hepatinica to patients whose level of serum aspartate transaminase and triglycerides reached 1.5 times the normal value . with this intervention , the blood biochemistry values returned to normal or were maintained . in conclusion , tripterygium wilfordii hook f might be an effective and safe treatment in patients with moderate to severe psv . twhf could be considered a suitable treatment option for certain patient populations , specifically patients who have contraindications to other therapies . increases in aspartate transaminase and triglyceride levels can be treated symptomatically . because there is a lack of long - term trials or reports of open studies running for more than 1 year , it is difficult to judge the full potential of twhf for use in psoriasis . further multi - center , large - sample clinical trials are needed to evaluate the long - term effects of twhf , including efficacy , safety , and tolerability .

background : few clinical trials have evaluated the efficacy and safety of tripterygium wilfordii hook f ( twhf ) compared with acitretin in psoriasis . we aimed to compare the efficacy and safety of twhf compared with acitretin in the treatment of moderate to severe psoriasis vulgaris.methods:adults with psoriasis area severity index ( pasi ) score 10 and psoriasis - affected body surface area 10% were randomized into a twhf ( 20 mg , 3 times a day ) or acitretin group ( 30 mg , once a day ) . the treatment course lasted for 8 weeks . patients were assessed at baseline and at 2 , 4 , and 8 weeks . laboratory tests were performed at baseline , week 4 , and week 8 . the data were analyzed using paired samples t - test or analysis of variance ( anova).results : a total of 115 patients was enrolled ( 58 twhf ; 57 acitretin ) . the median pasi score improved in the twhf group by 50.4% and in the acitretin group by 42.7% . there was no significant difference in median pasi improvement between two groups at 2 , 4 , and 8 weeks . there was also no significant difference in pasi 25 , pasi 50 , pasi 75 , and pasi 90 response between the two groups at 2 , 4 , and 8 weeks . there was a significant increase in the level of aspartate transaminase and triglycerides in the twhf group ( p = 0.026 and p = 0.011 , respectively ) . in the acitretin group , there was a significant increase in the level of alanine transaminase , cholesterol , and high - density lipoprotein ( p = 0.030 , p < 0.01 , and p < 0.01 , respectively).conclusions : there was no significant difference in treatment efficacy between the twhf and acitretin groups within 8 weeks , but there were fewer treatment - related adverse events in the twhf group .

I M Patients Study design Study procedures Statistical analysis Safety assessments Study end points R Patients Psoriasis area severity index responses Safety D

PMC3015832

fistulas inaccessible from the vagina may require abdominal repair ; we sought to evaluate the robotic - assisted laparoscopic approach for this procedure . a 41-year - old nulliparous woman presented with urinary incontinence following an abdominal hysterectomy , and office evaluation identified a vesicovaginal fistula . after discussion with the patient regarding the surgical options , the robotic approach was chosen to facilitate precise dissection , fine visualization , and suturing . a stent was placed from the bladder into the vagina , and no intentional cystotomy was made . the bladder was dissected away from the anterior vaginal wall at the fistula site , and the defects were closed independently with interposition of a fatty epiploica from the sigmoid colon . total operative time was approximately 4 hours , and robotic time was about 2.5 hours . the robotic - assisted laparoscopic approach is a viable option for successful repair of a vesicovaginal fistula in a patient in whom a vaginal approach is not indicated . most vesicovaginal fistulas in the united states today are the result of abdominal gynecologic surgery and may not have been detected or predictable based on the original surgery . patients typically complain of incontinence of urine , particularly upon standing . after confirming the absence of urinary tract infection , a careful speculum examination should be done to look for vaginal mucosa defects . additionally , use of medication to dye the urine while a tampon is in the vagina may confirm the presence of a connection . , imaging should be used to investigate for involvement of the upper urinary tract . if conservative options fail and after allowing postoperative inflammation to subside , traditional surgical options have included vaginal repair , usually using the latzko technique , or an abdominal incision for open repair . patients are typically offered an abdominal repair if the defect is large , multiple , involves other pelvic structures including the ureters , or if there is poor descent of the vaginal cuff causing the defect to be inadequately visualized from a vaginal approach . prior reports , however , have often included the use of an intentional cystotomy to facilitate dissection . because of the morbidity and recovery required for an abdominal incision , we sought to evaluate the role of the da vinci robotic system ( intuitive surgical , sunnyvale , ca , usa ) to facilitate repair of a vesicovaginal fistula . a 41-year - old , nulliparous woman presented with urinary incontinence after an emergent total abdominal hysterectomy with bilateral salpingectomy . there was no difficulty with dissection of the bladder or cystotomy noted at the time of the procedure . a phenazopyridine tampon test in the office 2 weeks after the surgery confirmed a connection from the bladder to the vagina . a 1 cm opening was identified on the base of the bladder above the trigone to the right of the midline and 2 cm posterior to the interureteric ridge . the ureteral orifices did not appear to be compromised by the fistula and outflow was seen from both . the patient declined prolonged drainage with a catheter after counseling that this might promote healing . given the difficulty in visualizing the lesion and the lack of cuff descent in this nulliparous woman , an abdominal rather than vaginal approach for repair was recommended . after discussion with the patient regarding the risks and benefits of an open abdominal procedure , a laparoscopic repair , or a robotic approach , the patient chose the robotic surgery . the similarities and differences between the 3 procedures were discussed with the patient , along with the attending surgeon 's experience with robotic surgery , which spans 4 years . the patient was aware that the surgery could be converted to laparoscopy or an open procedure should there be any difficulty with the robotic approach . the surgery was scheduled approximately 3 months after the hysterectomy had been performed . in the operating room , the patient was positioned in the dorsal lithotomy position similar to that used in standard laparoscopy . on cystoscopy , the fistula was noted , and a 5-french pollack stent was placed from the bladder through the fistulous tract into the vagina . double j ureteral stents were placed over guide wires , given the close proximity of the ureteral orifices to the fistula and repair . the hasson technique was chosen for umbilical port access due to the patient 's previous surgery . the camera was placed through a 12 mm port located superior to the umbilicus and approximately 18 cm superior to the symphysis pubis . twelve mm and 5 mm assistant ports were placed on the right side , and 8 mm robotic trocars were inserted on the lateral edge of the rectus muscles bilaterally ( figure 1 ) . lysis of omental and small bowel adhesions in the area of the previous incision was performed using hook cautery and the 30-degree up lens . port placement for robotic - assisted vesicovaginal fistula repair : c = 12 mm camera port ; r = 8 mm robotic ports located on lateral edge of rectus muscles ; s = 5 mm suction port ; a = 12 mm assistant port located superior and medial to anterior superior iliac spine . the vaginal cuff was identified by a sponge stick in the vagina and dissection proceeded along the anterior vaginal wall . the bladder was dissected away from the anterior vaginal wall by using hook cautery and the 0-degree lens for the robotic camera . fluid containing indigo carmine was instilled into the bladder and was seen coming from the fistula site . the bladder was repaired by using a running stitch of 3 - 0 vicryl on an rb needle using the da vinci robot . a second layer of closure was performed in an imbricating fashion with the same suture . the vagina was closed using a running stitch of 2- 0 monocryl on an sh needle . the ureteral stents were removed without difficulty , and backfill of the bladder again using indigo carmine failed to reveal any interruption of bladder integrity . a fatty epiploica from the sigmoid colon total operative time , including patient positioning , cystoscopy for ureteral stents , and fistula identification , was approximately 4.5 hours . she returned for a follow - up 3 weeks after surgery and reported no continuing symptoms of urinary incontinence . as in this patient , abdominal hysterectomy is the most common cause of vesicovaginal fistula in developed countries , while obstetric causes are much less common . although conservative management is successful in some patients , and others are candidates for vaginal repair , some patients may not be candidates or may be unsatisfied with these options . the laparoscopic approach is a viable option for successful repair of a vesicovaginal fistula in a patient in whom a vaginal approach is not indicated . as with other laparoscopic surgeries , this provides superior magnification of tissue , visualization of any additional pathology , and requires no large abdominal incision . additionally , laparoscopic and robotic surgery may result in a shorter hospital length of stay and improved cosmesis as compared with open abdominal surgery . however , this may be an obstacle to surgeons without laparoscopic suturing or advanced dissecting skills . the da vinci robotic tower includes 1 camera arm and 2 to 3 instrument arms that are controlled remotely by the surgeon sitting at a nonsterile console . once a surgeon is trained to use the robotic - assisted approach , the da vinci robot can be used to facilitate the laparoscopic repair . its advantages include improved dexterity with increased degrees of freedom giving the surgeon the sensation of having wrists rather than lever arms , enhanced magnification , 3-dimensional visualization providing depth perception similar to that of open surgery , increased surgeon comfort with a seat and decreased surgeon hand fatigue compared with traditional operative laparoscopy . a successful robotic - assisted technique has been described . in both of these previous reports of this surgical technique , intentional cystotomies were made in a healthy area of the posterior wall of the bladder to facilitate identification and repair . a stent placed cystoscopically from the bladder into the vagina , as explained here , may obviate the need to make an intentional cystotomy . this may decrease the risk of recurrence and length of postoperative catheterization . because robotic surgery is relatively new to gynecology , its ultimate role is still evolving . procedures that involve a high degree of precision and technical skill like suturing appear to be the first areas of application of this technology , given the advantages of robotic surgery over traditional laparoscopy . given the extent of gynecologic surgery that is performed laparoscopically at this point , it is reasonable to believe that robotic surgery could have an expanding list of uses . as the case volume grows , additional studies should be carried out to assess the benefits of robotic assistance over traditional laparoscopy or other surgical approaches . although the vaginal approach is the most common for repair of vesicovaginal fistulas , the robotic - assisted laparoscopic approach can also be a successful option in patients for whom the vaginal approach is not possible .

background and objectives : fistulas inaccessible from the vagina may require abdominal repair ; we sought to evaluate the robotic - assisted laparoscopic approach for this procedure.methods:a 41-year - old nulliparous woman presented with urinary incontinence following an abdominal hysterectomy , and office evaluation identified a vesicovaginal fistula . after discussion with the patient regarding the surgical options , the robotic approach was chosen to facilitate precise dissection , fine visualization , and suturing . a stent was placed from the bladder into the vagina , and no intentional cystotomy was made . the bladder was dissected away from the anterior vaginal wall at the fistula site , and the defects were closed independently with interposition of a fatty epiploica from the sigmoid colon . total operative time was approximately 4 hours , and robotic time was about 2.5 hours.results:at 3 months after surgery , the patient had no recurrent symptoms.conclusions:the robotic - assisted laparoscopic approach is a viable option for successful repair of a vesicovaginal fistula in a patient in whom a vaginal approach is not indicated .

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

PMC4349997

currently for super - obese individuals , bariatric surgery is the only evidence - based approach that results in clinically significant and sustainable weight loss . with the increasing prevalence of obesity , there is an increased demand for bariatric surgery . subsequently , a large number of patients shall be presenting with the complications of bariatric surgery . a 42-year - old caucasian female underwent laparoscopic roux - en - y gastric bypass in february 2007 . she subsequently developed gallstones , a known complication of bariatric surgery due to rapid weight loss . she presented to our unit in february 2012 with a week long history of right upper quadrant pain associated with vomiting . blood tests revealed deranged liver function tests : alkaline phosphatase of 169 u / l and alanine aminotransaminase of 188 u / l . a computed tomographic scan was organized confirming the appearances of a dilated common bile duct and the potential of a calcified stone in the common bile duct . she was managed conservatively with analgesia and ursodeoxycholic acid to attempt to dissolve the stones . her liver function tests and pain improved , and she was discharged with an outpatient magnetic resonance cholangiopancreatography ( mrcp ) . in march 2012 , her mrcp showed a dilated common bile duct of 10 mm and two small filling defects suggestive of gallstones . again , this confirmed the dilated common bile duct , but also showed free drainage of bile into the duodenum and no gallstones ; hence , therapeutic interventions were not undertaken . she was referred to the gastroenterologists for further advice , but as of june 2012 she was still symptomatic . ultimately , an endoscopic retrograde cholangiopancreatography ( ercp ) and sphincterotomy was deemed to be necessary to alleviate her symptoms . owing to the altered anatomy post roux - en - y gastric bypass , this was to be a technical challenge . the bypass of the distal stomach and proximal small bowel is achieved by anastomosis of the gastric pouch and the jejunum known as the roux alimentary limb . the biliary pancreatic limb is then anastomosed to a small bowel . as a result it is technically challenging to perform an ercp in the conventional manner , two subsequent port sites were used : a 5-mm port site in the right upper quadrant and a 10-mm port in the left hypochondrium ( fig . 1 ) . stay sutures were placed in a diamond - shaped configuration around the gastrotomy site these can allow both traction and counter - traction . a sterile ercp scope inserted manually through a left 10-mm port site all under laparoscopic visualization . there were no obvious gallstones in our patient ( fig . 2 ) . the gastrotomy site was closed with a two layer closure : the first layer as a running stitch and then as a horizontal mattress with 2.0 vicryl . she was admitted for an overnight stay and discharged the following day . upon review of the patient in a routine 6-week follow - up clinic , she was asymptomatic and was discharged back to the care of her general practitioner . with the increased demand for bariatric surgery worldwide , there will be an increased role of endoscopy to diagnose and manage complications . with altered anatomy , the transgastric approach can be used as an access point into the gastrointestinal tract . we have demonstrated that the technique when performed correctly can be a safe and viable option in most district general hospitals , which would certainly have a laparoscopic surgeon and a gastroenterologist on site .

the prevalence of bariatric surgery is increasing worldwide and as a direct consequence , there shall be an increasing number of patients presenting with the complications of bariatric surgery , often to non - specialist units . the authors report a case of a 42-year - old caucasian female who had previous laparoscopic roux - en - y gastric bypass , open cholecystectomy and abdominoplasty presenting with right upper quadrant pain in keeping with retained common bile duct stones . after the failure of conservative management , a laparoscopic - assisted transgastric endoscopic retrograde changiopancreatography and sphincterotomy was performed . we shall be describing our technique .

INTRODUCTION CASE REPORT DISCUSSION CONFLICT OF INTEREST STATEMENT

PMC4252934

success of endodontic treatment relies on the accurate determination of the working length and adequate enlargement of the root canal . showed that often canals are improperly cleaned and attributed this to inadequate instrumentation due to the fact that the root canal diameter is larger than the instrument caliber used in each particular case . in addition with shaping , determination of the anatomic diameter is also related to cleaning of the root canal system . determination of the anatomical diameter is based solely on clinician 's ability to detect the apical narrowing by tactile sense which is an empirical and inaccurate method . tan and messer . stated that traditional methods for determination of the anatomic diameter at the apical third have under estimated the real diameter of this region . without adequate scientific evidence decision ca thus the concept of cleaning the apical canal to three sizes larger than the first file to bind is not based on the scientific evidence . there are many factors that affect the determination of minimal initial working width at working length , these are ; canal shape , canal taper , canal curvature , canal content , canal wall irregularities and instruments for determining initial working width . preflaring of the cervical and middle thirds of the root canal improves anatomical diameter determination ; the instrument used for preflaring plays a major role in determining the anatomical diameter at the wl . thus , the objective of this study was to investigate the influence of cervical preflaring using different and currently used instruments on the accuracy of apical file size determination . tooth selection and preparation 80 extracted human permanent maxillary first molars displaying normal pulp chambers , patent root canals , fully formed apices without any sign of resorption were collected . maxillary molars with degree of curvature of the mesiobuccal root between 10 and 15 were utilized . all teeth were placed in 0.1% thymol solution and taken out 24 hours before use . the cusps of the teeth were cut horizontally to get a plane occlusal zone to determine the working length precisely . standard access cavities were performed and the pulp tissue was removed with a barbed broach , avoiding contact with the root canal walls . canals were then irrigated with copious 2.5% sodium hypochlorite solution ( qualigens fine chemicals , navi mumbai , india ) . sizing of canals : after final irrigation with normal saline , the canal was dried . to maintain the patency of canal , an iso 06/.02 k - file ( dentsply ) was inserted until it came out of the apex . after maintaining patency an iso 08/0.02 k - file was inserted with gentle pressure in watch winding motion until the tip of file was visible at the apex . the overall canal length was thus determined by placing tip of the file at the apex and working length was established 1 mm short of this length . cervical and middle third flaring- after working length determination , the teeth were divided in to eight groups ( n = 10 ) . one group was taken as control group that is without pre - flaring , other seven groups received pre - flaring of root canals . all rotary instruments were used in continuous rotary motion with the help of x - smart endomotor ( dentsply / maillefer , ballaigues , switzerland ) . the cervical and middle third of canals were pre - flared 3 - 4 mm short of the working length . gates - glidden drills ( dentsply maillefer , size iso 90 - 110 ) were used until the binding sensation was felt in the middle third . protaper files ( dentsply maillefer ) sx ( iso 20 , taper 3.5 - 19% ) , s1 ( iso 17 , taper 2 - 11% ) , s2 ( iso 20 , taper 4 - 11.5% ) were used in respective order . the flexmaster intro file ( vdw ) ( iso 22/0.11 ) was initially used for flaring followed by iso 25/0.06 and 25/0.04 files respectively . an iso size 25/0.08 hyflex cm file ( coltene ) was used to flare the canal followed by an iso 20/0.04 file . determination of initial apical file- after pre - flaring of all teeth was done , hand files were inserted in to the mesio - buccal root canal starting with k - file iso 08/0.02 at the working length . an iso 10/0.02 file was then inserted till the working length . at iso 10 , the file size was increased in increment of 5 iso units until slight friction was felt at the working length . the first file that had binding sensation at the working length was noted and fixed with methacrylate in the root canal . one millimeter of the root apex was cut horizontally with a microcutter so that the remaining tooth was at the working length . the apical sections were visualized using stereo microscope and images were recorded digitally for each specimen . the analysis of the images obtained was performed on a computer using the image pro plus software ( media cybernetics , usa ) . it was used to determine the diameter of the root canal and diameter of the initial apical file . the largest and smallest diameter of the root canal and the largest diameter of the instrument were recorded . the data were submitted to anova test and post hoc tests bonferroni multiple comparisons , to assess the effect of pre - flaring techniques on the discrepancies found between the diameter of the binding instrument and the anatomic diameter of the root canal . cervical preflaring and the type of instrument had a significant effect on initial apical file size determination . preflaring with la group burs leads to the most accurate determination of the initial apical file size . in this group , the maximal apical root canal diameter and the diameter of the initial apical file had the lowest discrepancy ( mean 0.015 mm 0.015 ) followed by hyflex group ( mean 0.017 mm 0.017 ) and there was no statistical difference between these groups . following hyflex group were race instruments ( mean 0.020 mm 0.020 ) . protaper group showed greater discrepancy than the former groups ( mean 0.028 mm 0.028 ) . after protaper came flexmaster group ( mean 0.035 mm 0.035 ) followed by k3 ( mean 0.048 mm 0.048 ) and gatesglidden ( mean 0.051 0.051 ) which showed comparable results [ figure 1 ] . stereomicroscopic pictures of transverse sections of root canals at the wl with the iaf fixed in the root canal to show the discrepancies of root canal diameter and diameter of the iaf of ( a ) control group ( b ) la group ( c ) hf group ( d ) rc group ( e ) pt group ( f ) fm group ( g ) k3 group ( h ) gg group in order of minimal discrepancies between the initial apical file diameter and apical root canal diameter . discrepancies ( in mm ) between the diameter of initial apical file and canals at the working length , for different groups the instrument binding technique for determining anatomical diameter at working length is not precise ; preflaring of the cervical and middle thirds improve the determination of the anatomical diameters at the working length and the type of instrument play an important role . the increase in file size after preflaring can be explained by realizing that , within a canal , irregularities and curvature produced contacts with the file and interfere with its progression toward the apex . early flaring , regardless of the method used , removes these contacts , opens the space and reduces file contact ; thus , a file progresses more easily towards the apex after flaring . comes to a stop only when the diameter of the canal begins to apply pressure against the instrument . the point in the apical region of the canal at which preparation and obturation should be terminated is determined by several variables . and the canal is supposed to be prepared has been a controversial issue in the endodontic field . grossman also stated that the canal should be enlarged at least three sizes greater than its original diameter . however this concept needs to be reviewed , as it is ineffective and may leave canal walls untouched when no preflaring is performed . mechanical ( that is rotary flaring ) reduces treatment time , but is accompanied by a risk of complications . over enthusiastic use , inappropriate size and excessive depth can result in lateral perforations , ledges and instrument breakage . the roots of the teeth in the present study were embedded in methacrylate to preserve the apical region and to avoid any destruction during root canal preparation and cutting the root . the apical foramen was not covered by methacrylate to be able to precisely measure tooth length and exactly cut 1 mm of the apex with the iaf fixed in the root canal . in the present study , cervical preflaring of the root canal significantly increased the accuracy of determining the initial apical diameter by the initial apical file compared with non - flared root canals . the size of initial apical file in the control group was three times smaller than that in la and hf groups . each system has its own unique design feature and hence has a characteristic preparation technique . taper of instruments , used for preflaring , is a determining factor in the accuracy for determination of initial apical file . from all specimens evaluated , the root canal preflared with la axxess instruments presented the least discrepancies . this could be attributed to the configuration , metal alloy properties and mode of operation . moreover the 0.06 taper , safe end and flute design of la axxess burs have been associated with complete removal of cervical dentin projections without causing canal perforations and transportations . the 0.08 taper of the hyflex cm files could be associated with the good results . they are manufactured by a unique process that controls the material memory , making the files extremely flexible but without the shape memory of other niti files . this gives the file the ability to follow the anatomy of the canal very closely . these advanced features of hyflex cm files increase its efficiency to remove the dentin projections from cervical and middle thirds of root canals more effectively and quickly . this is the first study to compare the effect of preflaring with this new and unique file system . similarly the greater taper of race instruments that is , 0.10 and 0.08 , effectively created the path for the initial apical file to progress without touching canal walls ( curvature ) and dentin projections , this could be attributed to its good performance in reducing the discrepancy between the apical canal diameter and initial apical file diameter . these results are in agreement with previous study , showing better results of race files over protaper and flexmaster . flexmaster intro file has a 11% taper but its simple k - file type design and less flute space for effective debris removal can be associated with its lack of performance as compared to previous instruments . these results are in agreement with previous study . showing better results of protaper over flexmaster . k3 files having 0.10 and 0.08 taper has a positive cutting and rake angle but its low flexibility as compared to other files could reflect its results in reducing the discrepancies between the initial apical file and apical canal diameter . although the results of k3 file are comparable with gates glidden drills . apical shaping is easier when early flaring is used , from the data presented , one can speculate that early flaring reduces the discrepancy between the initial apical file diameter and apical canal diameter . an appropriate apical sizing method can help operator to avoid unnecessary enlargement of the apex . there is further need of research to develop methods for optimal determination of root canal size in all dimensions while respecting the complexity of apical anatomy . although early preflaring of the root canal could not guarantee that the instruments bound only at the working length . clinically , it is not always possible to straighten the coronal two - thirds of root canal because sometimes the radius of root curvatures is long . however , it must not be forgotten that the capability of the endodontist in applying all available information is also a determinant for success . cervical preflaring plays a vital role in reducing the discrepancy between initial apical file diameter and apical canal diameter . taper , cross sectional design and flexibility of the instrument used for preflaring plays a vital role in determining its effect . la axxess burs and hyflex cm files showed best results in comparison to all groups compared in this study .

aim : to investigate the influence of cervical preflaring using different rotary instruments on apical file size determination.materials and methods : extracted human molar teeth were randomly divided in to eight groups ( n = 10 ) : control group ( cg ) ; la axxess group ( la ) ; hyflex group ( hf ) ; gatesglidden group ( gg ) ; protaper group ( pt ) ; race group(rc ) ; flexmaster group ( fm ) ; and k3 group ( k3 ) . patency was maintained and working length was established under magnification . all instruments were used according to manufacturer 's instructions . steriomicroscopic images were taken to determine the discrepancies in diameters . proplus software ( usa ) was used to determine the diameter of the root canal . anova test and post hoc tests bonferroni multiple comparisons were used for statistical analysis.results:canals preflared with la axxess burs showed the best results . control group that is , the canals with no cervical preflaring showed the maximum discrepancy between the initial apical file diameter and apical canal diameter.conclusion:cervical preflaring plays an important role in reducing the discrepancy between initial apical file diameter and apical canal diameter .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

PMC4672992

liver transplantation is the only treatment option for patients with end stage liver disease or advanced hepatocellular carcinoma . for the last 25 years , the number of waiting list candidates has gradually increased and exceeded the number of available grafts . the number of heart beating donors has decreased over the last decade . at the same time , numbers of marginal grafts , such as donation after cardiac death ( dcd ) , as well as old and fatty livers have increased . marginal grafts are often declined for liver transplantation because of the higher chance of primary graft non- or delayed function . in dcd grafts , development of ischemic type biliary strictures ( itbs ) is of special concern . with the conventional static cold preservation technique , itbs occur in about 10 - 40% of dcd grafts . in the majority of patients , itbs leads to re - transplantation or patient death . especially prolonged warm and cold ischemic times are risk factors for itbs . donor age , genetic predispositions ( such as ccr5 delta 32 ) , and the choice of preservation solution have also been discussed as additional risk factors . partial microthrombosis of the peribiliary vessels has been suggested as potential mechanism for itbs after liver transplantation with dcd grafts . prior to the clinical introduction of liver transplantation , ex vivo liver perfusions have been used to study hepatic metabolism and physiology . after liver transplantation found its way into the clinical setting in the 1960s , innumerable attempts have been made to use ex vivo liver perfusion as a preservation method by mimicking physiological nutrition and oxygenation conditions . its utility for preservation of marginal grafts has been investigated in the last decade , but it did not reach standard clinical care . we recently described a reduction in bile duct injury in dcd liver transplantation by ex vivo perfused preservation . the selection ranges from cellular solutions like whole blood from the donor animal or packed red cells in combination with human plasma , to acellular approaches like machine university of wisconsin solution , igl solution , or steen solution . all different techniques , solutions , and temperature settings aim at 1 ) stable perfusion conditions , 2 ) sufficient oxygenation , and 3 ) re - establishment of organ function . an enhanced preservation capacity as well as the ability of organ assessment and treatment during normothermic and subnormothermic perfusion faces higher technical complexity and costs compared to hypothermic perfusion . we have developed a subnormothermic ex vivo liver perfusion system over the last 4 years . the system can be used to 1 ) recharge the hepatic energy content , 2 ) to assess the graft s quality , and to 3 ) repair marginal livers prior to transplantation . the porcine study design of liver injury is based on a donation after cardiac death ( dcd ) model . after dissection of all liver vessels , cardiac death is induced followed by 45 min of warm graft ischemia . to simulate a graft transport in between the donor and recipient hospitals in a clinical setting , the graft is stored on ice for 4 hr after cold , dual flush . after cold storage , the organ is subnormothermic perfused for 6 hr in order to assess the perfusion stability . in a transplant model , the perfusion time could be shorter in order to recharge energy storage and to assess the organ viability . note : male yorkshire pigs , 30 - 35 kg , were utilized for this study . all animals received humane care in compliance with the principles of laboratory animal care formulated by the national society for medical research and the guide for the care of laboratory animals published by the national institutes of health . house male yorkshire pigs in research facilities for 1 week before perfusion / transplantation to reduce the level of stress and to accustom the animals to the housing conditions . less than 2 days of housing inside the facility will lead to a stress - induced physical reaction , which can alter the perfusion s outcome . injection of a mixture of ketamine ( 25 mg / kg ) , atropine ( 0.04 mg / kg ) , and midazolam ( 0.15 mg / kg ) . prior to intubation , ensure the pig breathes spontaneously 2 l of o2 dosed with 5% isoflurane . spray the vocal chords with 2% lidocaine 2 min before intubation to avoid vocal cord spasms . lower the isoflurane gas to 2% . set the ventilator to 14 - 16 breaths / min and a tidal volume of 10 ml / kg bodyweight . catheter in one of the ear veins to allow infusion of ringer s lactate solution ( 200 ml per hr ) . then scrub the pig and cover it with sterile drapes . use a towel to cover large and small bowels and move them to the left side . separate inferior vena cava ( ivc ) and distal aorta from each other ; ligate aorta branches to the back ; isolate and free renal arteries from adherent tissue . divide the falciforme ligament and the triangular ligament using cautery . release the portal vein by an incision of the peritoneum between pancreas and portal vein . tie off veins draining from the pancreas to the portal vein . dissect the coeliac trunk below the portal vein and follow it backwards to the aorta . surround the mesenteric artery with a 2 - 0 tie ; surround the splenic and left gastric arteries , which branch posteriorly to the coeliac trunk . dissect the aorta behind the diaphragm between heart and coeliac trunk ; place a 2 - 0 tie around the aorta . release the liver from the lower cava on the right side using electro cautery ; use scissors for the upper part between cava and liver . remove the gallbladder and cauterize bleeders from gallbladder bed . induce cardiac arrest by intracardial injection of 40 mval kcl 3 min after heparin administration . for the perfusion , collect 1.6 l of pig blood in cpda bags ( citrate , phosphate , dextrose , adenosine ) immediately after cardiac death . perform a soft spin ( 2,000 x g without brake ) . remove the plasma and the buffy coat under sterile condition ( biosafety cabinet class ii ) and store the erythrocytes in cpda bags for transfusion . tie off the previously set ties around femoral , renal , splenic , mesenteric , and left gastric arteries as well as the upper aorta . for a heart beating donor ( hbd ) model , perform the cannulation of aorta and portal vein under heart beating conditions . after 45 min warm ischemia , flush the liver with university of wisconsin ( uw ) solution using dual perfusion via aorta ( pressure bag ) and portal vein ( gravity driven ) . cut the liver out of the pig , leaving all remaining vessels long . during back - table preparation , clamp the upper ivc using a satinsky clamp and flush the liver a second time with about 0.5 l of uw solution retrogradely via lower ivc until the portal vein outflow is clear . perform an arterial back - table pressure perfusion with about 0.5 l of uw solution . cannulate the upper and lower parts of the ivc using 1/2 x 3/8 reducers with luer lock ; cannulate the portal vein and liver artery using 3/8 x 1/4 and 1/4 x 3/8 reducers with luer lock . place the liver in an organ bag , close the organ bag , and store the liver on ice until the perfusion has started . prepare the perfusion solution containing 2,000 ml steen solution , 400 ml washed erythrocytes , 550 mg sodium pyruvate , 100 ml amino acid solution ( 10% travasol ) , 10 mg calcium gluconate , 1,000 ie rapid acting insulin , 1 g cefazolin , 500 mg metronidazol , and 10,000 iu heparin . add other molecules for vasodilatation , immunosuppression , scavenging of reactive oxygen species , or liver cell treatment based on the particular study protocol . for the dialysis component , use standard dialysate containing 3.5 mm potassium , 25 mm bicarbonate , 27 mm glucose , as well as 275 mg / l pyruvate . set up the perfusion circuit ( schema see figure 2 ) . coming from the main reservoir as the starting and end point , the perfusion solution is driven by a centrifugal pump through an oxygenator . right after oxygenation of the solution , the circuit splits into a smaller line running to a dialyzer unit for electrolyte homeostasis and a bigger line running to a leukocyte filter for reduction of remnant white blood cells ( wbc ) . the solution that runs through the dialyzer returns to the main reservoir . after the leukocyte filter , the circuit splits up again into 2 equal lines . one line runs at high pressure ( around 60 mmhg ) directly into the aorta to perfuse the hepatic artery . the pressure of the portal perfusion depends on the elevation energy of the reservoir s solution level ( around 2 - 6 mmhg ) . all fluids are drained via the infra- and supra - hepatic cava back into the main reservoir . for gravity reduction and homogeneous perfusion , the liver is placed in a pool filled with temperature regulated water . it is separated from the water by an impermeable membrane and it swims in a perfusion suspension . collect all fluids from the circuit in a 3 l reservoir ( main reservoir ) and clamp the outflow.connect the outflow to a centrifugal pump followed by a commercial oxygenator.behind the oxygenator , split up the tubing into 2 lines . connect the second line to a leukocyte reduction filter.split up the line after the leukocyte reduction filter into an arterial line , which supplies perfusion solution to the hepatic artery , and a portal venous line delivering perfusion solution to a second reservoir , which drains into the portal vein by gravity outflow . clamp the portal inflow.connect the arterial line to a vena cava line draining into the main reservoir for fluid recollection.for collection of ascites or leakage of perfusion solution from the liver , prepare a suction line connected to the main reservoir . collect all fluids from the circuit in a 3 l reservoir ( main reservoir ) and clamp the outflow . connect the outflow to a centrifugal pump followed by a commercial oxygenator . behind the oxygenator , split up the tubing into 2 lines . split up the line after the leukocyte reduction filter into an arterial line , which supplies perfusion solution to the hepatic artery , and a portal venous line delivering perfusion solution to a second reservoir , which drains into the portal vein by gravity outflow . connect the arterial line to a vena cava line draining into the main reservoir for fluid recollection . for collection of ascites or leakage of perfusion solution from the liver , prepare a suction line connected to the main reservoir . release the outflow clamp from the main reservoir and fill the circuit with the perfusion solution . the perfusion solution will drive through the arterial line into the vena cava line back into the main reservoir . place the liver , with its convex side down to facilitate the access to the vessels , ideally in a gravity - free environment to avoid organ compression at the contact surface . cover the water bath with an impermeable membrane and place the liver onto that membrane . reduce gravity driven compression by submerging the liver with perfusion solution . reduce the speed of the centrifugal pump to 1,000 rounds / min and place two clamps at the connection of arterial and vena cava lines . release the clamp from the arterial line , pour perfusion solution into the arterial cannula to get rid of bubbles , and connect the line to the cannula . connect pressure lines to the luer locks of the arterial , portal , and vena cava cannulas . to mimic physiological conditions , apply treatments into the right vessel . inject glucose into the portal vein and not into the arterial line in order to establish a gradient mimicking an increased portal venous glucose gradient and inducing glycogen synthesis . after connecting the liver to the circuit , raise the temperature to 33 c within 60 min . aim for an arterial starting flow at about 250 ml / min at 40 mmhg . this may reach 700 ml / min during perfusion once the pressure is increased up to 70 mmhg . at starting temperature , aim for a portal vein flow of 500 - 600 ml / min at 3 - 5 mmhg . after raising the temperature , monitor the portal venous flow , which will increase up to 1,100 ml / min at 4 - 6 mmhg . avoid exceeding portal pressure above physiological values ( around 8 mmhg ) to protect sinusoidal fenestrations . avoid exceeding total flow above 2,000 ml / min in order to prevent damaging the organ . set the outflow to -2 mmhg by lowering the main reservoir to prevent liver congestion by functional outflow obstruction . add the dialysis component to the circuit in order to equilibrate the perfusion solution to predetermined values . set the dialysate flow to 500 ml / hr . take special attention to adjust the dialysis outflow so that the perfusion solution is neither diluted nor concentrated . within the first hour of perfusion ensure homogeneous oxygenation of the tissue to recover and maintain organ function by using a main gas mixture component of o2 ( 95 - 98% ) and co2 ( 2 - 5% ) . use variable gas during perfusion since the liver changes its metabolism and its ph demand during perfusion . maintain a low ph during the start of perfusion to protect the organ using the paradox ph concept and avoid severe tissue damage that can result from fast connections to physiological ph under reoxygenation , since after storage in uw solution , the organ has an acidotic ph below 7 . adjust the partial pressure of co2 continuously down to 25 - 30 mmhg so that the ph will reach a physiologic level within 1 hr . add sodium- or potassium bicarbonate to the circuit to achieve a physiological concentration of standard bicarbonate in the perfusion solution . monitor vascular flow and pressure and note a stable perfusion by a constant vascular resistance . keep the perfusion system stable for up to 8 hr . at the end of the ex vivo perfusion period , cool down the perfusion system to 20 c and , after disconnecting the circuit s tubing from the liver , flush the perfusion solution out the liver dually with ice cold uw solution . store the liver once again placed on ice in a sterile organ bag note : male yorkshire pigs , 30 - 35 kg , were utilized for this study . all animals received humane care in compliance with the principles of laboratory animal care formulated by the national society for medical research and the guide for the care of laboratory animals published by the national institutes of health . house male yorkshire pigs in research facilities for 1 week before perfusion / transplantation to reduce the level of stress and to accustom the animals to the housing conditions . less than 2 days of housing inside the facility will lead to a stress - induced physical reaction , which can alter the perfusion s outcome . injection of a mixture of ketamine ( 25 mg / kg ) , atropine ( 0.04 mg / kg ) , and midazolam ( 0.15 mg / kg ) . prior to intubation , ensure the pig breathes spontaneously 2 l of o2 dosed with 5% isoflurane . spray the vocal chords with 2% lidocaine 2 min before intubation to avoid vocal cord spasms . lower the isoflurane gas to 2% . set the ventilator to 14 - 16 breaths / min and a tidal volume of 10 ml / kg bodyweight . catheter in one of the ear veins to allow infusion of ringer s lactate solution ( 200 ml per hr ) . then scrub the pig and cover it with sterile drapes . use a towel to cover large and small bowels and move them to the left side . separate inferior vena cava ( ivc ) and distal aorta from each other ; ligate aorta branches to the back ; isolate and free renal arteries from adherent tissue . divide the falciforme ligament and the triangular ligament using cautery . release the portal vein by an incision of the peritoneum between pancreas and portal vein . tie off veins draining from the pancreas to the portal vein . dissect the coeliac trunk below the portal vein and follow it backwards to the aorta . surround the mesenteric artery with a 2 - 0 tie ; surround the splenic and left gastric arteries , which branch posteriorly to the coeliac trunk . dissect the aorta behind the diaphragm between heart and coeliac trunk ; place a 2 - 0 tie around the aorta . release the liver from the lower cava on the right side using electro cautery ; use scissors for the upper part between cava and liver . induce cardiac arrest by intracardial injection of 40 mval kcl 3 min after heparin administration . set cardiac arrest as the starting point of warm ischemia . for the perfusion , collect 1.6 l of pig blood in cpda bags ( citrate , phosphate , dextrose , adenosine ) immediately after cardiac death . remove the plasma and the buffy coat under sterile condition ( biosafety cabinet class ii ) and store the erythrocytes in cpda bags for transfusion . tie off the previously set ties around femoral , renal , splenic , mesenteric , and left gastric arteries as well as the upper aorta . for a heart beating donor ( hbd ) model , perform the cannulation of aorta and portal vein under heart beating conditions . after 45 min warm ischemia , flush the liver with university of wisconsin ( uw ) solution using dual perfusion via aorta ( pressure bag ) and portal vein ( gravity driven ) . cut the liver out of the pig , leaving all remaining vessels long . during back - table preparation , clamp the upper ivc using a satinsky clamp and flush the liver a second time with about 0.5 l of uw solution retrogradely via lower ivc until the portal vein outflow is clear . perform an arterial back - table pressure perfusion with about 0.5 l of uw solution . cannulate the upper and lower parts of the ivc using 1/2 x 3/8 reducers with luer lock ; cannulate the portal vein and liver artery using 3/8 x 1/4 and 1/4 x 3/8 reducers with luer lock . place the liver in an organ bag , close the organ bag , and store the liver on ice until the perfusion has started . prepare the perfusion solution containing 2,000 ml steen solution , 400 ml washed erythrocytes , 550 mg sodium pyruvate , 100 ml amino acid solution ( 10% travasol ) , 10 mg calcium gluconate , 1,000 ie rapid acting insulin , 1 g cefazolin , 500 mg metronidazol , and 10,000 iu heparin . add other molecules for vasodilatation , immunosuppression , scavenging of reactive oxygen species , or liver cell treatment based on the particular study protocol . for the dialysis component , use standard dialysate containing 3.5 mm potassium , 25 mm bicarbonate , 27 mm glucose , as well as 275 mg / l pyruvate . set up the perfusion circuit ( schema see figure 2 ) . coming from the main reservoir as the starting and end point , the perfusion solution is driven by a centrifugal pump through an oxygenator . right after oxygenation of the solution , the circuit splits into a smaller line running to a dialyzer unit for electrolyte homeostasis and a bigger line running to a leukocyte filter for reduction of remnant white blood cells ( wbc ) . the solution that runs through the dialyzer returns to the main reservoir . after the leukocyte filter , one line runs at high pressure ( around 60 mmhg ) directly into the aorta to perfuse the hepatic artery . the pressure of the portal perfusion depends on the elevation energy of the reservoir s solution level ( around 2 - 6 mmhg ) . all fluids are drained via the infra- and supra - hepatic cava back into the main reservoir . for gravity reduction and homogeneous perfusion , it is separated from the water by an impermeable membrane and it swims in a perfusion suspension . collect all fluids from the circuit in a 3 l reservoir ( main reservoir ) and clamp the outflow.connect the outflow to a centrifugal pump followed by a commercial oxygenator.behind the oxygenator , split up the tubing into 2 lines . connect the second line to a leukocyte reduction filter.split up the line after the leukocyte reduction filter into an arterial line , which supplies perfusion solution to the hepatic artery , and a portal venous line delivering perfusion solution to a second reservoir , which drains into the portal vein by gravity outflow . clamp the portal inflow.connect the arterial line to a vena cava line draining into the main reservoir for fluid recollection.for collection of ascites or leakage of perfusion solution from the liver , prepare a suction line connected to the main reservoir . collect all fluids from the circuit in a 3 l reservoir ( main reservoir ) and clamp the outflow . connect the outflow to a centrifugal pump followed by a commercial oxygenator . behind the oxygenator , split up the tubing into 2 lines . split up the line after the leukocyte reduction filter into an arterial line , which supplies perfusion solution to the hepatic artery , and a portal venous line delivering perfusion solution to a second reservoir , which drains into the portal vein by gravity outflow . connect the arterial line to a vena cava line draining into the main reservoir for fluid recollection . for collection of ascites or leakage of perfusion solution from the liver , prepare a suction line connected to the main reservoir . release the outflow clamp from the main reservoir and fill the circuit with the perfusion solution . the perfusion solution will drive through the arterial line into the vena cava line back into the main reservoir . place the liver , with its convex side down to facilitate the access to the vessels , ideally in a gravity - free environment to avoid organ compression at the contact surface . use a heat- and coolable water bath . set the starting temperature of the circuit and water bath to 20 c . cover the water bath with an impermeable membrane and place the liver onto that membrane . reduce gravity driven compression by submerging the liver with perfusion solution . reduce the speed of the centrifugal pump to 1,000 rounds / min and place two clamps at the connection of arterial and vena cava lines . then , cut the tubing in between the clamps . using a 3-way connector , release the clamp from the arterial line , pour perfusion solution into the arterial cannula to get rid of bubbles , and connect the line to the cannula . connect pressure lines to the luer locks of the arterial , portal , and vena cava cannulas . to mimic physiological conditions , apply treatments into the right vessel . inject glucose into the portal vein and not into the arterial line in order to establish a gradient mimicking an increased portal venous glucose gradient and inducing glycogen synthesis . after connecting the liver to the circuit , raise the temperature to 33 c within 60 min . aim for an arterial starting flow at about 250 ml / min at 40 mmhg . this may reach 700 ml / min during perfusion once the pressure is increased up to 70 mmhg . at starting temperature , aim for a portal vein flow of 500 - 600 ml / min at 3 - 5 mmhg . after raising the temperature , monitor the portal venous flow , which will increase up to 1,100 ml / min at 4 - 6 mmhg . avoid exceeding portal pressure above physiological values ( around 8 mmhg ) to protect sinusoidal fenestrations . avoid exceeding total flow above 2,000 ml / min in order to prevent damaging the organ . set the outflow to -2 mmhg by lowering the main reservoir to prevent liver congestion by functional outflow obstruction . add the dialysis component to the circuit in order to equilibrate the perfusion solution to predetermined values . set the dialysate flow to 500 ml / hr . take special attention to adjust the dialysis outflow so that the perfusion solution is neither diluted nor concentrated . ensure homogeneous oxygenation of the tissue to recover and maintain organ function by using a main gas mixture component of o2 ( 95 - 98% ) and co2 ( 2 - 5% ) . use variable gas during perfusion since the liver changes its metabolism and its ph demand during perfusion . maintain a low ph during the start of perfusion to protect the organ using the paradox ph concept and avoid severe tissue damage that can result from fast connections to physiological ph under reoxygenation , since after storage in uw solution , the organ has an acidotic ph below 7 . adjust the partial pressure of co2 continuously down to 25 - 30 mmhg so that the ph will reach a physiologic level within 1 hr . add sodium- or potassium bicarbonate to the circuit to achieve a physiological concentration of standard bicarbonate in the perfusion solution . monitor vascular flow and pressure and note a stable perfusion by a constant vascular resistance . keep the perfusion system stable for up to 8 hr . at the end of the ex vivo perfusion period , cool down the perfusion system to 20 c and , after disconnecting the circuit s tubing from the liver , flush the perfusion solution out the liver dually with ice cold uw solution . store the liver once again placed on ice in a sterile organ bag below , we present the results of 5 perfusion experiments with dcd - grafts after 45 min warm- and 4 hr cold ischemia prior to the start of the subnormothermic ex vivo perfusion . the main goal for an ex vivo liver perfusion is to ensure a sufficient oxygen supply to the organ . achieving constant vascular flows with stable pressures is a good indicator of adequate oxygenation . during an induction period of 1 - 2 hr the perfusion solution and the organ are warmed up to 33 c , which deceases the vascular resistance of the liver . once the target temperature of 33 c is achieved , flow values level at a constant , nearly physiological range for the rest of the 6 hr perfusion time ( figures 3a-3d ) . at the same time , the organ becomes metabolically active . figure 4a shows the venous po2 , a marker of oxygen consumption . within the initial 2 hr the venous po2 declines to a constant plateau . at this metabolically active state , h&e staining after 6 hr of perfusion reveals hepatocyte necrosis < 5 % with an intact lobular and sinusoidal structure ( figure 6 ) . pas staining at the same time point shows replenished cellular glycogen storage compared to exhausted storage in cold preserved dcd - grafts ( figure 7 ) . perfusion flows and pressure ( n = 5 , error bars show standard deviation ) . ( a , b ) hepatic artery ( ha ) flow and pressure : during the warming phase in the first 1 - 2 hr , the flow increases at stable pressures and is constant afterwards . looking at the decreasing portal venous pressure ( c ) , the increase of ha flow towards the end of the perfusion might be an autoregulatory reaction of the liver . ( c , d ) the portal venous ( pv ) flow increases corresponding to the ha flow during the first 2 hr of warming . figure 4 . monitoring parameters ( n = 5 , error bars show standard deviation ) . ( a ) the venous po2 as a marker of oxygen demand and metabolic activity decreases within the initial phase of warming due to activated cellular metabolism ; it remains stable afterwards . ( b ) bile production as a marker of metabolic activity starts at temperatures around 30 c and , thus , between the first and second hour of perfusion . ( c , d ) the dialyzer assures electrolyte homeostasis ; an initial hyperkalemia is quickly balanced . ast is a sensitive marker of hepatocellular injury ; the shallow increase suggests no significant injury during ex vivo perfusion . ( a ) sham liver sample before warm ischemia , one representative liver lobule with intact architecture . ( b ) liver sample after 45 min of warm ischemia , 4 hr of cold ischemia , and 6 hr of subnormothermic perfusion , the lobular architecture is intact without necrosis and only minimal cell swelling , the sinusoidal spaces are mildly dilated in comparison to the sham sample . in a pig model that mimics dcd liver transplantation , we demonstrated that subnormothermic liver perfusion with a cellular perfusion solution results in stable perfusion parameters , minimal hepatocyte injury , and active hepatic metabolism . ex vivo liver perfusion as preservation technique offers for the first time the opportunity to assess markers of graft function and injury during organ preservation and prior to transplantation . beside the macroscopic evaluation of the graft perfusion homogeneity , flow values provide a good indicator of the graft s viability and the extent of the ischemic injury it had suffered earlier . levels of hepatic enzymes like ast can be used to assess the degree and dynamics of hepatocellular injury . this thorough graft assessment may allow a reliable discrimination between transplantable and non - transplantable marginal organs . we chose a subnormothermic temperature of 33 c in our perfusion system because the temperature is sufficient to allow metabolism as well as atp and glycogen synthesis . at the same time , it provides a decreased oxygen demand in comparison to normothermic perfusion settings which provides additional safety against ischemic injury . in general , perfusion temperatures above 30 c have shown to minimize cold ischemic injury and provide sufficient metabolic activity . contrary to other groups , we did not use whole blood as perfusate , but a normo - osmotic albumin solution ( steen ) with washed and filtered red blood cells . by excluding the plasma components as well as thrombocytes and leukocytes , the perfusion solution is designed to minimize pro - inflammatory signaling during the ex vivo perfusion . in addition to the graft assessment , stable perfusion conditions over several hours allow graft treatment . however , almost no treatment regime has made its way into clinical practice , yet . one reason seems to be the lack of opportunity to apply those treatments during cold storage . a metabolically active liver on an ex vivo perfusion system is optimal for applying any kind of treatment . in this regard , not only treatments to ameliorate reperfusion conditions like attenuation of kupffer cell activity or scavenging of reactive oxygen species are conceivable but also treatments like gene therapy to condition the graft , e.g. , against hepatitis c recurrence . other potential strategies could include reduction on steatosis during the ex vivo perfusion period . in summary , ex vivo liver perfusion is a novel strategy to minimize cold ischemic injury and to assess marginal liver grafts prior to liver transplantation . the ex vivo perfusion setting provides unique opportunity to repair and condition grafts prior to transplantation .

the success of liver transplantation has resulted in a dramatic organ shortage . in most transplant regions 20 - 30% of patients on the waiting list for liver transplantation die without receiving an organ transplant or are delisted for disease progression . one strategy to increase the donor pool is the utilization of marginal grafts , such as fatty livers , grafts from older donors , or donation after cardiac death ( dcd ) . the current preservation technique of cold static storage is only poorly tolerated by marginal livers resulting in significant organ damage . in addition , cold static organ storage does not allow graft assessment or repair prior to transplantation.these shortcomings of cold static preservation have triggered an interest in warm perfused organ preservation to reduce cold ischemic injury , assess liver grafts during preservation , and explore the opportunity to repair marginal livers prior to transplantation . the optimal pressure and flow conditions , perfusion temperature , composition of the perfusion solution and the need for an oxygen carrier has been controversial in the past.in spite of promising results in several animal studies , the complexity and the costs have prevented a broader clinical application so far . recently , with enhanced technology and a better understanding of liver physiology during ex vivo perfusion the outcome of warm liver perfusion has improved and consistently good results can be achieved.this paper will provide information about liver retrieval , storage techniques , and isolated liver perfusion in pigs . we will illustrate a ) the requirements to ensure sufficient oxygen supply to the organ , b ) technical considerations about the perfusion machine and the perfusion solution , and c ) biochemical aspects of isolated organs .

Introduction Protocol 1. Animals 2. Organ Retrieval 3. Representative Results Discussion Disclosures

PMC3692199

it is well known that the anterior mandible contains several anatomical landmarks such as intra bony vascular canal , which is named the mandibular incisive canal ( mic ) and lingual foramen . the lingual foramen is situated in the midline of the mandible , at the level of , superior or inferior to the mental spines . these anatomical landmarks in the anterior jaw have regained attention for optimizing surgical planning and avoiding complications . the mandibular intermental foramen region is generally considered as a safe area , involving few risks of damage to vital anatomic structures during surgical procedure . however , these safety recommendations are not based on knowledge of the position and course of some anatomical landmarks . the descriptions of lingual foramen and their bony canals dimensions and locations are important to consider during anterior dental surgery ( implant placement , genioplastic , or grafting procedures ) for avoiding various complications . some of these complications are as follow : intraoperative bleeding , nerve injury , pulp canal obliteration , and neuropraxia of the mandibular incisive nerve . the reported short- and long - term neurosensory disturbances include alteration or loss of pulp sensitivity in the lower front teeth . nowadays , dental implants are regarded as a standard option for the prosthetic rehabilitation of edentulous patients . in most cases , life - threatening hemorrhagic episodes may occur , due to perforation of the lingual cortex while placing dental implants in the anterior third of the mandible . several studies indicated that if the lingual periosteum is ruptured an extensive hematoma develops within this region and progressive swelling of the floor of the mouth may cause upper airway obstruction . cone - beam computed tomography ( cbct ) has been shown to be superior to panoramic radiographs in displaying the mandibular lingual foramen and their bony canals variations . image quality of cbct systems and their relatively lower dose and cost when compared to conventional computed tomography have allowed more accessible three - dimensional assessment of craniofacial structures in dental practice . cbct allows comparatively higher resolution ( spatial resolution of 0.1 mm homogeneous voxel ) than spiral ct , which liang et al . performed an assessment of the superior and inferior genial spinal foramina and canals in a study . the purpose of this study was to assess the course and anatomical variations of lingual foramen and its bony canals with cbct imaging in iranian population . this was a cross - sectional study and cbct images were obtained from patients who were referred to esfahan school of dentistry for preoperative implant placement planning between 2010 and 2011 . the sampling method was consecutive and the sample size was 102 ( d = 0.25 , = 0.05 , and = 0.2 ) . inclusion criteria include age above 18 years old and exclusion criteria include bone pathology in the mandible region and syndromic patients . all patients had informed consent for participation in this study . all cbct ( galileos , version 1.7 ) images were taken using a standard exposure and patient positioning protocol . the acquisition parameters were as follows : tube volume , 85 kv ; tube current , 1042 ma ; acquisition period , 14 s ; effective radiation time was between 2 and 6 s ; reformatted imaging time 2.5 min ; and voxel size was 0.3 0.3 0.3 mm . basic observations consisted of the number of lingual foramina and characteristics of its bony canal including the buccal and lingual canal diameters , the canal length , and canal slope . the distance between the terminal end of lingual canal at the buccal and lingual sides from the inferior border of the mandible and alveolar crest were measured [ figures 1 and 2 ] . dimensional measurements on a reformatted cross - sectional image of mandibular cbct scatterplot diagram between age and lower lingual foramen distance to crest . this plot shows a negative linear effect between age and distance of lower lingual foramen to crest ( p = 0.031 , r = 0.248 ) in this study , we divided participants into three age groups for statistical analysis : under 35 , 3555 , and above 55 years . afterward , we evaluated the effect of patient age and gender on the dimensional measurements of the anatomical landmark mentioned above . all data were gathered and statistically analyzed by spss version 16 . a five percent level of significance the t test and analysis of variance ( anova ) were used to determine the effect of age and sex on gathered data and pearson correlation was applied to obtain the relation between age and measured dimensions . cbct imaging of the mandible , from 102 patients which included 57 males ( 54% ) and 55 females ( 46% ) , were investigated . the mean age was 52.37 ( sd : 13.33 ) years , range 2191 years . 73.5% of patients showed a dentate anterior mandible , and 26.5% of the patients who were referred for cbct imaging were edentulous . from the 102 mandibles investigated , 102 ( 100% ) had at least one lingual foramen . 54 mandibles ( 52.9% ) had two foramina at the lingual side of the mandibular midline and 20 mandibles ( 19.6% ) showed three foramina in the mandibular midline and three mandibles ( 2.9% ) showed four foramina in the mandibular midline . the average length of the superior lingual canals was 7.83 ( sd 2.25 ) mm . the mean diameter of the opening of the superior lingual canals at the lingual side ( superior lingual foramen ) was 1.12 ( sd 0.31 ) mm and at the labial side , it was 0.68 ( sd 0.17 ) mm . the mean distances between the openings of the superior lingual canals at the labial side and lower border of the mandible , mandibular crest , and buccal plate were 10.08 ( sd 2.06 ) , 18 ( sd 5.63 ) , and 4.73 ( sd 1.85 ) mm , respectively . the average length of the inferior lingual canals was 6.33 ( sd 1.65 ) mm . the mean diameter of the opening of the inferior lingual canals at the lingual side was 0.9 ( sd 0.39 ) mm and at the labial side , it was 0.57 ( sd 0.18 ) mm . the mean distances between the opening of the inferior lingual canals at the labial side and lower border of the mandible , mandibular crest , and buccal plate were 6.43 ( sd 2.07 ) , 21.89 ( sd 4.12 ) , and 4.8 ( sd 1.87 ) mm , respectively . the mean distances between the superior and inferior lingual foramina from lower border of the mandible were 14.12 ( sd 2.49 ) and 4.27 ( sd 2.65 ) mm , respectively . also the mean distances between the superior and inferior lingual foramina from mandibular crest were 14.39 ( sd 4.82 ) and 24.27 ( sd 5 ) mm . in our study , 96% courses of the superior lingual canals were running downward to the labial and 3% of the canals were running horizontally and 1% of the canals were directed upward to the labial side . from all the inferior lingual canals , 21.47% of them had courses running downward to the labial , 2.68% of the canals were running horizontally , and 77.8% of the canals tables 1 and 2 show the distinctive dimensional measurements of the superior and inferior lingual foramina and their bony canals regarding to age and sex of the patients . as it can be seen in table 1 , the distance between lingual foramen and alveolar crest was significantly larger in less than 35 years old group . also males had significantly larger distances between buccal end of lingual canal from inferior and buccal plate [ table 2 ] . the differences between dimensional characteristics of lingual foramen and canal with age groups the differences between dimensional characteristics of lingual foramen and canal with sex furthermore , we evaluated the correlation between the mentioned measurements and patient 's age . as a result , we did not find any significant correlation rather than a correlation between age and lower lingual foramen distance to crest with p value = 0.031 and a0.248 pearson correlation [ figure 3 ] . with the increasing use of implants and grafting procedures for anterior jaw bone , the number of reported postoperative complaints has been rising . however , the lingual foramen is well identified on oral radiographs and thus clearly described in textbooks related to radiographic anatomy . knowledge of lingual foramen could be important for presurgical considerations of implant installation in the midline of the mandible . some studies assume a vascular content , its being an anastomosis of the sublingual branch of the right and left lingual arteries . the artery could be of sufficient size to provoke a hemorrhage intraosseously or in the connective soft tissue , which might be difficult to control . previous studies have been performed about frequency , diameter , and other anatomical features of lingual foramen and its canals . the purpose of the present study was to investigate the prevalence and anatomical variations of lingual foramen among iranian population . in our study , 102 mandibles were investigated and all of the images had at least one lingual foramen . study , in which lingual foramen was seen in 82% of the spiral ct images . one possible explanation for this discrepancy is that the reformatting procedure with some ct scans lacking a reformatted cross - sectional slice exactly at the mandibular midline . furthermore , it is possible that the 1-mm slice thickness may have masked smaller diameter structure on the mandibular midline . recently , cbct , which has an approach different from spiral ct , has come to be used widely . cbct allows comparatively less radiation and higher resolution ( spatial resolution of 0.3 mm homogeneous voxel ) than spiral ct . therefore , the cbct measurements for the mandibular lingual foramen and canal are considered to be reliable . the superior and inferior lingual foramen frequencies in present study were 99% and 74.5% , respectively . yet , our results provide no evidence for those of kawai et al . , which reported that the superior and inferior lingual foramen occurred with frequencies of 86.8% and 83.8% , respectively . the location of lingual foramen and canal is important to avoid complications during surgery in that region . for instance , the mean distances between the superior and inferior lingual foramina from lower border of the mandible were 14.12 ( sd 2.49 ) and 4.27 ( sd 2.65 ) mm , respectively . while kawai et al . on japanese mandibles showed that distances of the superior and inferior lingual foramina from the inferior mandibular plane were 11.43 ( sd 1.56 ) and 4.42 ( sd 2.64 ) mm , respectively . as it can be obtained , the mean distance between the superior and inferior lingual foramina from lower border of the mandible was greater in iranian population rather than other populations in previous studies . the vertical distance from the alveolar crest to the opening of the superior lingual canal at labial side was 18 ( sd 5.63 ) mm . this means that a long implant is needed to injure the blood vessels . in type a and b ridges , lekcholm et al . recommended placements of implants no longer than 13 mm in atrophied mandibles ( types c and d ) , the vertical and horizontal dimensions are shorter , and implant length should be considered carefully . also , the mean height of the opening of the superior and inferior lingual canals at the labial side from lower border of the mandible were 10.08 ( sd 2.06 ) and 6.43 ( sd 2.07 ) mm , respectively , in order to their appearance , which is similar to liang et al . , which reported that the mean height of the superior and inferior lingual canals from the lower cortical border were 11.5 ( sd 2.8 ) and 7.4 ( sd 2.4 ) mm , respectively . according to the results of the study , the mean length of the superior lingual canals was 7.83 ( sd 2.25 ) mm and the mean length of the inferior lingual canals was 6.33 ( sd 1.65 ) mm . from the 50 dry mandibles investigated by liang et al . , they showed that the mean length of the superior and inferior lingual canal was 6.8 ( sd 2.3 ) and 6.1 ( sd 2.6 ) mm , respectively . in a previous study performed by liang et al . , they showed that 72% of the canals had courses running downwards to the labial side and 28% of the canals were directed upward to the labial side . in the study we conducted , the majority of the superior lingual canals were running downward to the labial side . and most of the inferior lingual canals were directed upward to the labial side , which is similar to kawai et al . study . these canal directions may explain the slightly vertical - oval morphology of midline lingual foramina . thus , it is probable to realize in intraoral radiographs depending on differences in the angle of the projected x - rays . in images of the anterior region of the mandible obtain using the intra oral bisecting method , the mental spine is generally observed as a radio opaque region and occasionally a radio lucent pit can be observed near the spine . in our result , the course of the superior and inferior lingual canal was approximately constant . we recommend that one can confirm the mandibular lingual canal from intra oral bisecting images when the course of the mandibular lingual canal parallels the projection angle of the x - rays . regarding the vertical angulations of the x - ray beam in anterior bisecting images ( 15 ) , the probability projection of the superior lingual canal section and its distal end ( inferior lingual foramen ) is more than inferior lingual canal , because the x - ray beam is parallel to cortical border of canal . the mean diameter of the superior and inferior lingual foramen in our observation were 1.12 ( sd 0.31 ) and 0.9 ( sd 0.39 ) mm , respectively , while in a previous study they were 0.9 ( sd 0.4 ) mm and 0.8 ( sd 0.4 ) mm , respectively . although smaller canals with a diameter of less than 1 mm are rare in causing a major hematoma , larger canals could be mentioned in the radiologic reports and considered during the preoperative planning procedure . our results showed that the mean diameter of the superior lingual foramen was more than 1 mm . so the superior lingual foramen must be encountered with greater caution during operations to avoid bleeding complication . in our study , two lingual foramens were more frequent ( 52.9% ) , this has also been shown in the previous studies . however , the results are in disagreement with those of liang et al . and tepper et al . studies , in that , they found single foramen was most frequent . only those patients with a single lingual foramen ( 24.5% occurrence in our study ) will benefit from the inferior location of this foramen , allowing deeper flap surgery or implant placement without risk of damage to the canal . our study demonstrated that up to four lingual foramens have been detected , which support those of katakami et al . the data indicate that when there was only a single midline lingual foramen ( 24.5% ) , it was normally above the genial spine . from a clinical view , the location , not the number , of the midline lingual foramina is important to avoid complications . in this study the mean diameter of the opening of the superior and inferior lingual canals at the labial side was 0.68 ( sd 0.17 ) and 0.57 ( sd 0.18 ) mm , respectively . a previous study demonstrated that the mean diameter of the opening of the superior and inferior lingual canals at the labial side was 0.4 ( sd 0.3 ) and 0.5(sd 0.3 ) mm , respectively . from these data , we can suggest that may be there is no significance difference of the mean diameter of the opening of the superior and inferior lingual canals at the labial side between various studies . of 389 consecutively taken cone - beam computed tomograms of the mandible by arx et al . , there was no statistically significant influence on the vertical diameter of the lingual foramina by gender ( p = 0.34 ) or age ( p = 0.45 ) . also according to the results of our study , there were no significant difference on the diameter of lingual foramen by sex and gender . but we demonstrated that there is greater distance between the inferior lingual foramina to the alveolar crest and also superior lingual foramina to the inferior cortex in male population . furthermore , the results indicated that the distance of buccal end of lingual canal to inferior and buccal plate was also greater in size in male population . we showed that there is a significant difference between gender and distance of lingual foramen to alveolar crest . the results indicated that we did not find any moderate or strong correlations between age of the participants and the obtained measurements . but we only found a weak negative correlation between age of the participants and the distance of lower lingual foramen to crest in our study . this indicates that with increase in age the distance of lower lingual foramen to crest decreases . the most probable explanation for this correlation is that the crest has atrophied due to aging . in this present study , we found some variations in mentioned anatomical landmarks in isfahan population in comparison with previous studies . due to these findings , we suggest that according to different anatomical positions and measurement for lingual foramen and its bony canal in every individual , it is important to consider this point during preoperative planning for surgery and especially , for implant placement in the anterior mandible . furthermore , cbct imaging was able to show the anatomical features of the lingual foramen and its bony canal , to avoid post operative complications .

background : some studies have been performed on assessing the anatomical variations of lingual foramen and its bony canals , in many different countries but no study has been performed in iran yet . the purpose of this study is to assess the anatomical variations of lingual foramen and its bony canals with cone - beam computed tomography ( cbct ) imaging in isfahan.materials and methods : this was a cross - sectional study in which cbct images taken from 102 patients referred to the radiology department of head and neck in esfahan ( iran ) university between 2010 and 2011 . the presence of the lingual foramen and its bony canals , the locations , sizes , and length were assessed . the distances between the terminal end of lingual canal at the buccal and lingual side from the inferior border of the mandible and alveolar crest were measured . we also evaluated the effect of patient age and gender on the dimensional measurements of the anatomical landmark mentioned above t test , analysis of variance ( anova ) , and pearson 's correlation were used for statistical analysis and p value lower than 0.05 was considered significant.result:all of the cbct images taken showed the presence of lingual foramen . of all the participants , 52% of them had two foramens in their images . the mean diameters of the upper and lower lingual foramen were 1.12 and 0.9 mm , respectively.conclusion:these anatomical landmarks in isfahan population vary from previous studies . all of the images had at least one lingual foramen which demonstrates high prevalence of this anatomy among isfehanian population . therefore , it is recommended to use cbct imaging for preoperative evaluation prior to installing dental implants .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

PMC4641194

the incidence of b - cell neoplasms in europe has been estimated at approximately 21 per 100,000 . optimization of conventional cytostatic regimens through addition of tumor - specific anti - cd20 monoclonal antibodies ( mabs ) or dose intensification followed by autologous / allogeneic stem cell transplantation has significantly improved treatment outcome of b - cell neoplasms over the last years . however , many patients eventually succumb either to treatment - refractory disease or to severe treatment - related side effects [ 3 , 4 ] . this necessitates the development of target - directed anticancer therapies with increased antitumor efficacy , yet acceptable systemic toxicity . antibody - drug conjugates ( adcs ) harness the targeting function of monoclonal antibodies towards tumor - associated antigens ( taa ) to deliver potent cytotoxic drugs . adcs have progressed to phase iii trials and the first such compounds approved were gemtuzumab ozogamicin and brentuximab vedotin for the treatment of acute myeloid leukemia and relapsed hodgkin and anaplastic large cell lymphoma , respectively . with only modest complete remission rates of 30% and unexpectedly severe postapproval toxicity that in part outweighed its clinical benefit more recently trastuzumab emtansine ( t - dm1 ) has been approved for the treatment of metastasized her2-positive breast cancer . for the treatment of hematologic malignancies several other adcs , targeting cd79b , cd74 , cd33 , cd30 , cd22 , and cd19 , are currently in clinical development . prerequisite for the antitumoral activity of adcs is sufficient cellular internalization of the compound upon taa - binding , followed by the intracellular release of the carried payload . the b - cell lineage restricted receptor cd22 , being overexpressed in the majority of b - cell non - hodgkin lymphomas ( b - nhl ) , as well as in b - cell precursor acute lymphoblastic leukemia ( bcp - all ) , is a particularly attractive target for adc approaches . this is due to the very rapid and sustained internalization of the targeted receptor [ 11 , 12 ] and its absence on hematopoietic stem cells . inotuzumab ozogamicin ( cmc-544 ) , an anti - cd22-calicheamicin adc , has been extensively studied in patients with both indolent and aggressive b - cell nhl as well as acute leukemias . several phase i and ii studies conducted with inotuzumab ozogamicin demonstrated in part highly significant clinical activity across all explored entities . however , in 2013 an ongoing phase iii study in patients with aggressive b - nhl was discontinued since an interim analysis of overall survival demonstrated no statistically significant superiority of cmc-544 in combination with rituximab over the comparator arm . the press release reporting on the study termination concluded that hematologic cancers are a complex group of diseases , with more than 70 different types of lymphomas , leukemias or myelomas that require unique treatment options . therefore , clinical development of anti - cd22 adcs with alternative payloads remains of utmost importance . the murine anti - cd22 igg1 mab rfb4 and a disulfide antibody fragment derivative , dsfv - rfb4 , have been covalently linked to plant toxins or genetically fused to bacterial toxins , respectively [ 1519 ] . from these compounds however , administration of bl22 was associated with severe adverse effects such as immunogenic reactions and in a few cases development of capillary leak syndrome . as a consequence , a higher affinity antibody fragment derivative for linkage to the bacterial toxin has been developed and the compound ( ha22 , cat 8015 ) exhibited a more favorable toxicity profile , yet similar potent activity as its predecessor in a phase i trial in patients with chemotherapy - resistant hairy cell leukemia . valuable payload alternatives to bacterial toxins are ribonucleases from the pancreatic ribonuclease ( rnase ) a superfamily with near absence of immunogenicity . onconase ( onc , ranpirnase ) , a 12 kda basic single - chain protein , originally isolated from oocytes of rana pipiens , kills tumor cells with an ld50 of 10 m which is comparable to the potency of maytansinoids and auristatins . the antitumor effects of onc can be ascribed to trna- [ 24 , 25 ] , dsrna- , and mirna - cleavage [ 26 , 27 ] , as well as to transcriptional gene regulation interactions . in phase i / ii clinical trials for treatment of various solid tumors and malignant mesothelioma onc was immunologically well tolerated and displayed acceptable and reversible systemic toxicity [ 29 , 30 ] . after conjugation to the murine anti - cd22 igg2a - mab ll2 , onc caused only mild off - target toxicity in lymphoma xenografted mice , and the toxic total cumulative dose ( tcd ) was reached only at concentrations of > 300 mg / kg body weight . in comparison , a pseudomonas exotoxin - ll2 immunoconjugate caused 100% lethality in mice at a tcd of 7 mg / kg . thus , onc seems to present a promising payload for anti - cd22 immunoconjugates by combining high antitumor potency and low systemic toxicity . in this study we report on the generation , purification , and in vitro characterization of different onc - based anti - cd22 immunoconjugates . as antibody targeting moiety we reengineered the previously humanized rfb4 scfv into a humanized igg1 . we tested two different chemical conjugation strategies using various crosslinkers for noncleavable and thiol - cleavable linkage and different onc payload formats . sophisticated purification procedures were used for obtaining active adcs with distinct molar drug to antibody ratios ( dars ) . we show that immuno - rnases were able to kill targeted lymphoma and leukemia cells in a dar - dependent manner . all tumor cell lines were purchased from atcc ( manassas , va , usa ) . onconase ( ranpirnase ) was a kind gift from kuslima shogen ( alfacell corporation , new jersey , usa ) . the rnase substrate poly - ru was acquired from amersham biosciences ( little chalfont , uk ) ; the fluorogenic substrate 6-fam - darudgda - bhq-1 ( 6-carboxyfluorescein - darudgda - black - hole - quencher-1 ) was from biomers.net ( ulm , germany ) . fitc- ( fluorescein isothiocyanate- ) conjugated secondary antibodies were obtained from jackson immunoresearch ( laboratories , west grove , pa , usa ) ; the control antibody rfb4 was from santa cruz biotechnology ( dallas , texas , usa ) . benchmark prestained protein ladder , dmem ( dulbecco 's modified eagle 's medium ) , 2-mercaptoethanol , nupage lds sample buffer ( 4x ) , novex himark pre - stained protein standard , novex tris - acetate sds running buffer , and novex nupage 38% tris - acetate gels were purchased from life technologies gmbh ( darmstadt , germany ) . runblue lds sample buffer , runblue rapid sds run buffer , and runblue 12% sds gels were acquired from expedeon ( harston , uk ) . fbs ( fetal bovine serum ) , pbs ( phosphate buffered saline ) , penicillin , rpmi-1640 ( roswell park memorial institute ) medium , sodium azide , and streptomycin were obtained from sigma - aldrich ( st . louis , mo , usa ) . alamarblue , 2-it ( 2-iminothiolane ) , smcc ( succinimidyl 4-(n - maleimidomethyl ) cyclohexane-1-carboxylate ) , spdp ( n - succinimidyl 3-(2-pyridyldithio ) propionate ) , and spectra multicolor broad range protein ladder were procured from thermo fisher scientific ( waltham , ma , usa ) . aps ( ammonium persulfate ) , coomassie brilliant blue r-250 , dmso ( dimethyl sulfoxide ) , dtt ( dithiothreitol ) , g418 ( geneticin disulfate ) , glycerol , isopropanol , mes ( 2-(n - morpholino ) ethanesulfonic acid ) , nacl ( sodium chloride ) , rotiphorese gel 30 acrylamide / bisacrylamide solution , sds ( sodium dodecyl sulfate ) , toluidine blue , tris - hcl ( tris(hydroxymethyl)aminomethane hydrochloride ) , and zellutrans / roth dialysis membranes were provided by carl roth gmbh ( karlsruhe , germany ) . cdm hd ( chemically defined medium high density ) serum replacement and the hollow fiber cell culture bioreactor were purchased from fibercell systems ( frederick , usa ) . amicon ultra centrifugal filter units , edta ( ethylenediaminetetraacetic acid , disodium salt dihydrate ) , and millex - gv sterile filter units were purchased from merck kgaa ( darmstadt , germany ) . n , n , n - tetramethylethylenediamine ) was obtained from fluka biochemika ( buchs , switzerland ) . cell culture plates were purchased from greiner bio - one ( kremsmnster , austria ) . all columns used for purification were provided by ge healthcare ( little chalfont , uk ) . for generation of a humanized anti - cd22 igg1 , variable domain genes of the previously humanized scfv sgiii were synthesized ( entelechon gmbh , bad abbach , germany ) with splice donor and acceptor signal sequences and cloned into eukaryotic expression vectors containing regulatory elements of the immunoglobulin locus , a human constant heavy 1 chain , and a human constant chain , respectively . heavy chain and light chain plasmids of the humanized antibody construct were linearized with ahdi and sfii , respectively , and transfected into sp2/0-ag14 mouse myeloma cells by electroporation ( 230 v , 975 f ) . cells were grown by limiting dilution in selective media ( dmem , 10% fbs , 50 m 2-mercaptoethanol , and 1 mg / ml g418 ) for 2 - 3 weeks to obtain single - cell clones . positive clones were identified by flow cytometric analysis and monitored for igg secretion rates by dot blot using a purified mab as reference standard . the highest producing clone for hurfb4 igg was expanded to t-175 flasks , adapted to high glucose dmem culture media supplemented with 10% cdm hd serum replacement , and subsequently inoculated into a hollow fiber cell culture bioreactor . iggs were purified from cell culture supernatants by protein a chromatography using a hitrap rprotein a ff column and dialyzed against pbs ( ph 7.4 ) . purity was assessed by analytical size exclusion chromatography on a superdex 200 10/300 gl column as 95% . intermolecular protein conjugation of onc and mab hurfb4 was performed using as heterobifunctional cross - linking agents either succinimidyl 4-(n - maleimidomethyl ) cyclohexane-1-carboxylate ( smcc ) or n - succinimidyl 3-(2-pyridyldithio ) propionate ( spdp ) for noncleavable and thiol - cleavable linkage , respectively . smcc and spdp solutions were freshly prepared in dmso and diluted 1 : 10 in pbs ( ph 7.4 ) shortly before use . to generate smcc - based immunoconjugates purified hurfb4 igg antibody ( 1 mg / ml ) was incubated with 40-fold molar excess of smcc in pbs ( ph 7.4 ) containing 5 mm edta for 30 min at room temperature . onc ( 2 mg / ml ) was simultaneously reduced to des(30 - 75)-onc in pbs ( ph 7.2 ) in the presence of 5 mm dtt and 0.2 nm edta for 120 min at 15c . smcc and dtt were removed by dialysis against pbs ( ph 7.4 ) containing 5 mm edta . the maleimide activated antibody was added to a 4.0-fold molar excess of des(30 - 75)-onc and the reaction mixture was incubated for 30 min at room temperature . in order to prepare spdp - based immunoconjugates , purified hurfb4 igg antibody ( 1 mg / ml ) was incubated with a 40-fold molar excess of 2-iminothiolane ( 2-it ) in pbs ( ph 8.0 ) in the presence of 5 mm edta for 60 min at room temperature . onc ( 2 mg / ml ) was simultaneously incubated with a 2.0-fold molar excess of spdp in pbs ( ph 7.4 ) in the presence of 5 mm edta for 60 min at room temperature . excess reagents were removed using pd - minitrap g-25 columns equilibrated with pbs ( ph 7.4 ) containing 5 mm edta . both modified proteins were combined and incubated overnight at 4c for conjugation using a 10-fold molar excess of pyridyldithiol - activated onc . the smcc - based immunoconjugate preparations were subjected to preparative size exclusion chromatography ( sec ) on a superdex 200 10/300 gl column equilibrated and eluted with pbs ( ph 7.4 ) at a flow rate of 0.5 ml / min . preparative sec of the spdp - based immunoconjugate preparations was performed on a hiload 16/60 superdex 200 pg column with a flow rate of 0.3 ml / min using 20 mm mes , 50 mm nacl at ph 6.0 as elution buffer . antibody - onc conjugate populations isolated by sec were subjected to preparative ion exchange chromatography ( iex ) on a mono - s 5/50 gl column at a flow rate of 1 ml / min . spdp immunoconjugates with different onc - mab ratios were separated using 20 mm mes ( ph 6.0 ) with a linear nacl gradient ( 50 mm1 m ) as elution buffer . purified protein samples were concentrated using centrifugal filter units , sterile - filtered , and stored at 4c . purity and identity of concentrated immunoconjugates were analyzed by calibrated analytical sec using a superdex 200 10/300 gl column . concentrations of homogenously purified proteins were calculated from the absorbance at 280 nm measured on a nanodrop nd-1000 spectrophotometer ( thermo fisher scientific , waltham , ma , usa ) using the beer - lambert law with the molar absorption coefficient and the molecular weight calculated for each individual compound . the calculated value for the unmodified igg was 203,900 mcm , for onc 10,010 mcm , and for the chemically linked immuno - rnases carrying one , two , or three onc payloads 213,910 mcm , 223,920 mcm , and 233,930 mcm , respectively . the molecular weight was set at 150,000 g / mol for the unmodified igg , 11,840 g / mol for onc , and 161,840 g / mol ( oar 1 : 1 ) , 173,680 g / mol ( oar 2 : 1 ) , and 185,520 g / mol ( oar 3 : 1 ) for the immunoconjugates . the extinction coefficient and the molecular weight for immunoconjugates with determined sizes of 250290 kda were calculated for an adc with an average payload of 10 rnases and set at 304,000 mcm and 268,400 g / mol , respectively . to compare antigen binding activity , cytotoxicity , and ribonucleolytic activity of generated immunoconjugates , igg and onc determined protein concentrations protein samples were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ) performed under nonreducing conditions on precast 38% tris - acetate gels using tris - acetate buffer and under reducing conditions on precast 12% bis - tris gels using tris - mops buffer followed by detection by staining with coomassie brilliant blue . novex himark pre - stained protein standard and spectra multicolor broad range protein ladder were used as protein standards . coomassie stained protein bands on reducing sds - page gels of spdp - based adcs were captured on an epson perfection v750 pro scanner ( seiko epson corporation , nagano , japan ) and analyzed by photoshop elements 10 software ( adobe systems incorporated , san jose , usa ) . after converting to greyscale and applying the invert filter , mean densities of onc protein bands specific binding of hurfb4 igg was determined by flow cytometry using the human cd22-positive b - cell lines daudi , raji , and ramos and the human cd22-negative t - cell line jurkat . bound hurfb4 igg and chemically linked immuno - rnases were detected using a fitc - conjugated rabbit antibody specific for the fc region of human igg . the mouse monoclonal control antibody rfb4 was detected by staining with an fitc - conjugated goat anti - mouse igg . fluorescence recordings were made on a facscanto ii flow cytometer ( bd biosciences , san jose , ca , usa ) and median fluorescence intensity ( mfi ) was calculated using the facsdiva software ( bd biosciences , san jose , ca , usa ) . background fluorescence was determined using cells incubated with fitc - conjugated secondary antibody under the same conditions . equilibrium - binding curves were determined by incubating 5 10 raji cells in triplicate with serial dilutions of either murine rfb4 , hurfb4 igg , or chemically linked immuno - rnases for 2 h at room temperature in 100 l pbs - facs buffer containing 2% fbs and 0.1% sodium azide . after two washes with 200 l facs buffer bound antibodies were detected as described above . background fluorescence was subtracted from measured median fluorescence and relative affinities were determined according to the levenberg - marquardt algorithm for nonlinear regression using graphpad prism 5.0 ( graphpad software , la jolla , ca , usa ) . ribonucleolytic activity of smcc - based immunoconjugates was investigated by zymogram gel electrophoresis as previously described . briefly , samples were prepared in zymogram loading buffer containing final concentrations of 62.5 mm tris - hcl , ph 6.8 , 10% glycerol , and 2.5% sds and separated under nonreducing conditions on 12% polyacrylamide gels containing 0.3 mg / ml poly - ru in the separating gel as a substrate for onc . after electrophoresis the gel was washed twice using 10 mm tris - hcl , ph 8.0 , containing 20% isopropanol to remove sds , and placed in renaturating buffer containing 100 mm tris - hcl , ph 8.0 for 12 h ( 4c ) . rnase activity was detected by staining with 0.2% ( w / v ) toluidine blue in 10 mm tris - hcl , ph 8.0 for 10 min , followed by destaining with 10 mm tris - hcl , ph 8.0 , until visualization of the ribonucleolytic activity bands was obtained . ribonucleolytic activity of spdp - based immuno - rnases and onc was quantified using the fluorogenic substrate 6-carboxyfluorescein - darudgda - black - hole - quencher-1 ( 6-fam - darudgda - bhq-1 ) . increase in fluorescence after cleavage of substrate was monitored over time using an infinite f200 pro microplate reader ( tecan group , mnnedorf , switzerland ) with a 485/535 nm ( excitation / emission ) filter set . reactions were carried out in black 96-well plates in 100 mm mes - naoh buffer ( ph 6.0 ) containing 100 mm nacl and 6-fam - darudgda - bhq-1 ( 5 nm ) at ( 25 2)c in a total reaction volume of 200 l per well . buffer served as negative control and an excess concentration of rnase a was used as positive control . values of kcat / km were calculated using(1)kcatkm=f/tfmaxf0e , where f/t represents the initial reaction velocity , f0 the initial fluorescence intensity before addition of rnase , fmax the fluorescence intensity after complete cleavage of the substrate by excess rnase a , and [ e ] the rnase concentration . human lymphoma and leukemia cells were maintained in rpmi 1640 supplemented with 10% fetal bovine serum , 100 u / ml penicillin , and 100 g / ml streptomycin . daudi were seeded at a density of 2 10/100 l , while nalm6 and jurkat were seeded at a density of 1 10/100 l into 96-well flat - bottom plates and incubated with various concentrations of protein or buffer as control at 37c , 5% co2 for 72 h in a total volume of 110 l . in order to determine the viability , cells were incubated with 10 l alamarblue per well at 37c , 5% co2 for 4 h. absorbance was measured at 570 nm ( reference : 620 nm ) using an infinite f200 pro microplate reader ( tecan group , mnnedorf , switzerland ) . cell viability was expressed as percentage of viable cells treated with protein related to buffer control . the concentration required to inhibit cell viability by 50% relative to buffer - treated control cells was defined as ic50 ( half maximal inhibitory concentration ) and was determined from semilogarithmic plots . at least two independent assays with each assay containing triplicates were performed . we have previously grafted the specificity of the murine anti - cd22 monoclonal antibody ( mab ) rfb4 into human vh and vl frameworks preselected for stability from a human antibody phage display library . the resulting humanized hurfb4 scfv ( originally designated sgiii ) displayed excellent antigen binding and stability properties . to generate a humanized igg1 derivative ( mab hurfb4 ) from the scfv the variable domain encoding genes of the humanized vl chain and vh chain were cloned into immunoglobulin expression vectors containing a human constant heavy 1 chain and a human constant chain , respectively . the humanized antibody was produced from stably transfected sp2/0 mouse myeloma cell lines under serum - free conditions in a hollow - fiber culture system and purified from culture supernatants to homogeneity by protein a chromatography . the purified hurfb4 igg demonstrated specific binding to the human cd22-positive b - cell lines daudi , raji , and ramos , but no binding to the human cd22-negative t - cell line jurkat ( data not shown ) . flow cytometric affinity measurements confirmed that hurfb4 igg retained the same high apparent affinity of 0.27 0.02 nm as its murine ancestor mab rfb4 ( figure 1 ) . bivalent binding of the humanized igg increased the apparent binding affinity by 36-fold when compared to the parental monovalent scfv hurfb4 . for conjugating onc to hurfb4 igg we first employed the membrane permeable crosslinker succinimidyl 4-(n - maleimidomethyl ) cyclohexane-1-carboxylate ( smcc ) . we therefore reduced onc under mild conditions and simultaneously modified the -amino groups of igg - lysine residues via the n - hydroxysuccinimide ( nhs ) ester reactive group of smcc . the smcc - modified igg was reacted with free accessible sulfhydryl groups of des(30 - 75)-onc via the maleimide group of smcc , generating nonreducible thioether bonds . this conjugation approach yielded 41% nonconjugated igg ( 150 kda ) and 22% multimeric immuno - rnase conjugates ( 300 kda ) ( data not shown ) . although reductive unfolding of onc into a single stable intermediate des(30 - 75)-onc has been described [ 34 , 36 ] , reduction of the 30 - 75 disulfide bond in onc for chemical conjugation has not yet been investigated . to assess if this site of conjugation interferes with the ribonucleolytic activity of onc the 300 kda immunoconjugates were subjected to zymography . although multimeric immunoconjugates were enzymatically active ( figure 2(a ) ) and retained specific antigen binding ( figure 2(b ) ) they were not cytotoxic ( figure 2(c ) ) . therefore we next used the cleavable n - succinimidyl 3-(2-pyridyldithio ) propionate ( spdp ) crosslinker reacting with amino groups of onc . lysine residues of the hurfb4 igg were modified by traut 's reagent ( 2-iminothiolane , 2-it ) to provide an additional sulfhydryl group for subsequent immunoconjugation . preparative size exclusion chromatography ( sec ) of the spdp - immunoconjugate preparations yielded three distinct peaks ( figure 3(a ) ) corresponding to 17.3% adc multimers ( 58.6 ml ) , 70.2% immuno - rnase adcs ( 67.9 ml ) , and 12.5% pyridyldithiol - activated onc excess ( 106.4 ml ) as confirmed by sds - page under nonreducing and reducing conditions ( figures 3(b ) and 3(c ) ) . multimerization ( 300 kda adcs ) was most likely attributed to small amounts of onc molecules carrying two pyridyldithiol reactive groups , resulting in the cross - linking of two 2-it - modified igg molecules . sizes of immuno - rnase adcs were between 162 and 186 kda ( figure 3(b ) ) , corresponding to onc to antibody ratios ( oars ) between 1 : 1 and 3 : 1 . the small differences of the molecular weights of immuno - rnase adcs with distinct oars did not allow a preparative separation by size exclusion chromatography ( figure 3(a ) ) . to separate immuno - rnase adcs by their number of cytotoxic payloads we thus took advantage of the highly basic nature ( pi > 9.5 ) of onc and subjected the conjugate pool eluting at 67.9 ml in sec ( figure 3(a ) ) to a polishing step by cation exchange chromatography . by increasing the ionic strength of the elution buffer slowly from 140 to 300 mm nacl we were able to separate intact mab species with low onc payloads ( figures 4(a)4(c ) ) . immuno - rnase conjugates eluting at 142174 mm nacl , 194221 mm nacl , and 248270 in ion exchange chromatography migrated under nonreducing conditions on sds - page with estimated sizes of 162 kda , 174 kda , and 186 kda , respectively ( figure 4(a ) ) . the average molecular weights of 162 kda , 174 kda , and 186 kda were confirmed by subsequent calibrated sec and correspond to distinct immuno - rnase adcs with oar of 1 : 1 , 2 : 1 , and 3 : 1 , respectively . minor adc multimer contaminants with sizes > 300 kda could be separated by sec ( figure 4(b ) ) . analysis of immuno - rnase conjugates ( defined amount of 1 g each ) on reducing sds - page gels showed an oar depending increase of the staining intensities of onc protein bands ( 12 kda ) , while staining of the protein bands of the respective igg heavy and light chains remained unchanged ( figure 4(c ) ) . when compared to the 162 kda immunoconjugate measured densities of onc bands increased 1.9-fold for the 174 kda adc and 2.5-fold for the 186 kda adc , which corresponds very well to the relative amounts of one , two , or three onc loads per adc of 6.2 pmol , 11.5 pmol , and 16.2 pmol , respectively . it has been shown that conjugation procedures requiring pretreatment of onc with reducing agents such as dithiothreitol ( dtt ) can diminish ribonucleolytic activity up to 60% . by employing a conjugation strategy that avoids reduction of onc we were able to fully maintain the catalytic activity of the ribonuclease in the adc format ( table 1 ) . as a consequence , the 162 kda immuno - rnase adc with an oar of 1 : 1 showed exactly the same ribonucleolytic activity as onc alone . the 174 kda immuno - rnase adc exhibited 1.9-fold increased ribonucleolytic activity according to two onc payloads . the 186 kda immuno - rnase adc carrying three onc moieties exhibited a 2.9-fold higher catalytic efficiency than onc alone . thus , determination of ribonucleolytic activity additionally confirmed the separation of immuno - rnases with oars of 1 : 1 , 2 : 1 , and 3 : 1 . immunoconjugation had no significant impact on the biological activity of the antibody since antigen binding affinities of the immuno - rnase adcs ( kds 0.40.6 nm ) were comparable to the affinity of the native hurfb4 igg ( table 1 ) . specific antitumor activity of the spdp - based immunoconjugates with oars of 1 : 1 , 2 : 1 , and 3 : 1 was tested on human burkitt 's lymphoma daudi and pre - b acute lymphoblastic leukemia nalm6 cells in comparison with cd22-negative human acute t - cell leukemia cell line jurkat . the spdp - based immuno - rnases meditated a dose - dependent cytotoxicity towards targeted cd22-positive tumor cells ( figures 5(a ) and 5(b ) ) but not towards the cd22-negative leukemia cells ( figure 5(c ) ) . incubation of daudi and nalm6 cells with hurfb4 igg alone did not result in any cytotoxicity ( figures 5(a ) and 5(b ) ) . notably , a direct correlation between the cytotoxicity of the immuno - rnase adcs and the number of attached cytotoxic payloads became apparent . as anticipated , cytotoxicity successively increased with the number of conjugated payloads ( table 2 ) . in comparison with onc alone , displaying ic50 values of 1.5 m on daudi cells and 0.5 m on nalm6 cells , targeted delivery of three onc moieties per antibody by the most potent 186 kda adc resulted in enhanced cytotoxicity of 18,400-fold on daudi cells ( ic50 80 pm ) and 3,600-fold on nalm6 cells ( ic50 140 pm ) ( table 2 ) . spdp conjugation generated also conjugates with onc multiplicities eluting in gel filtration chromatography on a calibrated superdex 200 column at retention times correlating to sizes between 250 and 290 kda and most likely representing rnase loads of eight to twelve ( data not shown ) . although this mixture of immunoconjugate species could not be further separated , the effect of higher onc loading on in vitro potency was evaluated and compared with the immunoconjugates carrying low onc payloads ( figure 5(d ) ) . at equal molar doses of 10 nm , immunoconjugates with one , two , and three onc payloads reduced the mean viability of daudi cells by 38% , 64% , and 84% , respectively . although cytotoxic activity correlated with drug loading levels for immunoconjugates with low oar , immuno - rnase adcs with higher onc loadings displayed a significantly decreased cytotoxic activity of only 17% . cytotoxic payloads currently under clinical evaluation in adcs are antimicrotubule agents , dna minor groove binding agents , and alkylating agents . with biological activities in the ng / kg range these compounds represent a major safety challenge both in clinical product manufacturing and in systemic application to cancer patients . the safety and therapeutic index of adcs significantly depend not only on the reproducibility of exact attachment sites and number of attached payloads but also on the linker technology and conjugate homogeneity . improvements in linker stability have in fact accelerated the clinical development of new generation adcs and resulted in the recent approvals of brentuximab vedotin and ado - trastuzumab emtansine , respectively . despite implementation of sophisticated downstream purification protocols conjugation via lysines or cysteines of the antibody results in inherent heterogeneity of the final clinical product with 08 drug payloads per antibody on average . to decrease heterogeneity and achieve uniform drug - loading site - specific drug attachment employment of these technologies has resulted in adcs with defined dar of either two or four payloads per antibody [ 3840 ] . in this study we employed amphibian rnase onc as effector moiety for creating a novel protein - protein adc to target cd22-positive leukemia and lymphoma cells . prerequisite for the full enzymatic activity of onc is the formation of pyroglutamate at its n - terminus through hydrogen bonding with k9 [ 41 , 42 ] . it is therefore important that crosslinking of onc to the antibody moiety must not occur via k9 of the enzyme to preserve the catalytic activity of the ribonuclease . consequently , we explored two different conjugation strategies for analyzing the impact of the nature of the cross - linking bonds , the stoichiometry , and the efficiency of purification procedures for retaining enzymatic activity and cytotoxicity of the immuno - rnase conjugates . studies on the unfolding pathways of onc have shown that onc reductively unfolds via a single stable intermediate des(30 - 75)-onc [ 34 , 36 ] . it has been further shown that an onc variant lacking the disulfide bond 30/75 mimics the unfolding intermediate des(30 - 75)-onc and exhibits comparable cytotoxic properties as wild - type onc . mild reduction of the solvent - accessible c30/c75 disulfide bond of onc was therefore considered a feasible approach for generating active immuno - rnase adcs . although amine - to - sulfhydryl cross - linking with smcc resulted in enzymatically active adcs with sufficient binding activity , a major drawback of this conjugation approach was the uncontrolled formation of higher molecular weight immunoconjugates with impaired matrix - binding on iex . des(30 - 75)-onc carrying two sulfhydryl groups per molecule most likely caused multimerization by cross - linking smcc - modified igg molecules that resulted in abolishment of cytotoxic activity . in contrast , formation of spdp - linked immuno - rnase adcs via lysine residues seemed primarily to be a result of well - controllable 2-it and spdp cross - linking reactions , allowing for a preferential formation of immuno - rnase adcs with favorably low dars . high binding affinity to cd22-positive cells , well - preserved ribonucleolytic activity , and high cd22-specific cytotoxicity in vitro indicate no significant alterations of the molecules in critical regions , namely , the cdrs of the igg , as well as lysine residues k9 and k31 of onc . empirical evidence of random conjugation approaches has shown that the number of attached drugs has a significant impact on target antigen binding , systemic clearance , and antitumor efficacy of immunoconjugates [ 4448 ] . separation of isolated drug load species is at present only possible for dipeptide - linked adcs , such as anti - cd30 brentuximab vedotin through hydrophobic interaction chromatography ( hic ) [ 44 , 4951 ] . we have shown in the present study that iex matrix - binding of spdp - linked immuno - rnase adcs not only allowed for successful purification from nonreacted igg but also yielded homogenous adc species with one , two , and three rnase moieties per antibody molecule . moreover , our data revealed that the number of attached onc payloads is crucial for achieving significant in vitro cytotoxicity towards cd22-positive lymphoma and leukemia cell lines . in this respect , at least two onc payloads were required for achieving significant in vitro cytotoxicity and the spdp - linked immuno - rnase adc with a dar of 3 : 1 was most effective . although reasons for the significantly decreased in vitro potency of immunoconjugates containing eight to twelve onc payloads per anti - cd22 mab were not examined in closer detail our data are in line with data from other antibody - drug conjugates showing that higher drug loads increase the risk of reduced cytotoxic activity most likely due to conjugational involvement of lysine residues within the complementarity determining regions ( cdrs ) of the igg . in addition , it has been observed in mouse xenograft models that higher drug loading levels ( eight drugs per antibody ) can cause an accelerated systemic clearance of adcs which leads to a decreased therapeutic index when compared to conjugates with only four or even only two drugs per antibody . thus , similar to other optimized non - rnase - based adcs , it is apparent that the conjugational design of chemically linked immuno - rnases should aim at a low dar . another option for successfully obtaining immuno - rnases with stoichiometrically defined number of onc cargos through employment of the dock - and - lock method has recently been reported . preferably in a head - to - head comparison advantages and disadvantages of both methodologies should be addressed in future studies . current clinical and preclinical development strategies for cd22-targeting adcs and immunotoxins focus on the use of traditional cytotoxic payloads , such as calicheamicins , auristatins , maytansinoids , and truncated pseudomonas exotoxin . all of these cytotoxic agents have been associated with significant systemic toxicity either due to their off - target release through destabilization of the cross - linking bonds or because of immunogenicity , as in case of pseudomonas exotoxin . payload - dependent hemato- and hepatotoxicities , ranging from mild , reversible transaminasemia to even fatal venoocclusive disease ( vod ) , have been reported for the anti - cd22 calicheamicin immunoconjugate inotuzumab ozogamicin ( cmc-544 ) used in clinical phases i iii for the treatment of patients with relapsed / refractory b - cell malignancies [ 53 , 5658 ] . substantial postapproval safety risks attributed to vod led to the refusal of the marketing authorization in europe for gemtuzumab ozogamicin [ 5 , 59 , 60 ] in 2008 and its voluntary withdrawal from the us in 2010 . despite efforts to obtain a maximal adc stability through introduction of noncleavable thioether bonds or intracellularly cleavable dipeptide bonds preterm payload release with potentially increased toxicity remains a major clinical problem . as with other dipeptide - linked auristatin - based immunoconjugates [ 6163 ] , preliminary results of a phase ii trial of the anti - cd22-mmae immunoconjugate pinatuzumab vedotin in combination with rituximab in patients with relapsed / refractory non - hodgkin lymphoma reported on significant payload - related systemic toxicities including neutropenia and peripheral neuropathy . since similar toxicities were also common among patients within phase i / ii trials of naked auristatin payloads [ 65 , 66 ] , deconjugation of the payload from the antibody with systemic release of the neurotoxic microtubule inhibitors can thus be deduced . payload release from presumably noncleavable thioether bonds has been also demonstrated : during the thioether fragmentation reaction the cytotoxic moieties were shown to be transferred to the unpaired c34 cysteine residue of serum albumin . identification of albumin as a covariate affecting the pharmacokinetics of the recently fda - approved smcc - linked anti - her2 trastuzumab emtansine ( t - dm1 ) might explain at least in part payload - related systemic toxicities observed in late clinical development . lately , application of self - hydrolyzing maleimide drug linkers has been shown to reduce off - target bone marrow toxicities in rats through enhanced adc stability , which holds promise for future clinical development of such adcs . while adcs under current development represent a unique treatment option they create a series of challenges in engineering , chemistry , and safety . alternative payloads with reversible , easily manageable systemic toxicities , yet also high antitumoral efficacy , such as onc in the present study , represent valuable alternatives . in summary , we have developed novel spdp - linked immuno - rnase adcs with stoichiometrically defined number of cytotoxic onc payloads for the targeted therapy of cd22 malignancies . we have shown for the first time that the number of attached onc payloads well correlates with the tumor - specific cytotoxicity of the adc . because of their highly specific toxicity towards targeted tumor cells , a fairly well - controllable conjugation and purification procedure , and expected favorable safety and immunogenicity profile we believe that further preclinical development of the 3 : 1 dar spdp - linked immuno - rnase adc is warranted .

antibody - drug conjugates ( adcs ) have evolved as a new class of potent cancer therapeutics . we here report on the development of adcs with specificity for the b - cell lineage specific ( surface ) antigen cd22 being expressed in the majority of hematological malignancies . as targeting moiety a previously generated humanized anti - cd22 single - chain variable fragment ( scfv ) derivative from the monoclonal antibody rfb4 was reengineered into a humanized igg1 antibody format ( hurfb4 ) . onconase ( ranpirnase ) , a clinically active pancreatic - type ribonuclease , was employed as cytotoxic payload moiety . chemical conjugation via thiol - cleavable disulfide linkage retained full enzymatic activity and full binding affinity of the adc . development of sophisticated purification procedures using size exclusion and ion exchange chromatography allowed the separation of immunoconjugate species with stoichiometrically defined number of onconase cargos . a minimum of two onconase molecules per igg was required for achieving significant in vitro cytotoxicity towards lymphoma and leukemia cell lines . antibody - drug conjugates with an onconase to antibody ratio of 3 : 1 exhibited an ic50 of 0.08 nm , corresponding to more than 18,400-fold increased cytotoxicity of the adc when compared with unconjugated onconase . these results justify further development of this adc as a promising first - in - class compound for the treatment of cd22-positive malignancies .

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

PMC4915315

there are 2 major pathological types of lung cancer , which account for approximately 85% of lung cancers : small cell lung cancer ( sclc ) and non - small cell lung cancer ( nsclc ) . lung cancer can be further divided into 3 histological types : squamous cell carcinoma , adenocarcinoma , and large cell lung cancer . currently , the primary treatment method for lung cancer is surgery , supplemented by comprehensive treatment with chemotherapy , radiation therapy , and targeted therapy . lung cancer does not usually cause any symptoms in the early stages of the disease , and thus is often diagnosed in late stages . therefore , the identification of early diagnostic markers is important for the diagnosis and treatment of lung cancer . chemokines are a family of small proteins of molecular weights ranging from 8 to 17 kda , with chemotactic activity . studies have shown that chemokines guide cell migration along a concentration gradient during inflammatory reactions . the chemokine superfamily can be divided into the following branches : c , cc , cxc , and cxxxc . chemokines play important roles in the occurrence and development of cancers due to their chemotactic activity in cancer cells xcl2 is a small protein of the xc chemokine family , and is closely associated with another chemokine , xcl1 . it is mainly expressed in activated t cells , and is also expressed at low levels in inactivated t cells . in contrast to the typical structure of other chemokines , cx3cl1 has different spacing of the characteristic n - terminal cysteines . there are 3 amino acids separating the initial pair of cysteines in cx3cl1 , with none in cc chemokines and only 1 intervening amino acid in cxc chemokines . soluble cx3cl1 has the ability to recruit t cells and monocytes , and thus enhances binding and adhesion capacity of cells . studies have shown that cx3cl1 is highly expressed in the hippocampus region during short - term memory , which may be regulated by glutamatergic neurotransmission . the interaction between cx3cl1 and glutamate led us to investigate the expression of xcl2 and cx3cl1 in lung cancer . a total of 38 tumor samples were collected from 18 male and 20 female patients with nsclc who underwent surgical resection in zhejiang provincial people s hospital between july 2014 and december 2014 . based on the tnm classification for lung cancer staging ( 2009 ) by the union for international cancer control ( uicc ) , the clinical staging of selected patients was as follows : 23 cases of squamous cell carcinoma , and 15 cases of adenocarcinoma ; tem staging : 21 cases of stage i ii , and 17 cases of stage iii iv ; lymph node metastasis for lung cancer staging : 24 cases of n0 , 7 cases of n1 , and 7 cases of n2 . the study protocol was approved by the research ethics committee of zhejiang provincial people s hospital , and all patients gave their informed consent before study commencement . xcl2 and cx3cl1 antibodies were purchased from cell signaling technology ( ( beverly , ma , usa ) . total rna was extracted using trizol rna extraction kit ( invitrogen ; carlsbad , ca , usa ) according to the manufacture s instructions . aliquots of tissue or serum were mixed with equal amounts of trizol , and centrifuged at 12 000 rpm at 4c for 10 min . the supernatant was transferred into a new eppendorf ( ep ) tube , chilled on ice for 5 min , and mixed with 200 l of chloroform . the mixture was vortexed for 15 s and centrifuged at 12 000 rpm at 4c for 15 min . the top layer was transferred into a new ep tube and mixed with an equal volume of isopropanol . the mixture was allowed to precipitate at room temperature for 10 min , and centrifuged at 12 000 rpm / min at 4c for 10 min . the remnant was mixed with 75% ethanol and centrifuged at 7500 rpm at 4c for 5 min . rna was air - dried and dissolved in 20 l of depc - treated water . rnas were then transcribed into cdna , which was further amplified using a one - step rt - pcr kit . the reaction conditions were 94c for 45 s , followed by 35 cycles of refolding at 59c for 45 s and extension at 72c . briefly , sections were treated sequentially with xylene , absolute ethanol , and 95% , 90% , 85% , and 80% ethanol for 10 min each . the sections were then incubated with secondary antibodies , stained with hematoxylin solution , soaked in 1% hydrochloric acid alcohol , rinsed with running water anti - blue , dehydrated with an ethanol gradient , treated with xylene for transparency , and mounted with neutral gum . cytoplasm of xcl2- and cxc3cl1-positive cells were positively stained ( colored yellow or brown ) . the positive rate was calculated as the ratio of the number of positive cells to the area of visual fields using image j software , and compared to determine the difference in the expression level of both proteins . after 90 min of reaction at 37c , 100 l of biotinylated antibody was added to each well and allowed to react at 37c for 60 min . after 30 min of reaction at 37c , the plate was rinsed 5 times with 0.01 m tbs . the plate was incubated with tmb at 37c in the dark and the reaction was terminated by adding tmb stop solution after 30 min . all tests were repeated at least 3 times and are presented as mean standard deviation . a total of 38 tumor samples were collected from 18 male and 20 female patients with nsclc who underwent surgical resection in zhejiang provincial people s hospital between july 2014 and december 2014 . based on the tnm classification for lung cancer staging ( 2009 ) by the union for international cancer control ( uicc ) , the clinical staging of selected patients was as follows : 23 cases of squamous cell carcinoma , and 15 cases of adenocarcinoma ; tem staging : 21 cases of stage i ii , and 17 cases of stage iii iv ; lymph node metastasis for lung cancer staging : 24 cases of n0 , 7 cases of n1 , and 7 cases of n2 . the study protocol was approved by the research ethics committee of zhejiang provincial people s hospital , and all patients gave their informed consent before study commencement . xcl2 and cx3cl1 antibodies were purchased from cell signaling technology ( ( beverly , ma , usa ) . total rna was extracted using trizol rna extraction kit ( invitrogen ; carlsbad , ca , usa ) according to the manufacture s instructions . aliquots of tissue or serum were mixed with equal amounts of trizol , and centrifuged at 12 000 rpm at 4c for 10 min . the supernatant was transferred into a new eppendorf ( ep ) tube , chilled on ice for 5 min , and mixed with 200 l of chloroform . the mixture was vortexed for 15 s and centrifuged at 12 000 rpm at 4c for 15 min . the top layer was transferred into a new ep tube and mixed with an equal volume of isopropanol . the mixture was allowed to precipitate at room temperature for 10 min , and centrifuged at 12 000 rpm / min at 4c for 10 min . the remnant was mixed with 75% ethanol and centrifuged at 7500 rpm at 4c for 5 min . rna was air - dried and dissolved in 20 l of depc - treated water . rnas were then transcribed into cdna , which was further amplified using a one - step rt - pcr kit . the reaction conditions were 94c for 45 s , followed by 35 cycles of refolding at 59c for 45 s and extension at 72c . briefly , sections were treated sequentially with xylene , absolute ethanol , and 95% , 90% , 85% , and 80% ethanol for 10 min each . the sections were then incubated with secondary antibodies , stained with hematoxylin solution , soaked in 1% hydrochloric acid alcohol , rinsed with running water anti - blue , dehydrated with an ethanol gradient , treated with xylene for transparency , and mounted with neutral gum . cytoplasm of xcl2- and cxc3cl1-positive cells were positively stained ( colored yellow or brown ) . the positive rate was calculated as the ratio of the number of positive cells to the area of visual fields using image j software , and compared to determine the difference in the expression level of both proteins . after 90 min of reaction at 37c , 100 l of biotinylated antibody was added to each well and allowed to react at 37c for 60 min . after 30 min of reaction at 37c , the plate was rinsed 5 times with 0.01 m tbs . the plate was incubated with tmb at 37c in the dark and the reaction was terminated by adding tmb stop solution after 30 min . all tests were repeated at least 3 times and are presented as mean standard deviation . as shown in figure 1a and 1b , qrt - pcr analyses demonstrated that the expression of xcl2 and cx3cl1 mrna in lung cancer was significantly higher than that in normal tissues ( p<0.05 ) . it was shown that the expression of both chemokines in lung cancer was also significantly increased compared with normal tissues ( p<0.05 , figure 1c , 1d ) . it was shown that the expression level of xcl2 was significantly increased with higher pathological stages ( p<0.05 or p<0.001 , figure 2a2f ) . xcl2 expression in cancers with higher numbers of metastatic lymph nodes was also significantly increased compared with cancers with lower numbers of metastatic lymph nodes ( p<0.05 or p<0.001 , figure 2 g ) . moreover , xcl2 expression in t4 lung cancer was significantly higher compared with t3 cancer ( p<0.05 , figure 2h ) . results of immunohistochemical analyses showed that cx3cl1 expression was significantly increased with increasing pathological stages ( p<0.05 , figure 3a3f ) . cx3cl1 expression in n1 lung cancer was also significantly higher compared with n0 cancer ( p<0.001 , figure 3 g ) . moreover , cx3cl1 expression in t4 lung cancer was significantly higher compared with t3 cancer ( p<0.05 , figure 3h ) . as shown in figure 1a and 1b , qrt - pcr analyses demonstrated that the expression of xcl2 and cx3cl1 mrna in lung cancer was significantly higher than that in normal tissues ( p<0.05 ) . it was shown that the expression of both chemokines in lung cancer was also significantly increased compared with normal tissues ( p<0.05 , figure 1c , 1d ) . it was shown that the expression level of xcl2 was significantly increased with higher pathological stages ( p<0.05 or p<0.001 , figure 2a2f ) . xcl2 expression in cancers with higher numbers of metastatic lymph nodes was also significantly increased compared with cancers with lower numbers of metastatic lymph nodes ( p<0.05 or p<0.001 , figure 2 g ) . moreover , xcl2 expression in t4 lung cancer was significantly higher compared with t3 cancer ( p<0.05 , figure 2h ) . results of immunohistochemical analyses showed that cx3cl1 expression was significantly increased with increasing pathological stages ( p<0.05 , figure 3a3f ) . cx3cl1 expression in n1 lung cancer was also significantly higher compared with n0 cancer ( p<0.001 , figure 3 g ) . moreover , cx3cl1 expression in t4 lung cancer was significantly higher compared with t3 cancer ( p<0.05 , figure 3h ) . the occurrence and development of lung cancer is a multi - step , multi - factor process involving the deactivation of tumor suppressor genes and activation of oncogenes . numerous studies have shown that chemokines play important regulatory roles in the pathogenesis of a variety of tumors , as well as in the proliferation , apoptosis , and signal transduction of tumor cells by acting as either tumor suppressors or promoters . wang et al . demonstrated that serum chemokine levels in patients with nsclc are higher compared with healthy controls . jia et al . reported that chemokine concentration in nsclc patients was lower than in patients with benign lung cancer . wu et al . found that chemokine expression in nsclc is associated with tnm staging , lymph node metastasis , survival rate , and overall response rate . it has also been shown that the expression of chemokines is closely related to tnm staging and distant metastasis of nsclc . these findings suggest that the changes in chemokine expression are strongly associated with the pathological stages of lung cancer . chemokines play important roles in the occurrence and development of cancers due to their chemotactic effect on cancer cells . studies have demonstrated that ccr1 , the ccl5 receptor , and cx3cl1 synergistically induce liver metastases from colorectal cancer . high expression of xcl2 has been reported in a variety of cancers , including melanoma , nsclc , prostate cancer , and colorectal cancer . in our study , the expression of xcl2 mrna and protein in lung cancer was significantly higher compared with normal tissues . found that xcl2 is overexpressed in lung cancer and is associated with the prognosis of the disease . liu et al . demonstrated that cx3cl1 expression is gradually increased in benign lung tissue , primary lung tumors , and lymph node metastases , and that the overall survival rate of patients with stronger cx3cl1 expression is much lower compared with those with weaker cx3cl1 expression . in a study of cx3cl1 expression in nsclc , howard et al . demonstrated that cx3cl1 expression in cancer tissues is markedly higher than that in adjacent normal tissues it has also been shown that serum cx3cl1 expression is positively correlated with both serum vegf expression and microvessel density in tumors , suggesting that cx3cl1 overexpression is closely associated with the occurrence and development of lung cancer . in our study , in summary , the current study demonstrated that xcl2 and cx3cl1 expression in lung cancers were significantly higher compared to adjacent normal tissues . our findings indicate that both chemokines might be important targets in gene therapy for lung cancer , and their antagonists might have an anti - lung cancer effect .

backgroundchemokines are a family of small proteins secreted by cells with chemotactic activity , and they play important roles in cell adhesion . however , the expression of chemokine xcl2 and cx3cl1 in lung cancers in different pathological stages remains unclear.material/methodsxcl2 and cx3cl1 expression in lung cancers and adjacent non - cancerous tissues was detected by quantitative pcr and elisa . the relative expression of both chemokines in lung cancers in different pathological stages was compared by immunohistochemical assay.resultsthe relative expression level of xcl2 and cx3cl1 in lung cancer was significantly higher compared with adjacent normal tissues ( p<0.001 ) . the expression level of both chemokines was significantly increased with higher pathological stages , as indicated by immunohistochemical assay ( p<0.05 or p < 0.001 ) . their expression level in cancers with higher numbers of metastatic lymph nodes was also significantly increased compared with cancers with lower numbers of metastatic lymph nodes ( p<0.05 or p<0.001).conclusionsthe expression of xcl2 and cx3cl1 increases with increasing degree of malignancy , indicating that both chemokines might be important targets in gene therapy for lung cancer .

Background Material and Methods Sample collection Reagents Quantitative reverse transcription PCR (qRT-PCR) Immunohistochemical staining of XCL2 and CX3CL1 in lung cancer ELISA Statistical analysis Results XCL2 and CX3CL1 expression in lung cancer Comparison of XCL2 expression in lung cancers in different pathological stages Comparison of CX3CL1 expression in lung cancers in different pathological stages Discussion Conclusions

PMC4643172

because of an increased detection rate of small renal masses ( 4 cm or less ) , partial nephrectomy ( pn ) as a nephron - sparing surgery ( nss ) has been conducted in a number of centers as the gold standard of care . in cautiously selected patients with tumor sizes of < 7 cm , pn can be considered feasible with superior renal functional preservation and comparable oncologic and survival outcomes to radical nephrectomy . currently , the application of pure laparoscopic or robotic approaches for minimally invasive nephron - sparing surgery ( minss ) has emerged as an alternative modality to the existing open approach because minss can provide several additional advantages , such as better cosmetic results , shorter periods of convalescence , and decreased blood loss , as well as comparable oncologic and functional outcomes . despite advantages with laparoscopic partial nephrectomy ( lpn ) and robot - assisted laparoscopic partial nephrectomy ( ralpn ) , previously , we had devised the sliding loop technique ( slt ) by modifying the conventional sliding clip technique introduced by benway et al . , and established that slt was feasible for renorrhaphy in a porcine model . the comparative analysis between the two techniques showed that slt is superior to the sliding clip technique in terms of lowered risks of renal parenchymal dehiscence , as it allows more tension following renorrhaphy . in this paper , based on our feasibility experiments in animal models , we attempted to verify the clinical feasibility and safety of slt renorrhaphy in patients undergoing ralpn . this study design and the use of patients ' information stored in the hospital database were approved by the institutional review board ( irb ) at the seoul national university hospital ( snuh ) ( irb no . we were given exemption from obtaining informed consent by the irb because the present study is a retrospective study and personal identifiers were completely removed and the data were analyzed anonymously . our study was conducted according to the ethical standards laid down in the 1964 declaration of helsinki and its later amendments . we collected and reviewed the electronic medical records and surgical videos of 31 patients who had undergone ralpn for small renal masses by two surgeons ( cwj and ck ) at snuh between january 2014 and october 2014 . the decision to perform ralpn was mainly determined according to tumor characteristics and patient 's individual preferences , so patients were not randomized based on distinct institutionalized standards or guidelines . under general anesthesia , then , the patients were turned to the flank position at approximately 60 degrees . in all cases , four robotic arm approaches , consisting of a 12-mm camera ( 30 degree downward lens ) port and three robotic working ports ( 8 mm ) were commonly used with two additional trocars for assistance , consisting of a 12-mm trocar located at the periumbilical area and a 5-mm trocar located at a point about 8 cm toward the caudal side from where camera port was used . the placement of trocars and their locations in ralpn is presented in fig . 1 . gerota 's fascia was incised and the surrounding perirenal fat was removed to expose the renal mass . after the exact margin and depth of the tumor was confirmed with an intraoperative flexible ultrasound , the resection margin was outlined with electrocautery , with consideration for a sufficient margin of normal parenchymal tissue around the tumor . mannitol ( 0.5 mg / kg ) was intravenously administered before hilar vessel clamping to prevent ischemic renal injury . after clamping of the renal artery with laparoscopic bulldog clamps , tumor excision was performed with cold scissors . the excised tumor was located above the liver or spleen for later retrieval using endobag at the end of surgery . after replacing the equipment of the 1st and 2nd robotic arms with robotic needle drivers , in order to ensure hemostasis and to repair any collecting system openings , bed suturing of the tumor resection site was conducted with continuous running suture using about 15 cm of 3 - 0 v - loc 90 ( covidien , dublin , ireland ) barbed suture material ( fig . following bed suturing , biologic hemostatic tissue sealant ( i.e. , fibrilla ) was applied to the parenchymal defect site . the slt used for renorrhaphy in our study was a modification of the conventional sliding clip technique . however , the setting up of suture material and the method of creating tension are slightly different between slt and the sliding clip technique . we designed a suture material of approximately 13 cm in length with a loop , which had a length of about 1 cm after being tied three times at the end of each 1 - 0 vicryl ( ethicon inc . , somerville , nj , usa ) thread , and placed a tfe polymer pledget ( 7.0 mm3.0 mm ) ( ethicon inc . ) and a 10-mm weck hem - o - lok ( teleflex , research triangle , nc , usa ) clip below the knot ( fig . each prepared suture material was inserted through the renal capsule at intervals of approximately 1 cm , and the needle was passed through the premade loop ( fig . 2b ) . by pulling the thread toward the direction of needle passage with a robotic needle driver , then , the surgeon pushed the hem - o - lok clip perpendicularly toward the renal parenchyma using another robotic needle driver to tighten the renorrhaphy ( fig . 2c ) clips were placed at each thread just above the second hem - o - lok clip to maintain the tension of sutures ( fig . 2 ; video clip , supplementary material ) , secure tightness of renal parenchymal suturing can be obtained . following renorrhaphy , the renal artery was unclamped by a laparoscopic bulldog remover and the tumor resection bed was examined to check for additional bleeding . the excised tumor and perirenal fat were placed in an endobag and extracted via the 12-mm assistance port , which may be extended in cases of larger tumors . all trocars were removed under direct laparoscopic vision , and the fascia at the 12-mm camera and 12-mm port sites was closed with a thick nonabsorbable suture material to prevent the risk of herniation . this study design and the use of patients ' information stored in the hospital database were approved by the institutional review board ( irb ) at the seoul national university hospital ( snuh ) ( irb no . we were given exemption from obtaining informed consent by the irb because the present study is a retrospective study and personal identifiers were completely removed and the data were analyzed anonymously . our study was conducted according to the ethical standards laid down in the 1964 declaration of helsinki and its later amendments . we collected and reviewed the electronic medical records and surgical videos of 31 patients who had undergone ralpn for small renal masses by two surgeons ( cwj and ck ) at snuh between january 2014 and october 2014 . the decision to perform ralpn was mainly determined according to tumor characteristics and patient 's individual preferences , so patients were not randomized based on distinct institutionalized standards or guidelines . under general anesthesia , a nasogastric tube and foley catheter were inserted in the supine position . then , the patients were turned to the flank position at approximately 60 degrees . in all cases , basically , four robotic arm approaches , consisting of a 12-mm camera ( 30 degree downward lens ) port and three robotic working ports ( 8 mm ) were commonly used with two additional trocars for assistance , consisting of a 12-mm trocar located at the periumbilical area and a 5-mm trocar located at a point about 8 cm toward the caudal side from where camera port was used . gerota 's fascia was incised and the surrounding perirenal fat was removed to expose the renal mass . after the exact margin and depth of the tumor was confirmed with an intraoperative flexible ultrasound , the resection margin was outlined with electrocautery , with consideration for a sufficient margin of normal parenchymal tissue around the tumor . mannitol ( 0.5 mg / kg ) was intravenously administered before hilar vessel clamping to prevent ischemic renal injury . after clamping of the renal artery with laparoscopic bulldog clamps , tumor excision was performed with cold scissors . the excised tumor was located above the liver or spleen for later retrieval using endobag at the end of surgery . after replacing the equipment of the 1st and 2nd robotic arms with robotic needle drivers , in order to ensure hemostasis and to repair any collecting system openings , bed suturing of the tumor resection site was conducted with continuous running suture using about 15 cm of 3 - 0 v - loc 90 ( covidien , dublin , ireland ) barbed suture material ( fig . 2a ) . following bed suturing , biologic hemostatic tissue sealant ( i.e. , fibrilla ) the slt used for renorrhaphy in our study was a modification of the conventional sliding clip technique . however , the setting up of suture material and the method of creating tension are slightly different between slt and the sliding clip technique . we designed a suture material of approximately 13 cm in length with a loop , which had a length of about 1 cm after being tied three times at the end of each 1 - 0 vicryl ( ethicon inc . , somerville , nj , usa ) thread , and placed a tfe polymer pledget ( 7.0 mm3.0 mm ) ( ethicon inc . ) and a 10-mm weck hem - o - lok ( teleflex , research triangle , nc , usa ) clip below the knot ( fig . each prepared suture material was inserted through the renal capsule at intervals of approximately 1 cm , and the needle was passed through the premade loop ( fig . by pulling the thread toward the direction of needle passage with a robotic needle driver , a second hem - o - lok clip was placed on the thread . then , the surgeon pushed the hem - o - lok clip perpendicularly toward the renal parenchyma using another robotic needle driver to tighten the renorrhaphy ( fig . 2c ) clips were placed at each thread just above the second hem - o - lok clip to maintain the tension of sutures ( fig . 2 ; video clip , supplementary material ) , secure tightness of renal parenchymal suturing can be obtained . following renorrhaphy , the renal artery was unclamped by a laparoscopic bulldog remover and the tumor resection bed was examined to check for additional bleeding . the needle was cut and extracted . the excised tumor and perirenal fat were placed in an endobag and extracted via the 12-mm assistance port , which may be extended in cases of larger tumors . all trocars were removed under direct laparoscopic vision , and the fascia at the 12-mm camera and 12-mm port sites was closed with a thick nonabsorbable suture material to prevent the risk of herniation . under general anesthesia , a nasogastric tube and foley catheter were inserted in the supine position . then , the patients were turned to the flank position at approximately 60 degrees . in all cases , basically , four robotic arm approaches , consisting of a 12-mm camera ( 30 degree downward lens ) port and three robotic working ports ( 8 mm ) were commonly used with two additional trocars for assistance , consisting of a 12-mm trocar located at the periumbilical area and a 5-mm trocar located at a point about 8 cm toward the caudal side from where camera port was used . gerota 's fascia was incised and the surrounding perirenal fat was removed to expose the renal mass . after the exact margin and depth of the tumor was confirmed with an intraoperative flexible ultrasound , the resection margin was outlined with electrocautery , with consideration for a sufficient margin of normal parenchymal tissue around the tumor . mannitol ( 0.5 mg / kg ) was intravenously administered before hilar vessel clamping to prevent ischemic renal injury . after clamping of the renal artery with laparoscopic bulldog clamps , tumor excision was performed with cold scissors . the excised tumor was located above the liver or spleen for later retrieval using endobag at the end of surgery . after replacing the equipment of the 1st and 2nd robotic arms with robotic needle drivers , in order to ensure hemostasis and to repair any collecting system openings , bed suturing of the tumor resection site was conducted with continuous running suture using about 15 cm of 3 - 0 v - loc 90 ( covidien , dublin , ireland ) barbed suture material ( fig . 2a ) . following bed suturing , biologic hemostatic tissue sealant ( i.e. , fibrilla ) was applied to the parenchymal defect site . the slt used for renorrhaphy in our study was a modification of the conventional sliding clip technique . however , the setting up of suture material and the method of creating tension are slightly different between slt and the sliding clip technique . we designed a suture material of approximately 13 cm in length with a loop , which had a length of about 1 cm after being tied three times at the end of each 1 - 0 vicryl ( ethicon inc . , somerville , nj , usa ) thread , and placed a tfe polymer pledget ( 7.0 mm3.0 mm ) ( ethicon inc . ) and a 10-mm weck hem - o - lok ( teleflex , research triangle , nc , usa ) clip below the knot ( fig . each prepared suture material was inserted through the renal capsule at intervals of approximately 1 cm , and the needle was passed through the premade loop ( fig . 2b ) . by pulling the thread toward the direction of needle passage with a robotic needle driver , then , the surgeon pushed the hem - o - lok clip perpendicularly toward the renal parenchyma using another robotic needle driver to tighten the renorrhaphy ( fig . 2c ) clips were placed at each thread just above the second hem - o - lok clip to maintain the tension of sutures ( fig . 2 ; video clip , supplementary material ) , secure tightness of renal parenchymal suturing can be obtained . following renorrhaphy , the renal artery was unclamped by a laparoscopic bulldog remover and the tumor resection bed was examined to check for additional bleeding . the excised tumor and perirenal fat were placed in an endobag and extracted via the 12-mm assistance port , which may be extended in cases of larger tumors . all trocars were removed under direct laparoscopic vision , and the fascia at the 12-mm camera and 12-mm port sites was closed with a thick nonabsorbable suture material to prevent the risk of herniation . the characteristics and perioperative outcomes of the study cohort are listed in table 1 . mean age and body mass index ( bmi ) were 48.5 years ( range , 31 - 74 years ) and 25.3 kg / m ( range , 19.9 - 39.2 kg / m ) , respectively . a total of 16 and 15 tumors were located on right and left side , respectively . mean operative time and console time were 163.3 minutes ( range , 90 - 240 minutes ) and 114.8 minutes ( range , 55 - 180 minutes ) , respectively . mean renorrhaphy time ( rt ) , which was defined as the interval from the completion of bed suturing to renal artery declamping , was 9.0 minutes ( range , 5 - 24 minutes ) . warm ischemic time ( wit ) showed a mean value of 22.6 minutes , ranging from 13 to 51 minutes . mean estimated blood loss was 330.7 ml ( range , 50 - 1,100 ml ) . slt renorrhaphy was successfully conducted in all patients without conversion to robotic radical nephrectomy or open surgery . af ter renorrhaphy , there were no adverse events , such as dehiscence of approximated renal parenchyma , renal parenchymal tearing , and significant bleeding in any patients . mean preoperative serum creatinine ( cr ) level and estimated glomerular filtration rate ( egfr ) were 0.91 mg / dl and 87.5 ml / min/1.73 m , respectively , while their values at 1 month postsurgery were 0.93 mg / dl and 83.9 ml / min/1.73 m. however , there was no statistically significant perioperative change in either serum cr ( p=0.184 ) and egfr ( p=0.173 ) . there were 3 cases ( 9.7% ) of intraoperative transfusions ( clavien - dindo grade ii ) with a mean of two packed red cell blood units . however , there were no severe postoperative complications , such as delayed hemorrhage and urine leakage , as of the latest follow - up in all patients . for the past several years , open partial nephrectomy ( opn ) has been established as the preferred therapeutic modality for the management of small localized renal masses , providing comparable oncologic and survival outcomes , and superior benefit for the preservation of renal function to radical nephrectomy . since the introduction of minss using pure laparoscopic or robotic approaches in the urologic field , pn applying these minimally invasive techniques has been conducted by experienced surgeons in several centers and shown to have comparable outcomes to opn with relatively better cosmetic benefits and improved convalescence . in particular , ralpn is an emerging procedure that can provide many potential advantages , such as three - dimensional images , magnified vision , prevention of hand tremor , and fully articulating instruments . therefore , ralpn has become regarded as a viable means of surmounting the technical limitations related to lpn . even though minss has its advantages renorrhaphy should be performed in a time - sensitive manner due to its impact on wit . in any case , the shortening of wit is generally considered as a crucial factor for the preservation of renal function in the context of pn . renal reconstruction after tumor excision in opn has usually been performed with traditional tied - suture renorrhaphy . under direct vision through the open approach , knot tied - suturing for renorrhaphy can be easily performed while maintaining the suture line tension with direct hand control under acceptable wit . under the limitation of wit , intracoporeal knot tied - suture renorrhaphy in minss may be technically difficult and time - consuming even for skillful laparoscopic surgeons because it is necessary to control the needle and proceed with renorrhaphy with the dominant hand while maintaining the suture tension with the nondominant hand . therefore , to simplify renorrhaphy and reduce wit in minss , many knotless suturing techniques for renorrhaphy have been developed and applied in clinical practice . in particular , because knotless suturing with tight tension can be accomplished using several surgical clips including lapraty and hem - o - lok clips , renorrhaphy techniques using these tools have been studied by a number of investigators . orvieto et al . used simplified surgical techniques , including closure of the renal defect using lapraty clips , for 41 patients who received lpn and reported a wit of 29.7 minutes , but there were three cases of open conversion and a complication rate of 13.2% . v - hilar suture renorrhaphy , which is a form of knotless suturing using hem - o - lok clips , has been applied for renal hilar tumors in ralpn and shown to provide feasible results . recently , unidirectional barbed suture material ( v - loc ) has been applied to renorrhaphy in lpn and ralpn , and shown promising results in terms of reducing wit by approximately 20% in comparison with conventional polyglactin ( vicryl ) running sutures . furthermore , as demonstrated by our study , barbed suture materials can be used for intracorporeal repair of renal parenchymal defects in minss . reported the comparative results between two renorrhaphy methods , which are conventional one layer , interrupted , figure - of - eight ( olif ) suture and two layer , continuous , unknotted ( tlcu ) suture using hem - o - lok clip , in retroperitoneal lpn . it was revealed that tlcu renorrhaphy could provide better several advantages than olif renorrhaphy in terms of wit , hospital stay , and preservation of renal function measured by egfr . contrary to the renorrhaphy approaches mentioned above , " off - clamp , non - renorrhaphy " technique was more recently introduced in lpn . in this technique , biologic hemostatic agents such as floseal and tisseel are used in the bed of the excised mass as a hemostatic method following renal mass excision without renal artery clamping , and the perirenal fat and gerota 's fascia reapproximation are performed using 3 - 0 vicryl continuous sutures without renorrhaphy . although zero ischemia time was achieved , a high postoperative complication rate ( 25% ) , including delayed bleeding requiring blood transfusion , urine leak , and perirenal abscess , may be a concern . the slt applied to renorrhaphy in our study was a modification of the existing sliding clip technique , which is at present the most commonly used renorrhaphy method , especially in ralpn . in the case of sliding clip renorrhaphy , suture tension is created by sliding nonabsorbable weck clips ( i.e. , hem - o - lok clips ) along the thread and adding lapraty clips to prevent the weck clips from sliding back . surgical clips used for renorrhaphy may slip or migrate and loosen the compression , causing approximated parenchymal dehiscence or rebleeding . to counter these drawbacks , we designed the suture material with a loop at the end of the thread , and identified the feasibility of slt through preclinical animal experiments using a porcine model . first , in our slt , the application of greater tension would be possible outside the renal parenchyma as well as at the site of the thread passed through the loop , while sliding clip technique provides tension only at the renal parenchyma through which the thread is passed . consequently , our slt can apply superior tension without injury , and allow tighter renal parenchymal suturing without dehiscence . in fact , it was confirmed in our previous porcine model experiment that mean distance between renal surfaces after slt renorrhaphy was significantly narrower than that of the conventional sliding clip technique ( 1.80 mm vs. 5.28 mm , p<0.001 ) . second , in the current study , no postoperative complications have been observed as of the last follow - up for any patient . although there were 3 cases of intraoperative transfusions , these resulted from causes irrelevant to slt , including small vein bleeding during hilar dissection and low preoperative hemoglobin level . in contrast , it was reported that there were postoperative perirenal hematoma and anemia as complications related to sliding clip renorrhaphy . third , our mean wit was 22.6 minutes , which is comparable to that ( 22.8 minutes ) of sliding clip technique and superior to other studies . there was no significant decrease in renal function as measured by serum cr level and egfr in this study . furthermore , sliding loop renorrhaphy could be rapidly conducted , as evidenced by the fact that mean rt was 9.0 minutes , accounting for less than half ( about 40% ) of the acceptable mean wit . therefore , slt in ralpn may be a feasible renorrhaphy method in the light of renal function preservation and time - saving advantages . the results of our study should be carefully interpreted in the light of several limitations . first , the study design was a case - series with a small volume , and was retrospective in nature , meaning some degree of bias was unavoidable . second , because the study included patients who underwent surgery performed by multiple surgeons ( cwj and ck ) , there may be technical variations between surgeons that may have an influence on surgical outcomes . in fact , there were statistically significant differences among surgery - related parameters , including rt , wit , operative time , and console time . however , our study might be meaningful as an initial preliminary report demonstrating the potential feasibility of slt in real clinical practice . further large - scale , prospective , long - term follow - up , and direct comparative studies including other techniques will be required to confirm the clinical applicability of slt . in ralpn , slt was identified as a potentially feasible and safe method for renorrhaphy in terms of tightened renal parenchymal closure , rapid performance , renal function preservation , and low risk of complications in real clinical practice . further large scale , prospective , longterm follow - up , and direct comparative studies with other techniques are required to confirm the clinical applicability of slt . an accompanying video can be found in the ' urology in motion ' section of the journal homepage ( www.kjurology.org ) . the supplementary data can also be accessed by scanning a qr code located on the fig . 2 of this article , or be available on youtube ( http://youtu.be/ucxubkqxqza ) .

purposeto report the initial clinical outcomes of the newly devised sliding loop technique ( slt ) used for renorrhaphy in patients who underwent robot - assisted laparoscopic partial nephrectomy ( ralpn ) for small renal mass.materials and methodswe reviewed the surgical videos and medical charts of 31 patients who had undergone ralpn with the slt renorrhaphy performed by two surgeons ( cwj and ck ) between january 2014 and october 2014 . slt renorrhaphy was performed after tumor excision and renal parenchymal defect repair . assessed outcomes included renorrhaphy time ( rt ) , warm ischemic time , perioperative complications , and perioperative renal function change . rt was defined as interval from the end of bed suture to the renal artery declamping.resultsin all patients , sliding loop renorrhaphy was successfully conducted without conversions to radical nephrectomy or open approaches . mean renorrhaphy and warm ischemic time were 9.0 and 22.6 minutes , respectively . after completing renorrhaphy , there were no adverse events such as dehiscence of approximated renal parenchyma , renal parenchymal tearing , or significant bleeding . furthermore , no postoperative complications or significant renal function decline were observed as of the last follow - up for all patients . the limitations of this study include the small volume case series , the retrospective nature of the study , and the heterogeneity of surgeons.conclusionsfrom our initial clinical experience , slt may be an efficient and safe renorrhaphy method in real clinical practice . further large scale , prospective , long - term follow - up , and direct comparative studies with other techniques are required to confirm the clinical applicability of slt .

INTRODUCTION MATERIALS AND METHODS 1. Patient selection 2. Surgical technique 1) From patient positioning to port placement 2) Step-by-step surgical procedures from kidney exposure to renal mass excision 3) Hemostasis and collecting system repair and renorrhaphy using SLT 4) Tumor retrieval and fascia closure RESULTS DISCUSSION CONCLUSIONS SUPPLEMENTARY MATERIAL

PMC4085210

joint contracture causes functional disturbances in patients activities of daily living , decreased movement ability , and impairs the joint function , with high incidence1 , 2 . therefore , preventing the development of joint contracture is a key goal of physical therapists and rehabilitation specialists . unfortunately , medical professionals are still struggling to find definitive therapies for the prevention and management of joint contracture . although a number of factors can lead to the development of contractures3 , differentiating between them in a clinical setting can be difficult . orthopedic surgeons generally classify contractures on the basis of changes causing restriction of the normal range of motion of a joint , a rigidity resulting from some extracapsular cause3,4,5 . in physical therapy , classifying cases in terms of the area causing the condition has little practical purpose . therefore , joint immobility causing restricted range of motion can be treated as a case of contracture2 . changes in the cartilage and the synovial membrane have been reported as intracapsular joint sequelae resulting from contracture6,7,8,9 . to date , significant discrepancies exist among reports , and thus , the current state of research is inadequate for drawing meaningful conclusions . we previously clarified organic changes in the joint composition and structure resulting from contracture10 , 11 : fibrosis of the cartilage surface , atrophy of adipose tissue in the synovial membrane , fibrosis in the lower layers of the synovial membrane , microvasculature expansion , and blood congestion . furthermore , evans et al.6 and yaoita7 investigated the effects of natural healing processes on these organic changes and reported that restrictions of range of motion and structural changes both recovered within a comparatively short time period . this study aimed to investigate histopathological changes to clarify temporal effects on restrictions to range of motion and joint components after joint immobilization in a rat knee - contracture model . fifty - four 8-week - old male wistar rats ( body weight 234269 g ) were used in this study . the rats were randomly divided into two groups : a fixation group ( n = 36 ) and a control group ( n = 18 ) . in the fixation group , right hindlimb knee joints were immobilized in full flexion by plaster casts , which were removed after four weeks . the research protocol followed kanazawa university animal experiment regulations and kinjo university experimental animal handling regulations . all animal handling and experimentation were undertaken with the approval of the ethics committees of both universities ( kanazawa university : ap-091379 and kinjo university : approval no . rats from each group were raised in cages ( similar to fixation group rats ) up to ages corresponding to the subdivisions used for the fixation group . they were labeled as 0wc , 4wfc , 8wfc , 16wfc , 24wfc , and 32wfc , corresponding to the times of examination after cast removal : 0 , 4 , 8 , 16 , 24 , and 32 weeks , respectively . fixation group rats were immobilized with plaster casts according to a modified version of previously described methods10 , 12 , 13 . pentobarbital sodium ( 5 mg/100 g body weight ) anesthesia was administered through the abdominal cavity . manchester , nh , usa ) were fitted to the animals backs . to prevent scratches and abrasions to the right hind limb resulting from the plaster casts , the right hindlimb was covered with a layer of gauze that centered on the knee . the casts extended from the pelvic girdle to the ankle joint , with the hip joint at maximal extension , the knee joint at maximal flexion , and the ankle joint at maximal plantar flexion . the cast area was exposed from the distal ankle to the foot to confirm the absence of swelling and congestion . considering the bone growth during the cast period , the kneecap was exposed as well . rats were able to freely move in their cages using their left hind limb and both forelimbs . if swelling was observed in the fixed limb , the fixation procedure was repeated , and casts were immediately replaced to prevent continuance . if casts were observed to slip out of place or become loose , they were also replaced as soon as possible . the cast period was set at four weeks on the basis of the findings of previous studies , which confirmed that histological changes were evident in the joint structure and content at this point . following the cast period , fixation group rats were randomly divided into six subgroups , with six rats in each group : these were labeled 0wf , 4wf , 8wf , 16wf , 24wf , and 32wf , corresponding to the examination times after cast removal : 0 , 4 , 8 , 16 , 24 , and 32 weeks , respectively . the 0wf and 0wc groups underwent immediate assessments ( described below ) to measure the extension angle of their knee joints , to assess restrictions to range of motion , and to create histological specimens for analysis of the joints . the restriction of knee joint extension angle was measured and recorded on the basis of a preliminary experiment . an intra - abdominal pentobarbital sodium anesthetic was administered ( 5 mg/100 g body weight ) , and the rats were laid on their sides . using one hand to hold the femur in place , researchers used the other hand to hold the leg at 35 mm from the knee joint and guide the knee into maximal extension , the feeling experienced at the end of the range of motion . using a digital camera , images of the principal axis of extension of the knee joint ( the line running from the greater trochanter to the lateral condyle of the femur ) and the movement axis ( the line running from the head of the fibula to the lateral malleolus ) were captured in the sagittal plane . the images were loaded onto a computer , and the angles of the principal and movement axes were measured using three - point measuring instrument software ( version 1.1.2 ) . this protocol was used for all assessments . in addition , a digital force gauge ( imada dps-5 ) was attached at 35 mm from the knee joint , which was then extended to the angle measured . three technicians , each with more than five years of clinical experience , performed each procedure five times . the average value was 0.33 0.05 n. on the basis of these preliminary findings , all measurements of the restriction of knee joint extension angle were recorded by first attaching a digital force gauge at 35 mm from the knee joint , followed by application of 0.3 n of extension force and recording of the resulting angle . for the fixation group , these measurements were conducted before the experiment , immediately after cast removal and then weekly until the experiment was concluded . individual differences in the development of contractures were assessed using retention rate calculations , which were as follows : retention rate = [ ( the measured angle at each time - period ) ( the angle at development of contracture ) ] 100 . at the end of each experimental period , a group of six animals was euthanized with a pentobarbital sodium overdose , and their hindlimbs were severed at the hip joint . tissues were fixed over a 72-h soaking period in a 10% buffered neutral formalin solution , and then decalcified using the planck - rychlo solution for 72 h at 4 c . thereafter , an area approximately 10 mm above and below the knee joint was removed . tissues from the center of the kneecap , moving toward its inner regions in the sagittal plane , were removed . to preserve this removed region , a 5-mm margin was cut and stored in an embedding cassette . it was subsequently neutralized in a 5% anhydrous sodium sulfate solution for 72 h , rinsed under running water for 30 min , and soaked for approximately 3 h in 100% alcohol to remove any remaining fat . thereafter , samples were processed in an automatic dehydrating - and - paraffin - wax - embedding device into paraffin blocks and sliced into 3-m thick sections using a sliding microtome ( leica sm2000r ) . after these organizational segments had been attached to a slide glass and dehydrated , they were stained with hematoxylin - eosin ( he ) stain and enclosed in a hydrophobic mounting agent , to complete the histological specimen preparation . the specimens were observed using a microscopic digital camera ( olympus dp50 ) connected to an optical microscope ( olympus bx-51 ) to examine the entire knee joint structure . although it is possible to classify cartilage on the basis of the degeneration it has undergone in cases of osteoarthritis14,15,16 , histological classification of cartilage degeneration resulting from immobilization is a relatively new area of study . therefore , to quantify histological changes in the cartilage , we classified samples into one of the following four groups : group i , fibrosis and degeneration could not be identified in outer layers ; group ii , irregularities and fibrosis could be identified on the cartilage surface ; group iii , thinning and transformation into fibrous pseudocartilage could be identified ; and group iv , destruction and damage could be identified in the cartilage . for each group fitted with plaster casts , a comparative examination was conducted with the control group using fisher s exact test using the statistical software , r ( version 2.15.1 ) . following the cast period , knee joint extension angles revealed following restrictions : 73.2 9.9 in the 0wf group , 71.4 8.3 in the 4wf group , 71.2 8.1 in the 8wf group , 68.8 7.4 in the 16wf group , 73.6 7.9 in the 24wf group , and 70.2 11.2 in the 32wf group . using retention rate calculations , each group showed improvement over time , and the retention rate in the 4wf group was 7.1 3.8% at the end of the study . excluding the 4wf group , retention rates by the fifth week after cast removal were as follows : 8wf , 1.4 1.1% ; 16wf , 1.7 0.6% ; 24wf , 0.5 1.1% ; and 32wf , 1.2 1.2% . furthermore , retention rates of all groups were 0% by the sixth week . knee extension angles were measured over the subsequent weeks , but none of the individual subjects in any group revealed any recurrence of their restrictions ( table 1table 1 . changes in knee extension angle restrictionsimmediateremovalweek following fixation removal1th2nd3rd4th5th6thgroup 4wf100%69.414.6%42.510.4%19.65.9%7.13.8%group 8wf100%74.45.9%50.54.8%20.38.5%7.85.3%1.41.1%0%group 16wf100%71.16.0%46.09.6%16.26.9%6.71.7%1.70.6%0%group 24wf100%76.68.8%41.48.4%16.04.5%7.53.2%0.51.1%0%group 32wf100%75.67.5%44.63.7%22.04.3%10.53.0%1.21.2%0% ) . considering the relationship between age and histological changes , case control comparisons matched by age produced the following data ( table 2table 2 . abnormal findings in joint cartilage and the frontal synovial membranefibrosis ofcartilagedegenerationof cartilagethinning ofcartilageadhesion offrontal tibiaatrophy ofadipocytefibroblasthyperplasiagroup 0wf600566group 4wf600366group 8wf600366group 16wf620366group 24wf631166group 32wf652066numbers within the table indicate the number of specimens presenting the conditions out of a total of 6 . , fig . ( he stain , scale bar left : 1 mm , center and right : 100 m ) . the 0wf group : adhesions of fibrous hyperplasia tissue between the frontal tibial area and the frontal synovial membrane were noted ( , left ) . adipocyte atrophy and fibroblast hyperplasia appeared , as well as expansion and blood congestion in the microvasculature ( right ) . there were no clear differences in the extent of any of the conditions between the 4wf and 0wf groups . the space between adhesions was more expanded than in the 4wf group ( , left ) . the 16wf group : adhesions covered an even larger area than in the 8wf group ( left ) . some hyaline cartilage had been replaced with fibrous cartilage tissue ( center ) . findings related to conditions in the frontal synovial membrane were identical to those of the 0wf group ( right ) . the 24wf group : spaces between adhesions were bigger than in the 16wf group ( , left ) . cartilage degeneration and thinning were identified ( center ) . findings related to conditions in the frontal synovial membrane were identical to those of the 0wf group ( right ) . the 32wf group : no adhesions were evident ( left ) . cartilage degeneration and thinning were identified ( center ) . findings related to conditions in the frontal synovial membrane were identical to those of the 0wf group ( right ) ) . in the articular cartilage of the 0wf group , fibrosis was identified on the surface of all cartilage specimens , and in five of six specimens , adhesion of the forward synovial membrane and fibrous structure of the hyperplasia was observed in the frontal tibial area . all specimens revealed adipocyte tissue atrophy in the frontal synovial membrane , fibroblast hyperplasia in the lower layers , and expansion and blood congestion in the microvasculature . no significant changes were observed between 0wf , 4wf , 8wf , 16wf , 24wf and 32wf groups in terms of adipocyte tissue atrophy and size , fibroblast hyperplasia , and microvasculature changes . numbers within the table indicate the number of specimens presenting the conditions out of a total of 6 . histological findings ( he stain , scale bar left : 1 mm , center and right : 100 m ) . the 0wf group : adhesions of fibrous hyperplasia tissue between the frontal tibial area and the frontal synovial membrane were noted ( , left ) . adipocyte atrophy and fibroblast hyperplasia appeared , as well as expansion and blood congestion in the microvasculature ( right ) . there were no clear differences in the extent of any of the conditions between the 4wf and 0wf groups . the space between adhesions was more expanded than in the 4wf group ( , left ) . the 16wf group : adhesions covered an even larger area than in the 8wf group ( left ) . some hyaline cartilage had been replaced with fibrous cartilage tissue ( center ) . findings related to conditions in the frontal synovial membrane were identical to those of the 0wf group ( right ) . the 24wf group : spaces between adhesions were bigger than in the 16wf group ( , left ) findings related to conditions in the frontal synovial membrane were identical to those of the 0wf group ( right ) . the 32wf group : no adhesions were evident ( left ) . cartilage degeneration and thinning were identified ( center ) . findings related to conditions in the frontal synovial membrane were identical to those of the 0wf group ( right ) although findings in the 4wf group were largely identical to those of the 0wf group , adhesions were only found in three out of six samples . the same numbers of adhesions were observed in the 8wf group and the 4wf group , but the gap between fibers tended to become larger . in addition , the results of articular cartilage examination in the 16 wf were the same as for the 4wf and 8wf groups , but two of six specimens revealed replacement of the original hyaline cartilage with of a fibrous - type cartilage . although adhesions of the frontal tibial area were identified in three of six specimens , gaps between fibers greater than those in the 8wf group , and fibrous structures were sparser . however , only a partial adhesion was observed in the frontal tibial area of just one of six specimens . the gap between fibers was even greater than those observed in the 16wf group . in three of six samples , original cartilage was replaced with fibrous - type structures , and one of these revealed additional thinning in the cartilage . fibrosis of the articular cartilage but no adhesion was observed in the frontal tibial area in the articular cartilage samples of the 32wf group . however , the original cartilage was replaced with fibrous - type structures in five of six cases , and the degeneration was quite severe ; two of these revealed additional thinning the cartilage . all adhesions had disappeared in this group . finally , inflammatory cell infiltration was observed in all groups . on the basis of histological classification of the cartilage in the cast group compared with the control group , a significant difference was observed between both groups ( table 3table 3 . in this study , six weeks after cast removal , all rats showed complete recovery in their range of knee joint extension . however , although the degree of contracture - associated adhesions decreased over time , a replacement of existing cartilage with fibrous - type structures along with thinning of the cartilage was observed . moreover , at all stages , there were persistent changes in the frontal synovial membrane , atrophy and differences in the size of adipocyte tissues , fibroblast hyperplasia in lower layers , and expansion and blood congestion in the microvasculature . therefore , although the range of knee joint extension improved , the articular cartilage and frontal synovial membrane remained damaged . evans et al.6 evaluated the restoration of mobility in immobilized joints following the onset of contracture using an internal - fixation knee - arthrodesis contracture model in rats . their study reported that provided , the fixation lasted no longer than 30 days , changes in the range of motion were reversible . yaoita7 and maoka17 also reported that range of motion and organizational structures were restored comparatively quickly following short - term fixation of 30 days or less in a rat knee contracture model . although we found some differences in the time required to restore mobility and used a different fixation method , the complete restoration of mobility we observed agrees with prior research . the hyaline cartilage forms a structure around joints and functions as a load - bearing buffer , reducing friction between bones in the course of normal joint movement , and playing a role in maintaining mobility . hyaline cartilage does not contain blood cells , lymph ducts , or nerves , and does not directly experience inflammation18 . in a broader evaluation of joint changes resulting from contracture , evans et al.6 examined histological changes in rats with internal fixation knee arthrodeses . they reported that after 30 days of fixation , there was a proliferation of intra - articular connective tissue as well as fibrosis and blood congestion under the kneecap and in the intercondylar fossa . they also observed cartilage compression at contact points of the femur and tibial condyles , and miniaturization of chondrocytes , and they concluded that intra - articular connective tissue hyperplasia and adhesions within the cartilage were the cause of the restricted range of motion . these adhesions did not completely cover the cruciate ligament of the knee and its surroundings , rather they originated from fibrosis of the synovial membrane . similar to results of the study conducted by evans et al.6 , yaoita et al.7 , using internal fixation for the knee joint in rats , revealed thinning of cartilage tissue , disordered cell arrangement , and necrobiosis after 30 days of fixation . because of these structural changes , fibrous connective tissues and adipocytes filled the intra - articular area from the rear of the joint , extending the joint surface forward , parallel to the femur , narrowing the articular space . both the studies cited here discussed the process used to induce contracture in detail and highlighted resulting histological changes . however , their descriptions of the recovery process after the removal of fixation was limited to general statements about the range of motion being restored to its pre - experimental levels , and the joint being otherwise normal . we could find no other study in the literature which described the progress of contracture and histological joint changes over time . moreover , the hyperplasia we observed in the membrane tissue of the outer layer of articular cartilage is consistent with our previously published reports10,11,12,13 and could have resulted from joint immobilization . in addition , adhesions within the frontal synovial membrane and the frontal tibial area were consistent with the findings of watanabe et al.10 during long - term cast fixation . although these adhesions may be consistent with the work of evans et al . and this could be explained by differences in the fixation methods and observation areas among the studies , but the true reasons remain unclear and require further investigation . in the present study , as the time interval progressed , adhesions diminished ; moreover , by 32 weeks after cast removal , they had completely disappeared . yaoita , who noted that free movement after removal of fixation resulted in mechanical stress , though it is equally possible that the adhesions may have simply detached . in contrast with the resolution of adhesions , the number of cases , wherein normal cartilage was replaced with fibrous structures and thinning , increased as time progressed . ely et al.8 evaluated changes in joint components resulting from cast fixation in adult dogs , and reported a narrowing of the space between opposite cartilage structures along with thinning of the cartilage in load - bearing areas . in addition , hagiwara et al.19 evaluated knee articular cartilage thickness in rats after internal fixation at 150 , and reported that the thickness increased in movement areas , although it remained unchanged in contact and non - contact areas . the timing of the appearance of cartilage thinning in the study of hagiwara et al . clearly differs from that reported by ely et al . and yaoita . furthermore , the cartilage thickening observed by hagiwara et al . was not observed in any sample in the present study . first , because adhesions connect viscera and tissue that should be separated20 , the detachment of these adhesions through mechanical stress may have caused bleeding and inflammation21 . as a result , the normal repair process may have caused the replacement of normal cartilage with fibrous tissue structures . second , the articular cartilage derives nutrition from synovial fluid , and if this nutritional supply were obstructed by surface tissue hyperplasia , it might cause degeneration . this could then result in a decline in cartilage regeneration , necrobiosis , and decreasing thickness , resulting in the replacement of cartilage with fibrous tissues through the recovery process21 . these qualitative changes to the articular cartilage did not occur immediately after the onset of the contracture but after an amount of time had passed . this cartilage degeneration is a new finding , and future biochemical analyses will be required to clarify its causes . takemura et al.12 analyzed changes in the frontal synovial membrane resulting from contracture with regard to contractures induced by cast fixation . joint immobilization resulted in adipocyte tissue atrophy and size differences in the frontal synovial membrane , fibroblast hyperplasia in the lower layers , and expansion and blood congestion in the microvasculature . watanabe et al.10 and matsuzaki et al.11 reported identical findings , and these were unsurprisingly confirmed by our study . these changes may have occurred because of a decrease in the mechanical stress resulting from the contracture . to date , very few articles have prospectively considered the recovery process of the frontal synovial membrane following immobilization . and yaoita provided detailed descriptions of the recovery process , they only made general statements about histological improvement . conversely , takemura et al . and matsuzaki et al . failed to observe any improvement in frontal synovial membrane atrophy . in this study , we observed adipocyte tissue atrophy and size differences in the frontal synovial membrane , fibroblast hyperplasia in the lower layers , and expansion and blood congestion in the microvasculature . moreover , this occurred at all the examination periods ( up to and including 32 weeks after removal of cast fixation ) . even after the passage of a long time interval , no trends toward improvement were identified in the findings . to the best of our knowledge , although articles have reported the posterior synovial membrane changes that result from joint immobilization , our report is the first to observe atrophy of adipose tissue in the frontal synovial membrane . in addition , atrophy of structural fat such as the corpus adiposum infrapatellar as well as bone marrow is not thought to easily occur in such cases11 . therefore , future histological and biochemical investigations may be necessary to clarify how this occurs . the results of our present study are in agreement with previous research that joint mobility may completely recover after contracture . we further add that this may occur through time alone , and that damage to the articular cartilage and frontal synovial membrane did not improve within 32 weeks after fixation , the end of our observation period . briefly , free motion merely improves mobility and does not result in any organic improvements in the joint itself . when physical therapists passively mobilize joints to improve range of motion following contractures , they often experience a different sensation toward the end of the range of motion . this may result from an increased coefficient of friction because of the fibrosis and degeneration in the cartilage surface . the inadequate consideration of these issues in reports to date means future research focusing more closely on appropriate medical interventions to limit organic changes is required . furthermore , future research must respond to advances in our understanding of the fundamental scientific basis of physical therapy .

[ purpose ] the purpose of our study was to clarify temporal effects on restrictions to range of motion and the histopathological changes of joint components after joint immobilization in a rat knee - contracture model . [ subjects ] fifty - four male wistar rats were randomly divided into two groups : a fixation group , and a control group . [ methods ] in the fixation group , unilateral knee joints were immobilized at full flexion using a plaster cast for 4 weeks . at four weeks the animals were randomly divided into six subgroups , corresponding to the time of examination after cast removal : 0 , 4 , 8 , 16 , 24 , and 32 weeks . for comparison , control group animals of corresponding age were also examined . [ results ] although movement restrictions of the knee joint had completely recovered 6 weeks after the cast removal , cartilage and synovial membrane structures did not completely recover . [ conclusion ] these findings have not previously been reported , and as they form an addition to the fundamental scientific foundations of physical therapy , further research must examine these findings from a variety of perspectives .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION

PMC5234353

pharmacopuncture is a new acupuncture therapy based on a combination of meridian theory and herbal pharmacology . in pharmacopuncture , by injecting the extracts of herbs at acupuncture points , doctors gain a synergism of positive effects , i.e. , a physical stimulation due acupuncture and a chemical effect due to the herbal medicine . recently , in korean medicine , pharmacopuncture has been widely used to treat patients with such diverse diseases as herniated intervertebral discs , arthropathia , skin diseases , and especially obesity . for obesity care , many reports have been published concerning related acupuncture points , such as jusanli ( st36 ) and zhongwan ( cv12 ) , and pharmacopuncture extracts , such as atractylodes lancea , panax ginseng , sweet bee venom , etc . in addition , mahwang ( ephedra sinica)- cheonoh ( aconitum carmichaeli ) extract is known to be able to inhibit the generation of fat cells , as well as the activation of collateral obese metabolic diseases . as a perennial plant belonging to the family ephedraceae , mahwang , in korean medicine , can perspire , drive out cold , generate diuresis , and reduce edema . mahwang also contains many alkaloids , such as ephedrine , pseudo - ephedrine , tannin and flavonoids , which can pharmacologically activate sympathetic nerves to reduce appetite , inhibit cholesterol absorption , and accelerate the disintegration of body fat [ 5 , 6 ] . because of these effects , it has frequently been used as a herb prescription to treat obese patients and as an ingredient in food for short - term weight loss or for elevating exercise performance . as a perennial plant belonging to the ranunculaceae family , cheonoh is expected to be able to promote the blood - circulation system and the meridian system on the surface of the body and to stimulate metabolism just as mahwang can ; thus , cheonoh has frequently been used with mahwang as a herb prescription for the treatment of obese patients . reports have been published indicating that mahwangcheonoh pharmacopuncture is effective in promoting the disintegration of fat cells [ 4 , 9 ] . however , to the best of the authors knowledge , no reports on its toxicity have yet to be published . accordingly , the authors performed such toxicity tests and obtained significant results demonstrating that mahwangcheonoh pharmacopuncture is a safe , credible pharmacopuncture therapy for treating obese patients . mahwangcheonoh pharmacopuncture extracts were prepared at the korean pharmacopuncture institute ( seoul , korea ) in a clean room , were stored in a refrigerator at 2.1 - 5.3c , and remained stable for 6 months following production ( lot no . kpi-2013 - 03 ) . the animals used in this study were 6-week - old specific - pathogen - free ( spf ) sprague - dawley ( sd ) male ( 184.7 - 204.0 g ) and female ( 156.1 - 179.5 g ) rats purchased from orientbio inc . this study was approved by the institutional animal care and use committees of biotoxtech ( no . 130364 ) . when the animals were received , a visual inspection was done , and weights were recorded using electronic scales ( cp3202s , sartorius , gttingen , germany ) . the general symptoms and changes were checked once daily during the seven days of acclimatization , after which all animals were checked and found to be normal . all animals were bred in stainless - steel wire breeding boxes ( 260 mm ( w ) 350 mm ( d ) 210 mm ( h ) ) kept at a temperature from 22.1 to 24.1c , a humidity from 47.7% to 70.8% , and an illuminance from 150 to 300 lux , with a 12-hour light ( 7:00 am to 7:00 pm)/dark cycle . each breeding group was divided by three animals during acclimatization periods and was divided by one animal during observation periods . feed ( teklad certified irradiated global 18% protein rodent diet 2918c , harlan laboratories inc . , usa ) and ultraviolet ( uv ) sterilizer running water were provided freely . twenty male and twenty female rats with weights closest to the mean weight were selected . the selected animals were distributed into four groups ( five rats of each sex per group ) . the mahwangcheonoh pharmacopuncture extracts were injected into the animals via a muscular route , and the high dose was set at 1.0 ml per animal because no mortalities had occurred in a preliminary test ( biotoxtech study no . : b13474p ) in which a single dose of 1.0 ml of mahwangcheonoh pharmacopuncture extract had been injected into the muscles of male and female rats . based on this high dose , we set 0.5 ml per animal as the medium dose and 0.25 ml per animal as the low dose . for the control group , 1.0 ml of normal saline was injected . in the low - dose group ( g2 ) and the medium - dose group ( g3 ) , the mahwangcheonoh pharmacopuncture extracts were injected into the left femoral muscle . for the control group ( g1 ) and the high - dose group ( g4 ) , 0.5 ml of normal saline and 0.5 ml of mahwangcheonoh pharmacopuncture extract , respectively , were injected into the left and the right femoral muscles , for a total of 1.0 ml per animal . on the day of injection ( day 0 ) , general symptoms ( types of toxic indications , times of toxic expression , recovery times ) and any deaths were recorded at 30 minutes and at 1 , 2 , 4 and 6 hours after injection . from day 1 until day 14 after injection , body weights were measured on the day of injection ( before injection ) and on days 3 , 7 and 14 after injection . all animals were denied access to food and water ( fasted ) for at least 18 hours prior to necropsy . on the day of necropsy ( 15 days after injection ) , the animals were anesthetized with isoflurane , and blood was collected from the abdominal aorta . one ml of the collected blood was put into a tube containing ethylene diamine tetraacetic - acid ( edta ) , and the hematology parameters , the prothrombin time ( pt ) and the activated partial thromboplastin time ( aptt ) were measured using a hematology analyzer ( advia 2120i , siemens , germany ) . two ml of the collected blood were put into a tube containing 3.2% sodium citrate ; then , the plasma was collected after centrifugation at 3,000 rpm for 10 minutes . a coagulation examination was performed using coagulation time analyzers ( coapresta 2000 , sekisui , japan ) to measure the pt and the aptt . the blood biochemistry was determined by using serum taken from blood that had been collected from the abdominal aorta and had been centrifuged at 3,000 rpm for 10 minutes . a blood chemistry analyzer ( 7180 , hitachi , japan ) and an electrolyte analyzer ( avl 9181 , roche , germany ) were used for the measurements . for the histopathology , the organs and the tissues collected from all animals after necropsy were inspected . after necropsy , tissues surrounding the injection regions were excised and fixed in 10% neutral buffered formalin solution , after which local resistance tests were performed on those tissues . fixed organs and tissues sections the weights , as well as the hematological and the blood biochemical results , were analyzed by using sas ( version 9.2 , 9.3 , sas institute inc . a bartlett test was conducted to evaluate equal variance between the test and the control groups ( p < 0.05 ) . the one - way analysis of variation ( anova ) test was conducted when equal variance was recognized on the bartlett test ( p < 0.05 ) , and the dunnett s t - test was conducted when equal variance was recognized on the anova test ( both p < 0.05 , p < 0.01 ) . also , the kruskal - wallis test was conducted when equal variance was not recognized on the bartlett test ( p < 0.05 ) , and the steel - test was conducted when equal variance was recognized on the kruskal - wallis test ( both p < 0.05 , p < 0.01 ) . during the observation period , no abnormal symptoms or deaths were observed in either the male or the female rats in the test and the control groups ( table 1 ) during that period , the weights of the male rats in the control and the test groups changed from 192.6 8.7 g ( g1 ) , 195.6 5.6 g ( g2 ) , 195.3 4.3 g ( g3 ) , and 193.2 7.4 g ( g4 ) to 314.1 11.9 g ( g1 ) , 315.0 18.0 g ( g2 ) , 314.2 16.3 g ( g3 ) , and 318.5 16.2 g ( g4 ) , respectively . those of the female rats changed from 166.3 6.0 g ( g1 ) , 168.3 6.9 g ( g2 ) , 169.2 9.1 g ( g3 ) , and 167.4 7.4 g ( g4 ) to 227.5 10.1 g ( g1 ) , 235.4 9.9 g ( g2 ) , 230.5 15.3 g ( g3 ) , and 223.4 9.2 g ( g4 ) , respectively . however , no statistically significant changes in weights associated with injection were recognized ( figs . no significant differences were observed in the hematology between the test groups and the control group for either the male or the female rats ( tables 2,3 ) . the change observed in the lowdose female group ( g2 ) was a minor variation because it was observed sporadically and exhibited no a dose dependency . that change was determined to be an accidental change with no statistical significance ( tables 4,5 ) . the results on the local resistance tests were not affected by the injections . however , a weak inflammation was observed in the region of injection in a rat in the control group . because the inflammation occurred in the control group , it was determined to have no toxicological significance , but rather to be a spontaneous or accidental change without injection relevance . according to national health statistics , the obesity rate in korean adults , based on the body mass index ( bmi ) , has reached 31.5% , being 37.7% in males and 25.3% in females . also , the teenage obesity rate in korea has reached 11.5% . because of this , the people s interest in obesity has also increased . according to a survey , 85.4% of the people were aware of being overweight , and 60% indicated that they were actively trying to lose weight . moreover , during 2008 , the social and economic cost associated with the treatment of obese patients in the country was estimated to be approximately 1.6 billion us dollars . because of this , many medical workers can not but be interested in research on treatments for obesity . in korean medicine , much research has been done on the treatment of obese patients by using acupuncture , electro- acupuncture , moxibustion , herbs , subcutaneous acupuncture , and exercise therapy . among these possible treatments , pharmacopuncture injection of one - tenth the volume of a medicine dose directly into fatty areas or acupuncture points moreover , many pharmacopuncture solutions for obesity treatment have already undergone many safety and reliability tests in both experimental and clinical studies . pharmacopuncture extracts known to be effective in the treatment of obese patients are ephedra , atractylodes lancea , coix lacryma - jobi , astragalus propinquus , angelica , ginseng [ 14 , 15 ] , and the like . all of these have been found through laboratory research to be able to reduce effectively blood lipids or fat cells , and body weight , to have no toxicity , and to result in no abnormalities in the blood levels . mahwangcheonoh pharmacopuncture has also been proven to be effective in the treatment of obese patients [ 4 , 9 ] , but its toxicity and safety have not been adequately addressed . thus , this research was conducted to establish the toxicity , safety , and stability of this pharmacopuncture so that it could be added as another reliable pharmacopuncture for the treatment of obese patients . this study observed the general symptoms , weight changes , and hematological and biochemical blood changes caused by single - dose intramuscular injection of mahwangcheonoh pharmacopuncture in sd 6-week - old male and female rats . the rats were also subjected to necropsy to identify any changes in tissues or organs due to the injections . no deaths or abnormalities occurred any of the rats , and no significant differences in body weights were observed during the 14-day observational period following injection of the pharmacopuncture . finally , no abnormal changes due to the pharmacopuncture were observed on the histopathological examinations after the necropsy . based on the above results , the lethal single dose of mahwangcheonoh pharmacopuncture for rats is over 1.0 ml / animal . in addition , the mahwangcheonoh pharmacopuncture at that dose is stable and safe to use and has no toxic effects on viscera , muscles or functions . although this study has shown that mahwangcheonoh pharmacopuncture is safe to use at doses of 1.0 ml or less , no research on its side effects has yet been officially reported . in addition , because this research involved investigating the effect of a single - dose injection over a 14-day observation period , research on its long - term effects and on the effects of multi - dose injections is needed if mahwangcheonoh pharmacopuncture is to be firmly established as an effective treatment for obese patients . the objective of this study was to analyze the safety and the toxicity of mahwangcheonoh pharmacopuncture . the results after a single intramuscular dose and observations of the general symptoms over a 14-day period showed no abnormal symptoms or mortalities in either the test groups or the control group . in addition , no significant changes in weight , hematology , blood biochemistry , and histopathology between the test groups and the control group were found . based on these results , we consider the lethal dose of mahwangcheonoh pharmacopuncture to be far beyond 1.0 ml / animal ; thus , mahwangcheonoh pharmacopuncture may be considered a safe therapeutic agent for the treatment of patients with obesity . s.d . , standard deviation ; rbc , red blood cell ; hgb , hemoglobin ; hct , hematocrit ; mch , mean corpuscular hemoglobin ; mcv , mean corpuscular cell volume ; mchc , mean corpuscular cell hemoglobin concentration ; plt , platelet ; reti , reticulocytes ; wbc , white blood cell ; neu , neutrophils ; lym , lymphocytes ; mono , monocytes ; eos , eosinophils ; baso , basophils ; pt , prothrombin time ; aptt , active partial thromboplastin time . s.d . , standard deviation ; rbc , red blood cell ; hgb , hemoglobin ; hct , hematocrit ; mch , mean corpuscular hemoglobin ; mcv , mean corpuscular cell volume ; mchc , mean corpuscular cell hemoglobin concentration ; plt , platelet ; reti , reticulocytes ; wbc , white blood cell ; neu , neutrophils ; lym , lymphocytes ; mono , monocytes ; eos , eosinophils ; baso , basophils ; pt , prothrombin time ; aptt , active partial thromboplastin time . s.d . , standard deviation ; alt , alanine aminotransferase ; ast , aspartate aminotransferase ; alp , alkaline phosphatase ; ggt , gamma glutamyltranspeptidase ; glu , glucose ; bun , blood urea nitrogen ; crea , creatinine ; t - bili , total bilirubin ; t - chol , total cholesterol ; tg , triglycerides ; tp , total protein ; alb , albumin ; a / g ratio , albumin / globulin ratio ; p , phosphorus ; ca , calcium ; na , sodium ; k , potassium ; cl , chlorine . s.d . , standard deviation ; alt , alanine aminotransferase ; ast , aspartate aminotransferase ; alp , alkaline phosphatase ; ggt , gamma glutamyltranspeptidase ; glu , glucose ; bun , blood urea nitrogen ; crea , creatinine ; t - bili , total bilirubin ; t - chol , total cholesterol ; tg , triglycerides ; tp , total protein ; alb , albumin ; a / g ratio , albumin / globulin ratio ; p , phosphorus ; ca , calcium ; na , sodium ; k , potassium ; cl , chlorine . p < 0.05 , which implies the existence of a statistically significant difference from the control group based on the steel test

objectives : this study was conducted to analyze the single - dose toxicity and the safety of mahwangcheonoh pharmacopuncture extracts.methods:six-week-old sprague - dawley rats were used for this study . doses of mahwangcheonoh pharmacopuncture extracts were set at 0.25 ml ( low - dose ) , 0.5 ml ( medium - dose ) and 1.0 ml ( high - dose ) for the test groups . a dose of 1.0 ml of normal saline solution was set for the control group . during 14 days , general symptoms , mortalities , and changes in hematology , blood biochemistry and histopathology of all rats were observed.results:no death was observed in all test groups . any abnormal symptom was not observed in all of the groups . no significant changes in weight between the control group and the test groups were observed . in addition , no significant differences in the hematology signs , the blood biochemistry levels and the histopathological signs related to the mahwangcheonoh pharmacopuncture extracts injection were observed.conclusion:the findings of this study indicate that mahwangcheonoh pharmacopuncture at doses of 1.0 ml or less may be consider safe and non - toxic . so , it can be used for therapy of obesity sufficiently . but further studies on this subject must be performed to confirm and verify this conclusion .

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

PMC4541000

type 1 diabetes ( t1d ) is an autoimmune disease in which the immune system is activated against the insulin producing -cells present in the pancreatic islets . the elimination of these cells impairs the mechanisms that control the glucose blood level and , up to now , the possible and most efficacious treatment for t1d is based on insulin administration and pancreas or pancreatic islet transplantation [ 2 , 3 ] . while exercise represents a cornerstone in the treatment and prevention of type 2 diabetes mellitus , only recently its effects on glycemic profile and autoimmunity in t1d have been debated by several studies , due to the controversial data reported regarding the benefits of supervised exercise training on metabolic control in people with t1d [ 4 , 5 ] . in fact , managing blood glucose in t1d while exercising is complex and involves multiple factors such as type of insulin used , time between food intake and sport activity , duration , and intensity of physical effort , known to deter people from enjoying the benefits of sport at both recreational and competitive levels [ 6 , 7 ] . some speculative hypotheses instead point at the positive immunomodulatory effect of exercise with respect to both t1d and inflammation [ 8 , 9 ] . showed how metabolic dysregulation precedes the autoimmune attack against -cells and suggested lifestyles modification as front - line prevention strategy . during exercise , skeletal muscle releases myokines ( il-6 ) and the subsequent production of il-1 receptor antagonist ( il-1ra ) by monocytes and macrophages may frame a potent anti - inflammatory window . there are several studies investigating the effects of exercise in inbred rodents with naturally occurring type 2 diabetes or streptozotocin - induced diabetes [ 12 , 13 ] ; however , few studies have been conducted in a nod mouse model which spontaneously develops t1d [ 1417 ] and resembles the disease progression in humans . by using the nod model , we tested the effect of moderate - intensity exercise in inducing immunological changes that would result in the delay of t1d progression . in particular , we analyzed the differences in cytokine profiles between trained and untrained animals and correlated these modulations with t1d appearance . eight - week - old female nod mice ( jackson laboratory ) were first acclimated to a six - lane treadmill ( columbus , oh ) by running for 10 min at 10 m / min followed by 2 min at 20 m / min ( 0 slope ) , in three running sessions over a week before exercise testing . animals were then trained on a treadmill following a chronic exercise regime ( 30 min / day , 5 days / week at the speed of 12 m / min ) for 12 weeks . age - matched , control animals were left untrained . prior to and upon completion of the training period , mice underwent muscular performance evaluation determined by a submaximal incremental test ( peak oxygen uptake ( vo2 ) challenge ) , as previously described . to determine vo2 peak , mice were placed in a treadmill for 5 min at a 0 incline and 0 m / min . the nod mice were then challenged with 1.5 min intervals of increasing speed at a 15 incline . nonfasting glycemic levels of experimental mice were monitored 2 - 3 times a week using onetouchultra2 glucometers ( lifescan ) . animals were considered diabetic and euthanized upon 3 consecutive blood glucose readings above 300 mg / dl . animal studies were performed in compliance with iacuc and the us department of health and human services guide for the care and use of laboratory animals . cytokine profiles were evaluated on the serum of the nod mice before and after 8 weeks of exercise training and upon completion of the 12-week endurance training program . the serum content of g - csf , gm - csf , ifn- , il-1 , il-2 , il-4 , il-6 , il-10 , il-2p40 , il-13 , mcp-1 , mip-1 , mip-2 , vegf , and tnf- was analyzed simultaneously using the cytokine bead assay ( millipore ) and data were collected using the luminex . pancreas of 20-week - old nod mice was collected and fixed in buffered formalin 10% overnight at 4c and subsequently in optimal cutting temperature ( oct , tissue - tek ) compound and frozen in dry ice . 5 m sections were obtained from the cryopreserved blocks , permeabilized with 0.3% pbs triton x-100 , then incubated in universal blocker reagent ( biogenex san raman ca ) for 30 min , and washed in optimax wash buffer ( biogenex ) . sections were then incubated overnight at 4c with guinea pig anti - insulin ( biogenex ready to use polyclonal antibodies ) and mouse antiglucagon ( monoclonal clone k79bb10 diluted at 1 : 1000 , sigma ) primary antibodies and for 1 hr at room temperature with goat anti - guinea pig alexa fluor 488 ( invitrogen ) and goat anti - mouse alexa fluor 568 ( invitrogen ) secondary antibodies diluted in wash buffer at 1 : 300 . as negative control samples , slides were incubated with the preimmune serum from animal of the same species instead of the primary antibody . dapi ( invitrogen ) was used for nuclear staining and slides were mounted with prolong antifade ( invitrogen ) . pictures were acquired with a zeiss axiovert 200 m confocal microscope , accessorized with a hamamatsu camera , available at the imaging core facility of the diabetes research institute , university of miami . slides analyzed at the university of milan were acquired with nikon eclipse 50i microscopy and images were captured using nis - elements d 4.00 software ( nikon instruments europe bv , netherlands ) . data were displayed and analyzed using adobe photoshop cs4 . quantification of cellular composition ( i.e. , -cells , -cells , and nuclei ) was performed by using the cells count macrowritten for image j ( http://imagej.nih.gov/ij/ ) . glucose profiles were shown as median and quartiles given the nonparametric distribution of the data , except where differently expressed . muscular performance data were presented as the mean sem and t - tests were used to calculate statistical significancy . cytokines were analyzed by a two - way anova with repeated measures followed by bonferroni 's post hoc test . since the distributions of g - csf , gm - csf , and il-10 showed deviations from normality due to the presence of outliers , these cytokines were subjected to logarithmic transformation prior to the execution of anova . deterioration of glycemic profile was appreciable around the 10th week of training in all nod mice under investigation , with slight lower glycemia in the exercising mice with respect to the untrained ones ( figure 1(a ) ) , although the impact of diabetes was remarkable already at week 6 in the trained mice , as indicated by the interquartiles and the diabetic incidence . a similar pattern was detectable among the diabetes - free mice ( figure 1(b ) ) , with a better glycemic profile in the nod mice on training compared to the controls . in the second part of the experiment , from weeks 6 to 12 of training , among the diabetes - free mice , the average glycemic values resulted in being significantly lower in the trained animals with respect to the controls ( 110 1.4 versus 116 2 mg / dl , means and sem , p < 0.05 ) ( figure 2 ) . an exercise - induced weight loss was registered in the trained mice after 6 weeks of training as compared to the sedentary mice ( figure 4(a ) , p < 0.01 ) . from week 7 , the exercising mice continued to weigh less than their age - matched controls , and they remained leaner until the end of the experiment ( figure 4(b ) , p < 0.01 ) . at training week 7 , 4 out of 20 exercising mice became diabetic and that reflected the diabetic incidence percentage , although not significantly , between exercising ( 10% ) and control mice ( 3% ) ( figure 3(a ) ) . in detail , 2 out of 14 mice on training were diabetic , whereas , among the controls , 5 out of 18 mice were diabetic . on the other hand , a substantial number of animals remained diabetes - free at the end of the experiment ( 12 trained mice versus 13 controls ) ( figure 2 ) . the survival rate declined progressively in both groups , with no statistical difference between the exercising mice ( 70% ) and the controls ( 88% ) , upon completion of the 12-week training program . this final rate was indirectly linked to the early diabetic incidence occurring in the trained mice , starting from week 7 . in fact , those 4 mice that became diabetic after 7 weeks of training died concurrently during week 10 . overall , 6 mice of the exercising group died because of diabetes ; likewise 2 control mice faced the same fate . a submaximal incremental running test was performed at baseline and upon completion of the 12-week chronic training program to determine the acute exercise capacity of the nod mice , putatively conditioned by the endurance training per se and , above all , by the diabetes progression . to evaluate this , we differentiated the results coming from all nod mice ( trained versus controls , regardless of the diabetes diagnosis ; figures 5(a ) and 5(c ) ) and those arisen from the comparison on all diabetes - free nod mice ( trained versus controls , without diabetes ; figures 5(b ) and 5(d ) ) . at the end of 12 weeks of training , maximal running speed assessed during the submaximal test was found to be significantly decreased in all nod mice with respect to their pretraining values ( p < 0.05 ) , whereas no difference was found between trained and control animals when comparing their posttraining values ( figure 5(a ) ) . when diabetes - free mice were compared , trained mice worsened their maximal running speed at 12 weeks with respect to their baseline values ( figure 5(b ) , p < 0.01 ) . similarly , a significant reduction in the distance covered during the submaximal performance test was also found in the nod - exercising mice at week 12 with respect to their pretraining values ( figure 5(c ) , p < 0.01 ) and the age - matched controls also ( figure 5(c ) , p < 0.05 ) . however , even the control mice deteriorated their performance after 12 weeks as compared to the maximal distance covered at baseline ( figure 5(c ) , p < 0.05 ) . a significant decrease in the maximal distance was also found in the diabetes - free trained mice at week 12 as compared to week 0 ( figure 5(d ) , p < 0.01 ) . typical islet from a diabetic exercising animal after 6 weeks of training is shown ( figure 6(a ) ) in the pancreatic section triple stained for insulin , glucagon , and nuclei . immunostaining of pancreatic sections indicated the presence of larger infiltrates in control mice , along with a greater amount of -cells in the nod - exercising mice at the end of the 12-week training program ( figures 6(b ) , 6(c ) , and 6(f ) ; p = 0.052 ) . pancreatic infiltrates were ubiquitously detected in mice on training and controls , whereas no -cells were found at the end of the study in all diabetic mice . serum pro- and anti - inflammatory cytokines measured at the baseline , after 8 weeks of training and at the conclusion of the 12-week training program , fluctuated with no statistical significance throughout the timeline in all nod mice ( figure 7 ) . only some cytokines seemed to be aligned to an exercise - promoted anti - inflammatory pattern : il-6 and mip-1 resulted in being lower , even though not significantly , at the termination of the training program in the trained animals compared to the sedentary controls , and with respect to their pretraining values . with this in vivo approach , we wanted to evaluate how physical activity as endurance training can contribute in controlling t1d progression and analyzing the role played by exercise on the immune system . preliminary studies have shown the importance and impact of physical exercise on controlling autoimmune diseases , by modulating different immune parameters and components [ 11 , 1823 ] . most studies on the effects of exercise on immunity have shown that habitual , moderate - intensity training augments immune responses by increasing the expression of proinflammatory cytokines , including il-2 , il-1 , and tnf- , while exhaustive exercise tends to be immunosuppressive [ 2426 ] . on the basis of our data , nod mice appeared to be interesting for the studying of insulin - dependent diabetes although they remained a questionable model for running exercise studies with high significance for the autoimmunity . in fact , in nod mice , diabetogenesis is under complex polygenic control and the penetrance of these polygenes is greatly conditioned by wide disparate conditions ( microbial and physical environment , diet , etc . ) : some environmental pathogens may even offer more protection to nod mice against the t1d onset , by the simple activation of their immune system . in this perspective , our hypothesis to exploit the positive stimulation of physical exercise was tricky to be challenged . on a side , exercise , under adequate mild doses , would have counteracted the activation of diabetogenic innate immune responses or , at least , it would have exerted those preventive , protective effects in t1d by decreasing oxidative stress and preservation of -cell integrity . however , there is incomplete knowledge on how exercise acts in t1d , and therefore physical activity could be as stressogenic as many other detrimental conditions for which nod mice are highly susceptible to develop diabetes . in our study , the design failed to demonstrate a delay in the onset of t1d thanks to exercise ; on the contrary , an early diabetic incidence occurred in the exercising group ( n = 4 ) as early as week 7 of the training program , although not significantly . cumulative diabetic incidence was though consistent with data of the literature , confirming expected percentages of t1d penetrance in this model . certainly , diabetes progression affected per se the exercise capacity of the mice irrespective of the endurance training : physical exercise represented an additional stress to the nod - exercising mice , even at moderate intensity . however , those nod mice , able to tolerate the exercise stress , showed at the same time a slightly ameliorated inflammatory metabolic profile , one that may be contrasting the diabetes progression and the inflammation attack toward the pancreatic islets . overall , exercise neither was able to modulate and control the immune attack toward autologous -cells even if initiated prior to the t1d onset , nor had ability to rescue -cells once lost , in accordance with previous studies [ 12 , 13 , 29 ] . however , after 10 weeks of training , diabetic and nondiabetic trained mice showed better glucose profiles than the control mice ( figures 1 and 2 ) . today the mechanisms by which exercise regulates glycemia in a t1d context are still unclear . previously , very few studies documented the possibility to positively control and lower blood glucose by means of exercise in t1d animal models . some of them were designed on rat models , using streptozotocin - induced diabetes , which rules out autoimmunity , a leading feature resembling human t1d [ 12 , 13 , 20 , 2931 ] . nod - scid mice were also studied because they do not develop t1d , since they do not have a functional immune system . other studies relied on exogenous insulin treatments to reduce blood glucose , which provides disadvantages when interpreting data , because high glucose is toxic to the islet cells , and glucotoxicity represents a biasing factor [ 32 , 33 ] . in the present study , nod mice were not treated with insulin . exercise had glucose - lowering effects , reaching statistical significance only in the late window of the observed training period ( figures 1(a ) and 2 ) . the significant reduction of blood glucose was paralleled also in diabetes - free nod mice , particularly when referring to the mean glycemic values ( figures 1(b ) and 2 ) . this could suggest a preserved ability by which exercise downregulates blood glucose , in nod mice , when the immune attack has not led yet to frank diabetes , that is , by insulin - stimulated glucose uptake in the skeletal muscle . over the 12-week study , both groups exihbited an expected increase in body weight ( + 1525% ) ; however , from week 6 , exercising mice began to display significant reductions in body weight compared to controls ( 15% ) which remained significantly lower ( p < 0.01 ) at the termination of the study , despite only a 1.4 g difference ( figure 4 ) . unfortunately , we were not able to ascertain whether this discrepancy was due to a simple exercise - induced leaner mass rather than a more cachectic state : no visible signs or discomfort or stress was though detected , except for polyuria in the diabetic mice . in addition , the expression profiles of serum inflammatory cytokines were not mirrored in the infiltrating immune cells , which are important mediators for -cell demise in the nod model . once precised , some of the cytokines measured in the blood at the termination of the study fluctuated in an anti - inflammatory fashion ( il-6 , mip-1 ) in the trained mice compared to controls , although without reaching statistical significance . therefore , these indications must be collected thoughtfully , especially considering the lack of statistical confirmation . besides , other cytokines ( il-2 , il-4 , and il-10 ) seemed to act in a proinflammatory way at the end of the study in the exercised mice . however , the important anti - inflammatory cytokine il-10 was found higher in the serum of the trained mice over the remainder of the study ( until week 10 ) compared to controls ( figure 7 ) . as to the inflammatory milieu in nod mouse islets , the morphological examination indicated a tendency for a decreased estimate of infiltrates in the trained mice along with a greater amount of -cell areas with respect to the diabetic controls ( figure 6(f ) , p = 0.052 ) . plesner et al . reported the nonbeta islet endocrine cell remodeling in diabetic nod mice : that study suggested that infiltrating immune cells may restrict alpha - cell expansion in nod mouse islet in the diabetic state . here , a greater alpha - cells - infiltrates ratio was also found in the diabetic states of trained mice sacrificed at week 7 of our study ( figures 6(d ) and 6(e ) ) . whether the alpha - cell mass may be dependent on the stage of disease remains to be ascertained . moreover , it would be interesting to understand when ( and possibly to which extent ) exercise may represent a strategy to maintain normal islet architecture in diabetes . a further in - depth analysis is obviously required to address this ; however , a putative capability of the exercising mice to oppose the diabetic deterioration that progressively sacrifices -cells and damage the pancreatic islet remains intriguing . still , the mechanisms by which exercise might be responsible for these effects need to be elucidated . an alternate explanation would be that the selected intensity / amount of exercise for this running study was revealed to be too stressful for this nod mouse model and therefore inappropriate to test the working hypothesis and meet the soft and hard endpoints [ 38 , 39 ] . our explorative studies set the base to further evaluate the implementation of likely exercise protocols along with the dosage of several autoimmunity markers and cytokine and chemokine profiles . in nod mice moderate - intensity exercise seemed to exert glucose - lowering effects only in the late states of diabetes . further studies are needed to clarify the utility of the nod mouse model to mimic and investigate the exercise effects in t1d , immunomodulation , and inflammation . specifically , dose - response studies in which exercise will be administered to nod mice at various levels of intensity will be necessary to determine the optimal regimen of physical exercise having clear - cut preventive effects on the development of t1d . a better understanding of the impact of exercise on immune function will be of assistance in designing improved treatments for patients with autoimmune diabetes in the clinical arena .

the nonobese diabetic ( nod ) mouse represents a well - established experimental model analogous to human type 1 diabetes mellitus ( t1d ) as it is characterized by progressive autoimmune destruction of pancreatic -cells . experiments were designed to investigate the impact of moderate - intensity training on t1d immunomodulation and inflammation . under a chronic exercise regime , nod mice were trained on a treadmill for 12 weeks ( 12 m / min for 30 min , 5 d / wk ) while age - matched , control animals were left untrained . prior to and upon completion of the training period , fed plasma glucose and immunological soluble factors were monitored . both groups showed deteriorated glycemic profiles throughout the study although trained mice tended to be more compensated than controls after 10 weeks of training . an exercise - induced weight loss was detected in the trained mice with respect to the controls from week 6 . after 12 weeks , il-6 and mip-1 were decreased in the trained animals compared to their baseline values and versus controls , although not significantly . morphometric analysis of pancreata revealed the presence of larger infiltrates along with decreased -cells areas in the control mice compared to trained mice . exercise may exert positive immunomodulation of systemic functions with respect to both t1d and inflammation , but only in a stringent therapeutic window .

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

PMC3947774

high frequency of established coagulopathy ( 30% ) in multiply injured trauma patients has been reported , which accounts for up to 40% of all trauma - related deaths . the standard laboratory screening tests of coagulation are the prothrombin time ( pt ) and activated partial thromboplastin time ( aptt ) , which can be affected adversely by poor sampling technique ; they vary in their complexity , turnaround time for clinical decision - making , and result reproducibility and have a need for special preparation or personnel . thromboelastography ( teg ) is a noninvasive diagnostic assay to evaluate the viscoelastic properties during blood clot formation and clot lysis , yielding a graphical and numerical output relating to the cumulative effects of various plasma and cellular elements of all phases of the coagulation and fibrinolysis . numerous studies have reported the utilization of teg as a monitoring device for hemostasis and transfusion management in various clinical settings , for example , cardiac surgery , liver transplantation , identification of patients with overt disseminated intravascular coagulation , hypercoagulability , and prediction thromboembolic events in surgical patients . studies claim that teg is a point of care device for rapid diagnosis and differentiation of hypercoagulable and hyperfibrinolytic conditions . teg 's ability to assess hemostasis in whole blood renders it to be ideal for rapidly identifying patients with trauma induced coagulation and transfusion guidance , as trauma patients present a spectrum of different coagulopathies that can be identified by teg . animal studies have depicted that the conventional laboratory tests most clinicians use to evaluate clotting function in trauma patients are inadequate to document the significant changes , due to hypothermia and hemorrhage . as values of viscoelastic haemostatic assays ( vha ) parameters differ in different ethnic populations , there is a lack of standard protocol based on vha parameters in indian population to guide transfusion of blood component therapy in bleeding patients . the purpose of this study was to compare teg with respect to the conventional coagulation tests and assess whether teg can serve as a screening test or replace the conventional routine test for the identification of coagulopathy due to trauma ; therefore we retrospectively studied hemostasis in critically injured trauma patients , admitted in level 1 trauma care center . patients were segregated as hypercoagulable and hypercoagulable with respect to the laboratory established reference range , manufacturer given range , and teg coagulation index . based on the ability of the above teg ranges to identify coagulation abnormalities in comparison to the conventional coagulation tests this was a retrospective study conducted at the department of laboratory medicine , of a level 1 trauma care center , over a period of six months ( january june 2013 ) . trauma patients with severe trauma ( n = 150 ) admitted to our level 1 trauma care center were retrospectively considered for the study , if their teg was analyzed on admission . patients between the age group of 16 and 50 years were included in the study . patients receiving anticoagulation therapy with warfarin or antiplatelet agents , patients with known underlying coagulopathy , and patients with clinical evidence of brain death at the time of admission , or evidence of other severe comorbidities , such as liver disease , diabetes mellitus , and known history of hypertension , which may influence the outcome , were excluded . the samples used for teg analysis were drawn on arrival of the patient to the emergency department , prior to any fluid administration . platelet count was done on sysmex xe-2100 hematology analyzer ( sysmex , kobe , japan ) . prothrombin time ( pt ) and activated partial thromboplastin time ( aptt ) were estimated on a sta compact automated analyzer ( diagnosticastago , france ) . thromboelastometry was performed on tem - a automated thromboelastometer ( framarbiomedica , rome , italy ) , using whole blood nonadditive ( 360 l ) . all analyses were performed with teg disposable cups and pins as devised by the manufacturer and measurements were performed within 4 minutes of sampling . teg evaluates the physical properties of the clot , via the torsion in a pin connected with a mechanical electrical transducer , suspended in cup . as the blood sample clots , the changes in rotation of the pin are converted into electrical signals that a computer uses to create graphical output . reference values successfully established in our laboratory for four tem parameters : reaction ( r - time ) time ( time from start to initial fibrin formation ) ; k - time ( clot kinetics , measuring time taken for a certain level of clot strength is reached ) ; -angle ( clot kinetics of clot buildup and cross - linking ) ; and maximum amplitude ( ma ) ( absolute clot strength ) , for 95% of 200 healthy volunteers ( 100 male and 100 female ) , were r - time : 1.814.2 ( min ) , k - time : 0.77.3 ( min ) , -angle : 27.372.3 ( ) , and ma : 32.187.9 ( mm ) ( table 1 ) . all patients underwent detailed clinical evaluation using the patient 's record , and data was complied which included age , gender , date of admission , description of injury , glasgow coma score , and injury severity score . coagulopathy was defined as > 1.5 times control pt & aptt values and/or inr > 1.6 . depending on the shape of the teg tracing , the hemostatic condition of a patient was defined as hypocoagulable ( if 2 or more parameters are observed : prolonged r - time , prolonged k - time , decreased -angle and/or ma ) or hypercoagulable ( if 2 or more of the following parameters are observed : short r - time , short k - time , increased -angle and/or ma ) . the teg coagulation index ( ci ) , derived from a linear equation that combines all the teg variables , was also calculated based on which the haemostatic condition of a patient was defined as hypercoagulable ( ci > 3 ) ; hypocoagulable ( ci < 3 ) . all patients were categorized as normal and outliers based on the normal range for the ( 1 ) plasma based routine coagulation test , ( 2 ) our laboratory established reference range , and ( 3 ) manufacturer 's reference range for teg parameters . true positive ( tp ) , true negative ( tn ) , false negative ( fn ) , and false positive ( fp ) were defined with conventional coagulation tests as standard . sensitivity , specificity , and accuracy were described in terms of tp , tn , fn , and fp ( the percentage of all patients with disease present who have a positive test ) = tp/(tp + fn ) = ( number of true positive assessment)/(number of all positive assessments ) . specificity . ( the percentage of all patients without disease who have a negative test ) = tn/(tn + fp ) = ( number of true negative assessment)/(number of all negative assessments ) . accuracy . ( the efficiency of a test is the percentage of the times that the test gives the correct answer compared to the total number of tests ) = ( tn + tp)/(tn + tp + fn + fp ) = ( number of correct assessments)/(number of all assessments ) . the other two basic measures of diagnostic accuracy , that is , positive predictive value ( ppv ) and negative predictive value ( npv ) , which are related to sensitivity and specificity through disease prevalence , were also calculated . the predictive value of a test is a measure ( % ) of the times that the value ( positive or negative ) is the true value ; that is , the percent of all positive tests that are true positives is ppv and npv is the proportion of negative test results that are true negative : positive predictive value = tp/(tp + fp ) negative predictive value = tn/(fn + tn ) . positive predictive value = tp/(tp + fp ) negative predictive value = tn/(fn + tn ) . one hundred and fifty injured patients presented to the emergency department from january through june 2013 were included in the study . 55.3% had incurred injury to the head ( hi ) of which 64 ( 77.1 ) had severe hi . average injury severity score was observed to be 28.4 11.3 ; mean glasgow coma scale ( gcs ) was 7.85 3.2table 2 . pt , aptt , and teg parameters for all patients are shown in table 2 . fifty - one patients were defined as coagulopathic by the conventional coagulation test , with an average hb of 10.2 3.15 mm / hg , platelet count 144.7 87.6 lakh , pt 21.6 5.4 sec , aptt 38.8 14.9 sec , and inr 1.8 0.6 . thirty patients were defined as coagulopathic by teg based on their haemostatic conditions , out of which twelve were hypercoagulable and eighteen hypocoagulable . this was done on the basis of the reference range that was established in our laboratory for indian population ; however when we applied the reference range of the manufacturer , there were 105 patients who developed coagulopathy , out of which 53 were hypercoagulable and 52 were hypocoagulable . patients were also defined as coagulopathic based on the teg coagulation index ; we observed that 73 were hypercoagulable and 7 were hypocoagulable . on comparing the identification of coagulopathy by conventional test to teg ( with laboratory reference range ) we observed 15 patients to be true positive ( tp ) , 15 were false positive ( fp ) , whereas 84 were true negative ( tn ) and 36 were false negative ( fn ) ; based on the above defined formulas the sensitivity was observed to be 29.4% , specificity was 84.8% , ppv was observed to be 50% , and 70% was the npv . when the identification of coagulopathy by conventional to manufacturers range for teg indices was compared , we observed 38 patients to be true positive ( tp ) , 67 were false positive ( fp ) , whereas 32 were true negative ( tn ) and 13 were false negative ( fn ) ; based on the above defined formulas the sensitivity was observed to be 74.5% , specificity was 32.3% , ppv was observed to be 25.3% , and 71.1% was the npv . for teg coagulation index we observed 23 tp , 57 fp , 42 tn , and 28 fn patients , with sensitivity 45.1% and specificity 42.4% , table 3 . many tests provide information regarding coagulation status , including platelet count , prothrombin time , international normalized ratio , activated partial thromboplastin time , d - dimer , and fibrinogen levels . none is satisfactory in a trauma setting , as they take a considerable time to measure and represent a single point in a potentially ongoing process of bleeding . by the time the test results are available , the patient may already have entered an irreversible state of hypothermia , acidosis , and coagulopathy . as a result , there is growing interest in point - of - care testing , with capability for repeated measurements and rapid results within minutes . pt and aptt reflect the overall activity from plasma clotting factors involved in the extrinsic and intrinsic pathways , respectively . pt or aptt do not adequately reflect coagulation defects from hypothermia and hemorrhage , as pt and aptt are performed in platelet poor plasma , which exclude the interaction of platelets and other blood components , which significantly contribute to trauma coagulopathy . teg variables are interdependent , measuring the interaction of the coagulation cascade and platelets in whole blood rather than specific endpoints in centrifuged plasma samples . enabling readily available analysis of coagulation in a shorter period of time than laboratory coagulation tests , thus providing useful and prompt identification of coagulation disorders . some studies report teg to be a useful research tool in comparison to six common tests , hematocrit , platelet count , fibrinogen , pt , aptt , and fibrin split degradation , suggesting a strong relationship . similar results were found between teg and standard laboratory tests of coagulation by kang et al . , depicting a good correlation between r - time and aptt . however teg continues to be a second - level hemostasis test due to the lack of its quality assurance procedures and that teg is not validated , as far as international standards are concerned . as a consequence of the dependence of teg on manual procedures , its versatility in terms of the type of sample and different initiators that could be used resulting in difficulty to establish standards and reference values , it still persists as an expensive , nonvalidated point - of - care device . there is also very limited published commercial data on performance with regard to assay precision or inter - assay variability that again leads to skepticism . other limitations associated with this technology are that , although it is sensitive to a deficiency or excess of coagulation factors , specific clotting factor deficiencies can not be identified , have a relatively long measurement time , and require technical expertise . the present study was conducted to evaluate the efficiency of thromboelastography in a trauma care setup to assess the changes in the hemostatic conditions of severely injured patients , in comparison to the routine coagulation tests . incidence of coagulopathy was observed to be 34% by the conventional coagulation tests ; this decreased to 20% by our laboratory established range , indicating that routine assays recognize certain patients as coagulopathic which may not have the clinical presentation , and as teg analyzes the whole hemostatic condition of the patient , it depicts these patients as normal ; 70% developed coagulopathy according to the manufacturer 's reference range , suggesting that many trauma patients would be incorrectly identified as coagulopathic by the manufacturer 's reference values . we found teg to have a sensitivity of 29.41% and a specificity of 84.84% for trauma coagulopathy , using our reference range . in their study to establish normal values for canadian population , scarpelini et al . reported a specificity of 81% for thromboelastography . in a prospective study of 36 adult patients undergoing cardiopulmonary bypass ( cpb ) to determine the utility of teg versus platelet studies and standard coagulation tests to more effectively discriminate patients likely to benefit from platelet or fresh frozen plasma ( ffp ) transfusion , essell et al . reported a sensitivity of ( 71.4% ) and the specificity ( 89.3% ) of teg . sensitivity is the proportion of true positives that are correctly identified by a diagnostic test ; it shows how good the test is at detecting a disease . specificity is the proportion of the true negatives correctly identified by a diagnostic test , suggesting how good the test is at identifying normal ( negative ) condition . we observed that the range given by the manufactures has a higher sensitivity ( 74.5% ) but a low specificity ( 32.3% ) compared to our laboratory range , which indicates that the manufacturers range may identify coagulation abnormalities in a number of patients , because a test with high sensitivity tends to capture all possible positive conditions without missing anyone . however a low specificity suggests that these patients may not have clinical presentation of coagulopathy . also high sensitivity but low specificity may result in that many patients who do not have coagulation abnormalities being identified as coagulopathic are then subjected to further investigation , using laboratory established range . the major limitation of both sensitivity and specificity is that they are of no practical use when it comes to helping the clinician estimate the probability of disease in individual patients . positive and negative predictive values describe a patient 's probability of having a condition once the results of the tests for this condition are known . in our present study teg using the laboratory range had ppv of 50% and npv of 70% ; for the manufacturers range ppv of 25.3% and npv of 71.1% were observed . the higher the ppv , the more likely a positive result is able to predict the presence of coagulopathy . . it will therefore be wrong for clinicians to directly apply published predictive values of a test to their own populations , when the prevalence of disease in their population is different from the prevalence of disease in the population in which the published study was carried out . accuracy measures how correct a diagnostic test identifies and excludes a given condition and can be determined from sensitivity and specificity with the presence of prevalence ; it measures the degree of veracity of a diagnostic test on a condition . we report an accuracy of 66% for teg using laboratory established range and 46.6% for the manufacturers . limitations of the present study are that teg investigations were not done in duplicate , as is the case for classical coagulation assays ; we did not take into account the transfusion requirements ; thus we could not assess the transfusion guidance using out teg reference values . however , point of care testing for hemostasis is emerging and its role in patient management remains to be demonstrated in prospective studies . based on the current study , it is concluded that for specific diagnosis of underlying etiology of bleeding , conventional coagulation tests remain the most sensitive tests compared to thromboelastography ; however in emergency trauma situations , where immediate corrective measures need to be taken based on coagulation parameters and conventional coagulation tests may cause delay , teg can give highly specific results depicting the underlying coagulopathy . however it is suggested that the reference range of teg tests should be established for each lab and its results eventually should be confirmed by specific coagulation tests .

background . thromboelastography ( teg ) unlike conventional coagulation assays evaluates the dynamic interaction of clotting factors and platelets indicating an overall clot quality . literature assessing the efficacy of teg in identifying trauma associated bleeding is lacking . aim . to compare teg with conventional plasma based coagulation tests and assess whether teg can serve as a screening test or replace the conventional routine test . materials . retrospective data was collected for 150 severe trauma patients . patients with known evidence of severe comorbidities , which may influence the outcome , were excluded . detailed evaluation of the patient 's clinical and laboratory records was conducted . diagnostic characteristics such as sensitivity , specificity , and accuracy were calculated . results . fifty - one patients were defined as coagulopathic by the conventional coagulation test , 30 by the laboratory established range for teg indices and 105 by manufactures range . specificity and sensitivity for the laboratory established range for teg were 29.4% and 84.8% ; for manufactures range sensitivity was 74.5% , specificity was 32.3% . conclusion . we observed that conventional coagulation assays are the most sensitive tests for diagnosis of coagulopathy due to trauma . however in emergency trauma situations , where immediate corrective measures need to be taken , coagulation parameters and conventional coagulation tests may cause delay . teg can give highly specific results depicting the underlying coagulopathy .

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

PMC5363493

gene therapy for x - linked severe combined immunodeficiency ( scid - x1 ) has been successfully employed in the clinic by restoration of c expression in bone - marrow - derived cd34 cells using integrating vectors.1 , 2 in the earliest studies , using conventional -retroviral vectors , robust immune reconstitution was observed in the majority of infants , likely owing to the selective advantage of the transduced progenitors . however , despite successful phenotypic correction , 25% of treated infants developed t cell acute lymphoblastic leukemia ( t - all ) as a consequence of insertional mutagenesis . furthermore , patients acquired secondary genomic abnormalities including activating mutations in notch1.3 , 4 although the role of insertional mutagenesis via the activation of proto - oncogenes such as lmo2 has been clearly implicated in the development of t - all in scid - x1 patients following gene therapy , the absence of adverse events in patients treated similarly for adenosine deaminase deficiency ( ada - scid ) , despite also exhibiting integrations near these proto - oncogenes , remains incompletely understood . we have previously developed a mouse model of lentivirus - mediated correction of scid - x1 and observed lymphoid malignancies . crucially , the oncogenic events observed in mice receiving gene therapy were not attributable to insertional mutagenesis , suggesting alternative mechanisms for genotoxic risk in this model . one significant difference between positive control scid - x1 mice reconstituted with wild - type hematopoietic progenitor cells and scid - x1 mice receiving gene therapy with an equivalent number of vector - exposed c - deficient progenitor cells was the absolute number of cells received that were competent for lymphoid reconstitution . the gene therapy group received a lower dose on account of only a subset of vector - exposed c - deficient cells being transduced . we therefore hypothesized that reconstitution of scid - x1 mice with limiting numbers of hematopoietic progenitor cells might be a risk factor for lymphoid malignancy . in the current study , we sought to explore the relationship between hematopoietic progenitor cell dose and the incidence of lymphoid malignancy by transplanting sub - lethally irradiated crag mice with serially fewer wild - type sca1 progenitor cells , in the absence of gene transfer . the progenitor cell doses examined fell within a range that supported immunological reconstitution , and the resultant lymphoid compartment sizes varied by no more than 2-fold , despite up to 100-fold differences in hematopoietic progenitor cell dose . of greatest interest , however , was the observation of a robust inverse correlation between cell dose and the incidence of t - lymphoid malignancy , with progressively shorter disease latency at lower cell doses . all malignancies were wild - type donor cell - derived and carried activating notch1 mutations . replicative stress has been implicated in the development of b cell leukemia in mice and is a plausible antecedent to t - lymphoid oncogenesis in the current study . while encompassing this possibility , our findings fall within a conceptual framework provided by recent insights into murine thymocyte biology . most notable among these is the observation of sustained thymic output despite complete t cell progenitor deprivation ( thymus autonomy ) , with autonomous t cell production being supported by induction of a thymocyte self - renewal phenotype.7 , 8 , 9 , 10 this self - renewal phenotype , which arises in the double - negative ( dn ) 3 thymic niche and can also be induced by deliberate lmo2 overexpression , appears to carry an inherent risk of oncogenesis that is dependent upon acquisition of additional genetic lesions , such as notch1 mutations . moreover , competition from bone - marrow - derived progenitors has been shown to increase the turnover of thymocytes with a self - renewal phenotype , thereby reducing the probability of acquiring oncogenic mutations . directly relevant to the scid - x1 phenotype is the inability of c - deficient thymic progenitors to progress to the dn3 niche , thereby reducing their ability to provide competition to c - proficient progenitors . thus , data generated in our model are consistent with limiting thymic precursor supply , not only inducing a self - renewal phenotype in the murine thymus , but also simultaneously reducing competition in the dn3 niche with a resultant cell dose - dependent increase in oncogenic risk . there is currently no evidence that limiting thymic precursor supply is a risk factor for lymphoid malignancy in humans ; nevertheless , the possibility of cell competition acting as a tumor suppressor mechanism in the human thymus following vector - mediated insertional oncogene activation remains a compelling hypothesis . coupled with disease - specific differences in the progression of thymic precursors through t cell ontogeny , this hypothesis has the potential to help explain differing risks of oncogenesis . to explore the notion that limiting thymic precursor supply is a risk factor for oncogenesis , sub - lethally irradiated crag2 recipients were reconstituted with different doses of wild - type bone marrow hematopoietic progenitor cells ( figure 1a ) . we chose to use crag2 recipients because il2rg mice have been shown to have a leaky t cell phenotype that manifests with age and would therefore not be suitable for longitudinal studies . importantly , however , the donor cells were il2rg . to ensure all mice received an equivalent cell dose , the total cell number was fixed at 5 10 per recipient animal , and , at the lower cell doses , sca1 progenitor cells from il2rg mice ( c null ) were combined with those expressing c ; 5 10 ( high dose ) , 5 10 ( mid dose ) or 5 10 ( low dose ) , with the latter two doses representing a phenocopy of 10% and 1% gene transfer , respectively . in this model , only wild - type cells are capable of progressing past the double - negative 2 ( dn2 ) stage of thymic development . to investigate long - term oncogenic risk , mice reconstituted with a high , mid , or low dose of c - expressing progenitors were observed for up to 18 months . lymphoid malignancies were never observed in mice receiving the high dose , consistent with our previous study but detected exclusively in animals from the mid- and low - dose groups between 6.6 and 17.6 months post - transplantation ( figure 1b ) . the latency for oncogenesis was also significantly longer in the mid - dose group compared with low - dose group ( 12.8 3.5 and 8.9 1.4 months , respectively ; p = 0.02 ) . of the 31 mice that had received the mid cell dose , four developed malignancy , while the incidence of malignancy was not significantly greater than in mice transplanted with the high dose of progenitors , the survival rate was decreased ( p = 0.03 ) . in the low - dose group , both the incidence of malignancy and malignancy - free survival rates were significantly different when compared with the high - dose group ( p = 0.0017 and p = 0.0001 , respectively ) . of interest , the rate of malignancy in mice transplanted with the low dose of c - expressing cells ( 33.3% ) was similar to that observed in our previous study ( 28.6% ) . necropsy showed hepatosplenomegaly with extensive blast infiltration , and histological analysis revealed marked disruption of tissue architecture in the liver , spleen , thymus ( figure 1c ) , lung , and kidney ( data not shown ) . interestingly , enlarged thymi containing malignant cells were almost exclusively present in mice transplanted with the lowest dose of c - expressing progenitors , which was in contrast to mice that developed malignancy from the mid dose group , where the thymus was small or , in most cases , not macroscopically visible ( table 1 ) . abnormal t cell populations in 12 of the 14 mice that developed malignancy were identified in the spleen , liver , thymus , bone marrow , and peripheral blood . the phenotypes of these tumors were diverse , with cells expressing immature , mature , and atypical combinations of surface markers ( table 1 ) . in addition , two of the 14 malignancies ( 14.3% ) did not express the t cell markers cd3 , cd4 , or cd8 and were b220 positive ( m249 and m250 ) . peripheral blood assessed at 6 ( data not shown ) and 16 weeks post - transplantation ( figure 2a ) demonstrated lymphoid reconstitution with all three doses , with t , b , and natural killer ( nk ) cell compartments observed at the expected ratios . lymphoid reconstitution for mice receiving the mid dose of c - expressing cells was only approximately half ( 47% ) that of animals receiving the high cell dose , despite receiving 10-fold fewer c - expressing cells . for mice receiving the lowest dose of c - expressing cells , lymphoid reconstitution was comparable to that of mice receiving the mid dose , and when compared to mice receiving the high dose of wild - type cells ( i.e. , 100-fold more c - expressing cells ) , the lymphoid compartment was reduced to only 43% . the level of bone marrow chimerism in mice reconstituted with either the high or low dose of wild - type ( cd45.1 positive ) progenitors was determined by flow cytometry . the contribution of wild - type cells was found to be 64.9% 3.0% ( n = 8) in the high - dose group , indicating that the majority of cells engrafted in the bone marrow were derived from the wild - type c - expressing progenitors . for animals transplanted with the low dose of wild - type cells , the contribution was 3.4% 0.3% ( n = 7 ) , consistent with only 1% of the transplanted cells expressing c . to determine whether t cell receptor ( tcr ) diversity was affected by limiting thymic precursor supply , tcr v repertoire and spectratype analyses ( on selected highly expressed tcr v families ) analysis of the tcr v repertoire demonstrated that , for the majority of cases , both broad representation of all v families and the percentage of each v family was equivalent in peripheral blood samples taken from mice receiving each of the three c - expressing progenitor cell doses ( figure 2b ) . spectratype analysis revealed normal gaussian distribution of peaks in mice receiving the high and mid dose of c - expressing progenitors for the majority of tcr v families investigated ( figure 2c ) . for mice reconstituted with low cell numbers , the spectratypes appeared more restricted for some tcr v families . the observation of a relatively diverse tcr v repertoire for all cohorts is suggestive of increased expansion of immature t cells at the low compared with the high cell dose prior to tcr rearrangement . to assess the level of thymic t cell export occurring in vivo , the number of signal joint t cell receptor excision circles ( sjtrecs ) , a by - product of tcr rearrangement , was quantified in sorted splenic t cell populations at 16 weeks post - transplantation ( figure 2d ) . the numbers of sjtrecs isolated from mice receiving the high and mid doses were similar , implying that the transplanted cells had undergone comparable cell division after tcr rearrangement , which occurs at the double - positive ( dp ) stage in the thymus . in contrast , there was a modest , but statistically significant , reduction in the number of sjtrecs present in splenocytes from mice transplanted with the low dose of progenitors when compared with both the high and mid cell doses . this is indicative of increased t cell expansion in the cohort of mice receiving the low dose of c - expressing progenitors and is consistent with the observation of similar peripheral t cell reconstitution when compared with the mid dose group ( figure 2a ) . we next sought to investigate the size and composition of the thymocyte population following transplantation . thymocytes are classified into distinct stages based on expression of cell - surface markers , the earliest of which are dn for both cd4 and cd8 . this dn population contains four sub - stages ( dn1dn4 ) identified by the level of cell - surface cd25 and cd44 . thymocytes then enter the dp stage where they are positive for both cd4 and cd8 before becoming single positive ( sp ; positive for either cd4 or cd8 ) . at 6 weeks post - transplantation , for animals receiving either the high or low dose of wild - type progenitor cells , the total number of thymocytes was significantly reduced when compared to age - matched wild - type control animals not receiving bone marrow transplantation ( figure 3a ) . this demonstrates that transplantation even of high doses of wild - type progenitors leads to sub - physiological levels of thymocyte reconstitution . the difference between the high and low wild - type treatment groups was also significant ; notably , however , the difference in the total number of thymocytes between these groups was only 7-fold , despite the 100-fold difference in the number of transplanted c - expressing cells capable of progressing past the dn2 stage , confirmed by flow cytometry using ly5.1 and ly5.2 labeling to identify the c - negative and c - positive populations , respectively ( figure s1 ) , indicating increased expansion in the low - dose cohort . the contribution of dn , dp , and sp cells in the thymus was examined by flow cytometry , which revealed a significant reduction of dp cells in transplanted animals , proportional to the number of available wild - type progenitors from the bone marrow ( figures 3b and s2a ) . interestingly , this was concomitant with a significant increase in the proportion of mature cd4 and cd8 sp cells , that also expressed cd3 , in transplanted animals compared with wild - type controls , potentially indicating an increased need for early t cells to transition through the thymus ( p < 0.01 for comparisons between all groups ) . examination of the dn compartment showed that the proportion of cells in the dn1 stage was significantly increased in animals receiving the low dose of c - progenitors compared both with mice receiving the high progenitor cell dose and with wild - type control animals ( figures 3c and s2b ) . a significant reduction was also observed for cells at the dn3 stage in mice receiving the low dose of c - expressing progenitors . the most evident difference between animals receiving the low dose of c - expressing progenitors and those receiving the high progenitor dose or wild - type animals not receiving bone marrow transplantation was in the fold changes of thymocyte cell numbers within the dn niches . for the low - dose cohort , the ratio of dn2 to dn1 cells was significantly reduced ( figure 3d ) , possibly reflecting a rapid transition of cells from the dn2 niche , in an attempt to prevent lymphopenia in these animals . in striking contrast , there was a marked 6-fold increase in the ratio of dn4 to dn3 cells in mice receiving the low dose of c - expressing progenitors , which may be indicative of a homeostatic mechanism to maintain the peripheral compartment size . it is possible that cells within the dn4 niche ( i.e. , negative for cd4 , cd8 , cd25 , and cd44 ) could include progenitor cells of non - t cell lineages that reside in the thymus which may affect interpretation of dn ratios . it should also be emphasized , despite the differences observed in the relative proportion of each dn niche , particularly for animals receiving the low progenitor cell dose compared to wild - type control animals , that this is in concert with a marked reduction in the absolute number of cells in the thymus , and , therefore , all dn niches are diminished . genomic dna was isolated from malignant blasts of affected animals and qpcr performed using primers to detect wild - type c sequence , with wild - type sequence accounting for between 69.7% and 97.2% of the genomic dna sample and correlating with the majority of each sample containing malignant cells . importantly , this confirmed that all malignancies were derived from the transplanted c wild - type donor and not the c - deficient donor or recipient cell populations , which is supported by the expression of t and/or b cell markers in the samples ( table 1 ) . whole - genome sequencing ( wgs ) was performed on selected leukemic clones that contained a high proportion ( 75% ) of malignant cells ( figure 4 ; m97 , m137 , m189 , m191 , m209 , and m313 ) to identify potential common mutational signatures . microarray analysis was also performed on five of the six samples ( the quality of rna for sample m137 was not sufficient for analysis ) . of the 407 mouse orthologs contained in the sanger cancer consensus genes database ( http://cancer.sanger.ac.uk/census/ ) , 14 ( 3.4% ) displayed expression levels that were altered > 2-fold when compared to wild - type thymocytes ( brca2 , card11 , ccnd1 , gnaq , hmga1 , il21r , itk , jazf1 , notch1 , nr4a3 , pdgfrb , recql4 , rel , and tfrc ) . all sequenced samples except m313 ( adoptive transfer recipient ) showed evidence of substantial copy number decreases on the x chromosome , in comparison to sca1 bone marrow progenitors . also , substantial copy number gains on chromosome 15 were shown to be common for mice receiving the low dose of c - positive progenitors ( m137 , m189 , m191 , and m209 ) . interestingly , one sample ( m209 ) also displayed large copy number gains on chromosome 14 ( figure 4 ) . activating notch1 mutations were identified by wgs in each of the samples , comprising either a 5 deletion or mutations within the sequence encoding the proline , glutamate , serine , and threonine ( pest ) domain located in exon 34 ( figure 5a ) . sanger sequencing across the affected regions confirmed these mutations and gene expression profiling by microarrays identified notch1 upregulation in malignant samples ( 3.5-fold , false discovery rate [ fdr ] adjusted p value = 0.012 ) . of the six samples investigated , five contained the 5 deletion and five contained a mutation in exon 34 , with four of the six samples carrying both lesions . similar mutations have been identified in leukemia samples generated under conditions of thymic progenitor cell deprivation . to investigate the presence of activating mutations in the notch1 proto - oncogene in our other samples , dna sequence analysis was performed across the breakpoint of the 5 deletion as well as the pest , transactivation ( tad ) , or heterodimerization ( hd ) domains . further , activating notch1 mutations ( 5 deletions and/or pest domain mutations ) were identified in the remaining tumors ( those not subjected to wgs ) from mice transplanted with the low dose of c - expressing progenitors ( table 2 ; figures 5b and 5c ) . the impact of activating mutations in notch1 was investigated by using qrt - pcr directed at the downstream targets , c - myc , dtx1 , and hes1 . in the malignant blasts examined ( n = 5 ) , elevated levels of these transcripts were observed when compared to normal wild - type thymocytes ; c - myc , dtx1 , and hes1 were found to be upregulated by 6.85 1.80 , 2.47 0.54 , and 2.78 0.26-fold , respectively ( data not shown ) . upregulation of hes1 ( 9.78-fold , fdr = 0.021 ) and c - myc ( 3.25-fold , fdr = 0.073 ) in malignant blasts was also detected by microarray analysis . we have identified limiting bone marrow progenitor supply to the thymus as an initiator of lymphoid malignancy in mice . importantly , this oncogenic risk is cell dose dependent , occurs within cell dose ranges that support immunological reconstitution , and does not require the complete absence of progenitor cell migration from the bone marrow as previously observed in thymic grafting studies,8 , 10 making this feature of our model unique . based on existing evidence , the mechanism underlying oncogenesis is theorized to have two critical components . the first is the induction of a transformation - prone self - renewal phenotype in a subset of thymocytes as a homeostatic response to an inadequate supply of progenitors from the bone marrow.7 , 8 , 9 the second is reduced competitive pressure on this at - risk population for thymic niche space from differentiation - committed thymocytes progressing through lymphoid ontogeny . the proposed combined effect would be an increase in the number and residency time of transformation - prone thymocytes , leading to a correspondingly higher probability of cells within this population being subject to oncogenic second hit events , such as activating mutations in notch1 . in an effort to phenocopy gene therapy for scid - x1 in our model system , mice were reconstituted with serially fewer wild - type bone marrow progenitor cells , used as surrogates for gene - corrected cells , against a background of c - deficient progenitors adjusted to maintain a constant total cell dose in all treatment groups . in this context , the inverse correlation observed between the incidence of t - lymphoid malignancy and wild - type progenitor cell dose is consistent with a failure of c - deficient cells to progress to the point in t cell ontogeny , the dn3 niche , where competitive pressure on tumor - prone self - renewing thymocytes has been hypothesized to exert a tumor - suppressive effect . when we initially observed t - lymphoid oncogenesis , independent of insertional mutagenesis in our earlier gene therapy study in scid - x1 mice , we favored replicative stress as a possible tumorigenic mechanism . for this reason , an effort was made to examine the differing effects of limiting progenitor cell doses on the extent and location of the homeostatic cellular expansion during t cell ontogeny by assessing tcr diversity and number sjtrec in peripheral blood t cells , although an analysis of whether there were differences in the percentages of naive and memory peripheral t cells , with an increase in the latter indicative of homeostatic proliferation under conditions of lymphopenia , was not performed . despite 100-fold fewer cells capable of progression past the dn2 stage of thymic ontogeny , and competent for lymphoid ontogeny in the low - dose wild - type progenitor cell treatment group , reduction in the resultant peripheral t cell compartment size this is clearly indicative of higher levels of cellular replication during t cell ontogeny in these animals . while this represents a 50-fold change in the relative size of the input progenitor and output peripheral t cell populations , the relative difference in the absolute number of cell divisions required to achieve this , in an exponentially expanding population , is much smaller . accepting that the dynamics of thymic homeostasis are complex,14 , 15 this argues against the oncogenic risk observed at limiting progenitor cell doses being a simple function of the cumulative burden of somatic mutations acquired during individual cell division events . data from tcr diversity and sjtrec analysis in peripheral blood and splenic t cells , respectively , also provide similar arguments supporting this point . relatively well - preserved tcr diversity , in concert with trec - based evidence for a 2-fold replicative expansion of t cells following tcr rearrangement in mice receiving 100-fold fewer c - expressing progenitors , provides evidence of homeostatic expansion both before and after the dp stage of t cell ontogeny . while this supports a 50-fold relative expansion prior to , and a 2-fold expansion after the dp stage , in an exponentially growing population , the difference in the absolute number of cell divisions occurring before and after the dp stage is likely to be relatively small . further analyses , including whether there is increased proliferation of wild - type progenitors , compared to the immunodeficient populations , or a greater number of early thymic progenitors ( etps ) would contribute to the understanding of the extent and location of the homeostatic cellular expansion during t cell ontogeny in our model . early thymic progenitors , identified by high levels of ckit on their surface ( ckit cells ) , have been shown to support long - term thymopoiesis in intrathymic transplantation studies.13 , 16 if cell division itself was a strong risk factor for transformation , then one would expect a high proportion of such events to occur in more mature t cells , which is unsupported by existing evidence.7 , 8 , 9 , 10 , 11 when considering the possible relevance of these murine data to the oncogenic mechanisms operative in early human scid - x1 gene therapy trials , it is important to emphasize that murine cells are inherently more transformation - prone than human cells , and to our knowledge there is no evidence to suggest that limiting thymic precursor supply in humans carries oncogenic risk through the induction of thymocyte self - renewal . indeed , based on clinical experience in allogeneic stem cell transplantation for scid phenotypes including scid - x1 , qasim et al . have argued against oncogenic risk being associated with a lack of bone marrow progenitor supply to the human thymus . they point to the absence of reports of t cell malignancy in patients with long - term thymopoiesis , despite single - lineage donor t cell recovery , which is considered indicative of thymic , but not bone marrow engraftment . a further important point is that oncogenesis in early human clinical trials , using conventional -retroviral vectors to treat scid - x1 , chronic granulomatous disease ( cgd ) , and wiskott - aldrich syndrome ( was ) invariably involved insertional mutagenesis , while oncogenic adverse events have not subsequently been observed with sin -retroviral or sin lentiviral vectors lacking strong viral enhancer - promoter elements . nevertheless , we argue that insertional proto - oncogene activation in human hematopoietic progenitors , as reported in early scid - x1 trials , is likely capable of inducing transformation - prone thymocytes . this is supported by the observation that deliberate lmo2 overexpression in mice can induce thymocyte self - renewal without a homeostatic drive being exerted by a limiting or absent progenitor supply . once such cells arise in the human thymus , albeit by an iatrogenic mechanism , it is plausible that c - deficient human thymic progenitors , akin to their murine counterparts , also fail to exert a tumor suppressor effect through competitive pressure . thus , the parallels of our findings , and those of previously reported thymic transplant studies , to the human gene therapy experience occurs once the cells acquire a capacity for self - renewal , induced by either limiting precursor supply or by proto - oncogene activation . this also provides an alternative explanation for the suggested oncogenicity of c itself that has been the topic of much debate.21 , 22 an intriguing question arising from the tumor suppressor hypothesis is whether there might be disease - specific differences among disorders affecting t cell ontogeny in the capacity of thymocytes to exert tumor - suppressive effects through cell competition . for example , in gene therapy trials for ada - scid , using conventional -retroviral vectors to transduce bone - marrow - derived cd34 cells , there have been no reports of t cell leukemia , despite the frequent observation of integration events in lmo2 and other proto - oncogene loci . unlike c - deficient thymocytes , which fail to progress beyond the dn2 thymic niche , ada - deficient thymocytes are able to progress through thymic ontogeny when the thymic microenvironment is sufficiently metabolically detoxified , thereby providing a possible tumor suppressor effect in more distal thymic niches by competing with transduced cells carrying insertional proto - oncogene activation events ( figure 6 ) . while alternative hypotheses exist to explain the differing risk of oncogenesis in early gene therapy trials for scid - x1 and ada - scid , none provide a unifying conceptual framework . for scid - x1 these include evidence for cooperativity between lmo2 and c expression in the thymus with double transgenic mice showing accelerated development of t cell malignancy , strikingly in association with notch1 mutations . for ada - scid , these include the possibility that the progenitors transduced in bulk cd34 populations might be more differentiated than for scid - x1 , and that the level of vector - encoded ada expression might be insufficient to support the development of a t cell leukemic phenotype , which has been shown to be associated with increased ada expression . another notable difference between these two gene therapy trials was the use of sub - ablative busulfan conditioning in ada - scid patients , which is known to increase the level of bone marrow engraftment . it is therefore conceivable that increased bone marrow engraftment may lead to increased thymic seeding with a resultant reduction in oncogenic risk . increasing the level of myeloablative condition in our model might also reduce the incidence of malignancy for a given cell dose . a further challenge is to reconcile the concept of cell competition as a tumor suppressor mechanism in the thymus with the occurrence of t cell leukemias in a gene therapy trial for wiskott - aldrich syndrome ( was ) using a conventional -retroviral vector . six of ten treated patients developed t - all with dominant lmo2 clones identified in each case . several of the leukemias also had secondary mutations in the proto - oncogenes tal1 and lyl1 , both of which are known to be associated with t - all . similar to ada - scid , there is no absolute block to t cell ontogeny in the thymus in was , although abnormalities of t cell development have been implicated in this condition.28 , 29 , 30 of particular relevance , the was protein ( wasp ) has been shown to be important for double - negative to double - positive cell transition that may partly be explained by reduced cycling of dn3 cells . further , it has been suggested that in the absence of wasp , the decreased migratory responses and increased percentage of single positive cells implies retention of these cells in the thymus . it therefore remains plausible that this could result in reduced competitive pressure on thymocytes rendered pre - leukemic by insertional proto - oncogene activation . data derived from our model are certainly consistent with this possibility given our demonstration that lymphoid oncogenesis does not require a complete block in bone marrow progenitor supply and , at least in mice , is observed at wild - type progenitor cell doses that are sufficiently high to reconstitute the peripheral t cell compartment . ultimately the hypothesis that ada and was protein - deficient thymocytes have differing capacities to act as tumor suppressors in the thymus , through cellular competition , requires experimental evaluation and could be addressed in the model system we describe in the current report using c - deficient cells . ultimately , despite evidence that insertional proto - oncogene activation was necessary , but not sufficient , for development of t cell leukemias in early gene therapy trials for scid - x1 , the second hit events required for transformation , which we hypothesize are suppressed by cellular competition , show considerable similarity in mice and humans , most notably activating mutations in the pest domain of notch1.3 , 4 these , predominantly frameshift , mutations result in the production of a truncated protein and persistent activation of notch1 downstream targets5 , 31 , 32 and are consistent with those identified in our previous gene therapy model . interestingly , in contrast to the findings of martins et al . , mutations in the hd or tad domains were not present in our samples . this is likely due to the notch1 5 deletion being functionally similar to a hd mutation ( see below ) ; thus , the presence of a 5 deletion and hd mutation in the same cell would be mechanistically redundant . finally , consistent with the strong association of notch1 mutations and cases of t cell leukemia and lymphoma in the two malignancies not expressing t cell - surface markers ( m249 and m250 ) , notch1 mutations could not be detected by sequencing amplicon clones . , where all leukemias had an immature cd8 single - positive ( isp)/double - positive ( dp)-like surface phenotype and possibly reflect inherent differences between these two models , notably the source of thymic progenitors being either whole thymus or , in our model , more immature bone marrow progenitor cells . in samples with the 5 deletion , an aberrant notch1 mrna initiated from a cryptic promoter in exon 25 produces an active intracellular notch1 protein that is independent of ligand binding and -secretase cleavage35 , 36 , 37 and therefore constitutively expressed ( figure 5a ) . these mutations have been shown to result from illegitimate v(d)j recombination and , in our study , may have arisen from a failure of cells to exit the thymus as a result of reduced progenitor cell supply . notably , although 5 deletions in notch1 were not identified in scid - x1 patient samples , they did contain deletions in the tumor suppressor gene locus cyclin - dependent kinase inhibitor 2a ( cdkn2a ) , also shown to be the consequence of aberrant v(d)j recombination ; however , similar cdkn2a deletions were not found in the six tumor genomes analyzed by wgs in this study . further evidence of illegitimate v(d)j recombination was identified in our model by whole - genome sequencing , where both / and / recombination events were present in all six tumor genomes analyzed ( data not shown ) . this is in keeping with the observation that the t - all that developed in some patients in the scid - x1 trials arose from both the / and / t cell populations . in summary , our studies support the role of regenerating thymic precursors in the thymus leading to further genetic abnormalities that predispose to cancer . these findings help explain the differences in cancer risk in diseases treated with gene therapy based on the relative size of the thymic precursor pool and the need for regenerative thymic clones that are predisposed to malignant transformation . although there is currently no evidence that limiting thymic precursor supply is a risk factor for lymphoid malignancy in humans , our data provide a compelling conceptual framework that could explain why t cell leukemia was observed in early scid - x1 trials but not in ada - scid patients , despite both groups containing integrations near proto - oncogenes such as lmo2 . il2rg mice were obtained from the jackson laboratory , crag2 mice were kindly provided by professor stephen nutt ( walter and eliza hall institute , parkville , australia ) , and c57bl/6 mice were obtained from the animal resources centre ( arc ) . all experimental procedures were approved by the animal ethics committee of the children s medical research institute and the children s hospital at westmead . recipient crag2 mice were exposed to sublethal ( 6 gy ) irradiation using a gammacell 40 exactor ( mds nordion ) . bone marrow was harvested from 8- to 10-week - old il2rg , c57bl/6 , or c57bl/6 ly5.1 congenic mice , and sca1 progenitor cells were isolated using the easysep sca1 positive selection kit ( stemcell technologies ) , resuspended in 200 l rpmi 1640 medium ( life technologies ) and injected intravenously into recipient mice via the tail vein . at 15 weeks post - transplantation , individual tcr v families in peripheral blood were detected using qrt - pcr and primers specific for the tcr chain . the tcrv repertoire diversity of the six most highly represented tcr v families was subsequently analyzed by cdr3 spectratyping as outlined previously . at 16 weeks post - transplantation , t cells from reconstituted mice were separated using the easysep mouse cd90.2/thy 1.2 positive selection kit ( stemcell technologies ) and genomic dna isolated using the gentra puregene cell kit ( qiagen ) . signal joint t cell excision circles ( sjtrec ) were detected by using real - time qpcr with the following primer and probe set ; forward 5-cattgcctttgaaccaagctg-3 ; reverse , 5-ttatgcatagggtgcaggtg-3 ; probe 5-fam - cggcagggtttttgtaaaggtgctcactt - qsy-3. reactions were performed on a rotor - gene 6000 ( qiagen ) , and the number of sjtrec within each sample was determined by using the comparative ct method and a sample containing a known number of sjtrec copies , diluted in murine 3t3 cell genomic dna . the constant gene segment of the tcra gene ( c ) was used to compensate for variations in input dna with the following primer and probe set : forward 5-tgactcccaaatcaatgtg-3 ; reverse , 5-gcaggtgaagcttgtctg-3 ; probe 5-fam - tgctggacatgaaagctatgga - qsy-3. cells from the bone marrow , thymus , spleen , liver , and peripheral blood were stained and analyzed on a facscanto ( bd biosciences ) running facsdiva ( v.6.1.3 ) and flowjo ( v.7.6.1 , flowjo llc ) software . lymphocytes were identified and gated based on their forward - scattered light ( fsc ) and side - scattered light ( ssc ) profiles . cell suspensions were treated with ack lysis buffer ( 150 mmol / l nh4cl , 10 mmol / l khco3 , and 0.1 mmol / l disodium edta ) and then stained with the following conjugated antibodies : b220-apc - cy7 ( bd biosciences ) , cd2-fitc ( bd biosciences ) , cd3-pe ( bd biosciences ) , cd3-alexaflore 647 ( bd biosciences ) , cd4-fitc ( caltag laboratories ) , cd8a - apc ( caltag laboratories ) , cd8a - alexaflore 647 ( bd biosciences ) , cd25-fitc ( bd biosciences ) , cd44-pe ( bd biosciences ) , cd45-apc ( bd biosciences ) , ki-67-fitc ( bd biosciences ) , ly5.1-percp - cy5.5 ( bd biosciences ) , ly5.2-percp - cy5.5 ( bd biosciences ) , nk1.1-apc ( caltag laboratories ) , and sca1-alexaflore 647 ( bd biosciences ) . tissue was dissected from c57bl/6 control or transplanted crag2 recipient mice and fixed in 10% buffered formalin ( sigma - aldrich ) . paraffin - embedded sections ( 5 m ) were stained with h&e and evaluated by light microscopy . images were captured from a zeiss axio imager.a1 microscope ( carl zeiss microimaging ) using a spot enhanced camera ( diagnostic instruments , scitech ) running spot software ( v.4.0.1 ) . to determine whether malignancies were donor or recipient in origin , genomic dna was isolated using the gentra puregene cell kit and qpcr performed using primers to either the human c ( 5-ctttattgataacgatctatatccctcaccc-3 and 5-ctccactctgcagagtctatggaatcc-3 ) or bacterial neomycin phospho - transferase ( 5-cttgggtggagaggctattc-3 and 5-aggtgagatgacaggagatc-3 ) gene . reactions were performed on a rotorgene 6000 ( qiagen ) using the quantitect sybr green pcr kit ( qiagen ) and standardized using primers to the murine titin gene ( 5-aaaacgagcagtgacgtgagc-3 and 5-ttcagtcatgctgctagcgc-3 ) by the comparative ct method . genomic dna from six lymphoid tumors and one control sample of c57bl/6 sca1 progenitor cells were sequenced at the beijing genomics institute ( bgi ) , using hiseq2000 . whole - genome sequencing was achieved to 30 times coverage and a comparative analysis between the control sample and the tumors , to call tumor - specific somatic single nucleotide variants ( snvs ) , somatic copy number variations ( cnvs ) , short insertions / deletions ( indels ) , and structure variant ( sv ) mutations , was performed . bgi s cancer genomics bioinformatics pipeline was applied for sequencing analysis and variation detection ( http://www.bgitechsolutions.com/ ) . paired - end reads were aligned by burrows - wheeler aligner ( bwa ) to the mouse mm9 reference genome . all bam files were processed to identify duplicates using the picard markduplicates tool from the broad institute . snvs were detected using varscan , indels were called by samtools , cnvs were called by bgi s proprietary method cnv_detection , and svs were called by crest . the integrative genomics viewer ( igv ) 2.3 was used to visually inspect sequence reads . bam files and associated metadata in xml format have been uploaded to the european nucleotide archive ( ena ; http://www.ebi.ac.uk/ena ) under the study identification number prjeb8006 . total rna for expression analysis was isolated using the rneasy mini kit ( qiagen ) , and transcriptome profiling was performed by the ramaciotti centre for gene function analysis ( university of new south wales ) . briefly , total rna samples ( 500 ng / sample ) were amplified and labeled according to the manufacturer s instructions ( affymetrix ) . amplified rna was quantified using a nanodrop ( thermo scientific ) and distribution of the amplified material was analyzed in a 2100 bioanalyzer ( agilent ) . amplified crna was hybridized onto affymetrix mouse 2.1 gene arrays ; the arrays were scanned and expression measurements extracted using the affymetrix expression console v.1.3 software . microarray data have been deposited at arrayexpress ( http://www.ebi.ac.uk/arrayexpress/ ) under the accession number e - mtab-3159 . robust multi - array average ( rma ) was applied to normalize the expression data , and the remaining analysis was carried out using bioconductor package in the r statistical language 3.01 . p values were adjusted by benjamini & hochberg s method to account for multiple test correction . genes with expression change 2-fold and adjusted p value ( fdr ) 0.05 were considered as differentially expressed . mutations within the pest , tad , or hd domains of notch1 were identified by sequencing pcr products amplified using pfu dna polymerase ( promega ) . primers spanning exons 26 , 27 , and 34 , where the hd , tad , and pest domains are located , are described elsewhere . the presence of structural variations in notch1 identified by was was confirmed by pcr amplification using phusion high - fidelity dna polymerase ( new england biolabs ) and primers adjacent to the 5 and 3 break points ( 5- cagtcaggagtggtggatcc-3 and 5-ggcttggtctagtgctcctg-3 ) . in cases where the pcr product contained more than one amplicon , the pcr product was cloned into pgem - t easy ( promega ) , and the sequence of individual clones was determined . total rna was extracted using the rneasy mini kit ( qiagen ) from lymphoblastic cells or from wild - type thymocytes and cdna synthesized from 1 g total rna by using the superscript iii first - strand synthesis supermix kit ( life technologies ) . to determine whether mutations in notch1 affected expression of downstream target genes , qrt - pcr was performed on a rotorgene 6000 using c - myc ( 5-ctgtttgaaggctggatttcct-3 and 5-cagcaccgacagacgcc-3 ) , dtx1 ( 5-tgcctggtggccatgtact-3 and 5-gacactgcaggctgccatc-3 ) , and hes1 ( 5-aagacggcctctgagcaca-3 and 5-ccttcgcctcttctccatgat-3 ) primers using the brilliant iii ultrafast sybr green qpcr master mix ( agilent technologies ) . expression levels were standardized using primers to the -actin gene ( 5- ctagacttcgagcaggagatggc-3 and 5- tctgcatcctgtcagcaatgcc-3 ) by the comparative ct method . results were analyzed by the wilcoxon rank - sum ( mann - whitney ) ( http://www.socscistatistics.com/tests/mannwhitney/default2.aspx ) or fisher s exact test ( http://www.graphpad.com/quickcalcs/contingency1.cfm ) . for comparison of population survival , cohorts of mice were evaluated using kaplan - meier analysis ( graphpad prism v.5.04 ) . values represent the mean sem and results considered significant when p < 0.05 .

in early gene therapy trials for scid - x1 , using -retroviral vectors , t cell leukemias developed in a subset of patients secondary to insertional proto - oncogene activation . in contrast , we have reported development of t cell leukemias in scid - x1 mice following lentivirus - mediated gene therapy independent of insertional mutagenesis . a distinguishing feature in our study was that only a proportion of transplanted c - deficient progenitors were transduced and therefore competent for reconstitution . we hypothesized that reconstitution of scid - x1 mice with limiting numbers of hematopoietic progenitors might be a risk factor for lymphoid malignancy . to test this hypothesis , in the absence of transduction , scid - x1 mice were reconstituted with serially fewer wild - type hematopoietic progenitors . a robust inverse correlation between hematopoietic progenitor cell dose and t - lymphoid malignancy was observed , with earlier disease onset at lower cell doses . malignancies were of donor origin and carried activating notch1 mutations . these findings align with emerging evidence that thymocyte self - renewal induced by progenitor deprivation carries an oncogenic risk that is modulated by intra - thymic competition from differentiation - committed cells . although insertional proto - oncogene activation is required for the development of malignancy in humans , failure of c - deficient thymocytes to effectively compete with this at - risk cell population may have also contributed to oncogenesis observed in early scid - x1 trials .

Introduction Results Discussion Materials and Methods Author Contributions Conflicts of Interest

PMC4421556

a 40-year - old colombian man living in central colombia , bogota , was referred to local hospital for turbid white urine that had appeared 2 months earlier . milky. the phenomenon was intermittent : the patient would pass discoloured urine during 4 or 5 days without complaints of fever , burning or urgency . thereafter , the urine was clear or pink . this anxious man had sought medical attention in various facilities , and despite the fact that no urinary tract infection was found by urine cytology and cultures , he had been repeatedly treated by urinary antibiotics that did not produce any effect on his albinuria . causes of turbid white urine at first visit to our nephrology unit , the patient kept describing his urine as milky. we verified that his description was appropriate by collecting urine in various occasions . in fact , analysing the urine revealed a massive proteinuria of 5 g / l , microhaematuria ( 1520 red cells per ml ) and leucocyturia ( 1015 leucocytes / ml ) . . a closer examination of urinary cytology showed that the white cell population consisted of abundant lymphocytes . further investigations revealed a normal renal function , with a creatinine clearance in the order of 105 ml / min . proteinuria was profuse ( 6.7 g/24 h ) , but serum albumin concentration was normal ( 43 g / l ) , and so were his serum lipids ( total cholesterol : 4.9 mmol / l , triglycerides 1.9 mmol / l ) . no oedema , no hypoalbuminaemia , no hyperlipidaemia and fasting coincided with a clearing of the urine so it could not be a nephrotic syndrome . the rest of the laboratory work up was also non - contributive : leucocytes 5700/mm , ( neutrophils 64% , lymphocytes 23% ) , haemoglobin 16.5 g / dl , haematocrit 48.2% , platelets 285.000/mm . a cystoscopy that found white urine in the bladder ( thus excluding malingering ) did not disclose tumoural or infectious lesions . the patient observed that the hospital diet that he found meagre also coincided with a clearing of the urine . this remark prompted us to ask the department of nutrition to give him fat meals . with a lipid - rich diet urine turned white and clotty . with a fat - free diet it was found that milky urine was demulsified with chloroform and sudan dye made it turn red . a new cystoscopy was made with previous administration of a fat - rich diet and disclosed a punctiform hole in the posterior wall of the bladder with a leak of white fluid ( figure 3 ) . iodopovidone closed the fistula and the patient was discharged with instructions to avoid fat foods . cystoscopic view of white urine flowing from a hole in the bladder wall ( arrow ) . its differential diagnosis includes phosphates , calcium and urates that in excess give the urine a turbid , white or cloudy appearance , although the amounts of these minerals are rarely high enough to produce albinuria . the urine ph plays a role in provoking a turbid discolouration of the urine : claude bernard in his doctoral thesis presented in paris in 1843 explained that rabbits , a species feeding on vegetal , excrete alkaline turbid urine that turns clear by adding acid to it . when thinking of mineral crystal deposits , some clues are given by the urine ph : phosphaturia is related to alkaline urine and sediment analysis shows identifiable crystals . when chyluria is centrifuged , it remains white , which permits to differentiate it from mineral deposits . other substances that can induce albinuria are pus in severe urinary tract infections , or caseous material in urinary tuberculosis ( tb ) . once such causes have been ruled out , the diagnosis of chyluria is likely to be appropriate , but this has no reason to be in non - endemic regions of filariasis infection , which was the case in our patient who lives in central colombia . chyluria implies an abnormal communication between lymphatics and the urinary tract , mediated by any obstruction of intestinal lymph drainage resulting in lymphatic vessel dilatation and rupture into the urinary tract . this parasitic disease is frequent in geographic areas between latitudes 40 north and 30 south . about 10% of the population in these countries suffer from filariasis , and one - tenth of the affected patients present with albinuria caused by urinary - lymphatic fistulae . nonetheless , chyluria can be related to other rare non - parasitic diseases such as fungal infections , congenital abnormalities of the lymphatic vessels , malignancy , trauma and pregnancy . white urine emission alternates with macrohaematuria and is sometimes accompanied with renal colic produced by chyle clots . proteinuria is explained by the high concentration of albumin and fibrinogen in chyle ( 3060 g / l ) , and leucocyturia consists of lymphocytes . a lipid - rich meal is rapidly followed by albinuria , whereas a fat - free diet allows the urine to turn clear . firstly , it shows that chyluria may occur in any part of the world , and that european readers may come across a case of albinuria in a patient who never lived in an endemic parasitic region where w. bancrofti infection is common . secondly , diagnosing chyluria may simply be achieved by dietary manoeuvres , alternating a lipid - rich diet followed by a fat - free diet . however , albinuria and chyluria may be caused by congenital or acquired aetiologies of lymphatic fistulae . when fortune smiles , as it was in our case , the urologist may observe white urine running from a punctiform hole and treat albinuria by sclerotherapy or povidone injection into the fistula . however , a fat - poor diet must be pursued : the fistula might reopen , and besides , a patient whose bmi is 30 can not but benefit from it .

turbid white urine albinuria is defined as a urine discoloration described as milky or cloudy . one of the most frequent causes of turbid white urine is chyluria complicating filariasis ( table 1 ) . the extant causes of albinuria are non parasitic and rare . amongst their aetiologies stand excessive mineral sediment excretion such as calciuria and phosphaturia , massive pyuria and fungal infections , and rarely congenital malformations of the lymphatic vessels . malingering is also possible , in patients adding milk to their urine . we observed a case of albinuria in which the diagnostic work up led to diagnosing an exceptional cause of chyluria in a patient living in a region of colombia where filariasis is not endemic .

Case Discussion

PMC5364369

stress granules ( sgs ) are large cytoplasmic rna - protein assemblies that form during cellular stress , and play a pivotal role in the integrated stress response ( kedersha et al . , 2013 ) . following translational arrest , polysomes disassemble leading to a sudden increase in free mrna levels in the cytoplasm . it is believed that this free mrna subsequently nucleates the formation of sgs by binding of a large set of rna - binding proteins ( rbps ) ( jain et al . , 2016 ) . interestingly , several of these sg proteins have been linked to the neurodegenerative disorders amyotrophic lateral sclerosis ( als ) and frontotemporal lobar degeneration ( ftld ) , in which neuronal cytoplasmic fibrillar aggregates of rbps , e.g. , tdp-43 and fus , appear ( boeynaems et al . it is thought that these inclusion bodies play a key role in the disease pathogenesis ( ramaswami et al . , 2013 ) . moreover , mutations in several of these sg proteins are also the genetic cause of the disease in a subset of familial cases ( renton et al . , 2014 ) . hence , sgs have been suggested as a stepping stone towards pathological aggregation in als and ftld ( aguzzi and altmeyer , 2016 , king et al . , 2012 , based on previous observations from other rna granules and nucleoli ( brangwynne et al . , 2009 , brangwynne et al . , 2011 ) , recent studies have shown that the physical basis of sg formation is liquid - liquid phase separation ( llps ) ( burke et al . , 2015 , conicella et al . , 2016 , lin et al . , 2015 , , 2015 , murakami et al . , 2015 , patel et al . , 2015 ) . depending on in vitro conditions , recombinant tdp-43 , fus , and other hnrnps spontaneously demix from an aqueous solution and form liquid - like protein - rich droplets . it was shown that the intrinsically disordered prion - like domains ( prlds ) of these proteins by themselves are sufficient in inducing llps . interestingly , these liquid - like droplets can mature over time to more fibrillar states , and this process is accelerated by disease - causing mutations ( conicella et al . , 2016 , molliex et al . , 2015 , murakami et al . , 2015 , patel et al . , 2015 ) . other reports have shown that these prlds can also form reversible hydrogels , which have a high sheet content ( kato et al . , 2012 ) . it was recently suggested that cellular sgs consist of a stable core surrounded by a liquid - like shell , and atp - dependent remodeling seems necessary for maintaining sg fluidity ( jain et al . , 2016 , kedersha et al . , 2016 ) . these data point at the importance of a tight control of sg dynamics in preventing pathological aggregation . as mentioned above , prlds can undergo llps or form hydrogels in vitro ( burke et al . , 2015 , kato et al . , 2012 , lin et al . , 2015 , , 2015 , murakami et al . , 2015 , patel et al . , 2015 ) . this type of low complexity domain ( lcd ) shows a strong sequence similarity to yeast prions in that they are enriched in polar uncharged amino acids ( usually glutamine / asparagine ) and glycine ( king et al . , 2012 ) . degenerate tyrosine - containing repeats in the prlds of fus and other hnrnps , which can mediate hydrophobic and interactions , seem to be crucial in promoting hydrogel formation ( kato et al . , 2012 ) . the llps of these prlds indeed is inhibited by aliphatic alcohols , which interfere with hydrophobic interactions ( molliex et al . , phase separation based on hydrophobic interactions is also likely to underlie nuclear speckle and nuclear pore mesh formation ( frey et al . , 2006 , moreover , we find that pathogenic arginine - rich dipeptide repeats in c9orf72 als / ftld also undergo llps in vitro and in cells , hereby uncovering a potential new toxic pathway in the disease . sg proteins also contain other domains that might be non - redundant in phase separation . besides canonical rna - binding domains , e.g. , rrm , these proteins are also enriched in disordered arginine - rich sequences , e.g. , rgg boxes ( aguzzi and altmeyer , 2016 ) ( figure 1a ) , which can bind rna or poly(adp - ribose ) and seed llps or aggregation of prlds ( altmeyer et al . , 2015 , burke et al . , 2015 , lin et al . , 2015 , molliex et al . , 2015 , patel et al . , 2015 , schwartz et al . , 2013 ) . while globular rrms have a clear rna - binding function , these disordered arginine - rich motifs are more promiscuous and also serve as protein binding modules ( dormann et al . , 2012 , thandapani et al . , 2013 ) . given the strong conservation of both disorder and arginine content in rbps ( varadi et al . , 2015 ) , we hypothesize that arginine - rich domains could play a role in llps in physiological and pathological processes . in fact , 40 amino - acid - long arg / gly - rich peptides from the rgg boxes of fus , hnrnpa1 , and fmrp spontaneously phase separate in the presence of a molecular crowder ( figures 1b1d , s1a , and s1b ) . this emerging basic mechanism of llps puts particular emphasis on recent observations from c9orf72 hexanucleotide repeat expansions , which are the most common genetic cause of als and ftld ( dejesus - hernandez et al . a potential toxic mechanism is the generation of five species of dipeptide repeats ( dprs ) through aberrant translation of the repeat rna ( ash et al . several groups have shown that two of them , namely , glycine - arginine ( gr ) and proline - arginine ( pr ) repeats , are highly toxic ( boeynaems et al . , 2016a , freibaum et al . , 2015 , jovii et al . , 2015 , we wondered whether these peptides exhibit a behavior similar to the rgg peptides in vitro . synthetic gr20 and pr20 , but not gp20 , strongly phase separated following the addition of a molecular crowder ( figures 1c and 1d ) . interestingly , the extent of phase separation for all examined peptides is significantly correlated with arginine content ( figures 1c and s2a ) , which indicates that arginines are likely to be the mediators of phase separation for these peptides . to further investigate such arginine - mediated phase separations , we used the pr dpr as a model . as shown for other proteins ( molliex et al . , 2015 , nott et al . , 2015 , patel et al . , 2015 ) , phase separation of pr20 is temperature dependent and reversible ( figures 2a , 2b , and s1a s1c ) . pr20 droplets are highly circular ( figure 2c ) , suggesting that they minimize surface tension . when we applied shear stress to these droplets , we indeed observed plastic deformation with minimization of their surface area after relaxation ( figure 2d ) . there was also a conservation of volume after the fusion of two droplets ( figure 2e ) . all these observations indicate that pr20 droplets behave as a liquid . besides temperature , llps of pr was also sensitive to protein concentration , molecular crowding , and the length of the peptide ( figures 3a and 3b ) . in agreement with the polar nature of the peptide , we found that llps was not perturbed by an aliphatic alcohol , but was hindered by increasing the salt concentration ( figures 3c , 3d , and s1d ) . additionally , the fluorescence of the hydrophobic dye 8-anilino-1-naphthalenesulfonic acid ( ans ) showed maximum intensity at 495 nm and was reduced following llps of pr20 ( figure s1f ) ( hawe et al . , 2008 ) . these observations argue against hydrophobic interactions and indicate the importance of electrostatic forces via arginines in llps . phase separation of like - charged inorganic polyelectrolytes has been reported before , and due to charge repulsion , this process is only conceivable in the presence of counteranions in solution ( brangwynne et al . we reasoned that phosphate ions in our buffer could be a likely candidate in stabilizing pr - pr interactions . in accordance , when we incubated pr in pure water with the molecular crowder and added different salts , the extent of phase separation depended on the charge of the anions ( figure 3e ) . indeed , addition of polyu rna promoted llps in a dose - dependent manner even without molecular crowders being present ( figures 3f , 4a , and s1e ) ; and this was also the case for other arginine - rich peptides ( figure s2a ) . similar to the peg - induced llps , pr20-rna droplets were highly circular ( figure 4a ) . they also continuously increased in size over time , as measured by dynamic light scattering ( dls ) ( figure 4b ) , mimicking observations for cellular sgs ( wheeler et al . , 2016 ) . due to its polyvalent nature , polyu was much more effective than phosphate and crowding agent peg in promoting llps . besides purely electrostatic interactions , arginines can also engage in interactions with aromatic side chains ( brangwynne et al . , 2015 ) . in support of the contribution of this mechanism , incubating pr20 with poly - tyrosine induced clustering of the insoluble poly - tyrosine polymers ( figures 3 g and s1h ) . disordered linear binding motifs often undergo induced folding following ligand binding ( tompa , 2016 ) , but they can also remain predominantly disordered when bound in so - called fuzzy complexes ( tompa and fuxreiter , 2008 ) and when phase separated , as already observed in the case of fus lcd droplets ( burke et al . , 2015 ) . cd spectroscopy clearly illustrated the random coil nature of the dispersed pr20 peptide , and this was also the case for other arginine - rich peptides ( figures s2b however , after titration with polyu rna , the pr20 cd spectrum was lost , i.e. , there is no signal for the peptide within droplets ( figure s2 g ) . therefore , we applied two other approaches to monitor the structural state of pr20 in droplets , both supporting its largely disordered ( fuzzy ) state . fluorescence anisotropy of peptide tagged with a fluorophore ( figure s2h ) is very low ( 0.02 ) , and even after the addition of polyu rna it only increases to about 0.2 , far from the value measured in the highly viscous glycerol ( 0.38 ) , the latter being close to the theoretical upper limit ( 0.4 ) for an immobile fluorophore . global hydrogen - deuterium exchange ( hdx ) experiments ( figures s2i and s2j ) show full accessibility of the free peptide , which is compatible with its disordered state . following the addition of polyu rna , accessibility drops only slightly ( to 80% of its initial value ) , indicating that the peptide is still largely accessible , and suggesting that it stays disordered after llps . to check whether the pr - rna droplets could have any internal fibrillar structure , we performed cryo - transmission electron microscopy ( tem ) , and could not discern any visible structures in the droplets ( figure 4c ) . further , we performed fluorescence recovery after photobleaching ( frap ) analyses with the fluorescently tagged pr20 ( figures 4d and 4e ) , and found in full - droplet bleaching experiments that demixed pr readily interchanges with pr in the dispersed phase ( figure 4d ) . aging of the pr - rna droplets had no effect on intradroplet fluorescence recovery ( figure 4e ) . this result is in contrast to findings for prld llps , which undergo a liquid - to - solid transition likely through sheet formation ( conicella et al . , 2016 , we took soluble hela cell lysate , to which we added increasing concentrations of pr30 . the pr30 peptide spontaneously demixed , and could be separated from the solution by gentle centrifugation . the resultant pellet was subsequently washed and analyzed by gel electrophoresis ( figure 5a ) . unexpectedly , this pellet was largely resistant to washing steps , suggesting that pr induced the precipitation of different cellular proteins to the insoluble fraction . the demixing of pr was also largely independent of the presence of rna in the lysate , as addition of rnase only had a modest effect on the extent of protein precipitation ( figure s3 ) . to see whether weak interactions could still be involved in the process , we performed mild crosslinking with paraformaldehyde as recently used to identify both the stable core and liquid - like shell of cellular sgs ( jain et al . , 2016 ) . the pr fraction seemed to be enriched for a specific set of proteins as was evident from the band pattern . mass spectroscopy ( ms ) analysis on both crosslinked and uncrosslinked samples identified 874 proteins that were detected and quantified in both conditions . we performed gene ontology ( go ) enrichment analyses to identify overrepresented biological processes and cellular compartments in our hit list . significantly overrepresented terms centered on rna and protein metabolism ( go biological process ; figure 5b ) and different cellular liquid - like compartments ( go cellular compartment ; figure s3 ) . indeed , when comparing the protein content of the pr30 precipitate with those of stress granules and nucleoli , we found a strong overlap with both cellular liquid compartments ( figure 5c ) , while this was not the case for an unrelated organelle ( figure s4 ) . the pr30 precipitate was also enriched for metastable aggregation - prone proteins ( figure 5d ) . given that 60% of identified proteins have been implicated in rna metabolism ( figure s4 ) , we evaluated overrepresentation of rna binding domains . these included globular rrm or dead / h box domains and disordered rgg boxes ( figure 5e ) . besides rgg boxes , r motifs were also highly abundant ( figure 5f ) , once more illustrating the importance of arginine - rich binding modules in llps . lastly , we found that the pr30 precipitate is enriched for disordered ( figure 5 g ) and aggregation - prone supersaturated proteins ( figure 5h ) ( ciryam et al . , 2013 ) . quantitative analysis revealed that 190 proteins were more abundant after crosslinking ( figure s5 ) , which suggests that weak interactions between these proteins and the pr30-induced insoluble fraction could be stabilized by our mild crosslinking protocol . interestingly , this set of cross - linking - sensitive proteins was enriched for different protein families involved in stress granule biology , such as hnrnps ( molliex et al . , 2015 ) and aminoacyl transferases ( david et al . , 2011 ) although both 40s and 60s ribosomal proteins were highly abundant in our protein set identified by ms , only 40s , but not 60s , constituents were overrepresented in the crosslinking - sensitive fraction . cellular sgs also contain 40s , but exclude 60s subunits ( kedersha et al . , our in vitro cell - free findings , we overexpressed a codon - optimized pr100 construct in hela cells ( figure 6a ) . as previously reported , pr100 predominantly localized to the nucleolus ( kwon et al . , 2014 , tao et al . , 2015 , wen et al . , 2014 , yamakawa et al . , 2015 ) . yet , the evidence whether arginine - rich dprs affect sgs has remained ambiguous ( tao et al . we found cytoplasmic pr100 granules positive for sg markers after overexpression of pr100 ( figure 6a ) , and this process was dose and length dependent ( figure s6 ) . pr100 expression was more efficient at inducing sgs than pa100 expression , showing that these sgs do not just originate from the transfection protocol ( figure 6b ) . pr100-induced sgs were also responsive to known sg modifying compounds , i.e. , arsenite and puromycin treatment enhanced sg formation , whereas cyclohexamide reduced their number ( figure 6c ) . by using a mutant mef cell line carrying a non - phosphorylatable form of eif2 , we found that pr100 requires the integrated stress response for sg induction ( figure 6d ) . also the presence of g3bp1/2 seemed to be required for this , based on the use of knockout cell lines ( figure 6e ) ( kedersha et al . when studying these pr100-induced sgs in more detail , we observed that they are slightly less dynamic than arsenite - induced sgs , based on frap analysis of a g3bp1-gfp fusion protein ( figure 6f ) . moreover , the protein content of pr100 sgs was strongly altered compared to arsenite sgs . while there was no difference in the sg enrichment of a general sg marker ( yb-1 ) , both a late sg marker ( ddx6 ) and als - related proteins carrying a prld ( ataxin-2 and tdp-43 ) were significantly enriched in pr100 sgs compared to arsenite sgs ( figure 6 g ) . the current hypothesis for the formation of aggregates in als / ftld patients is a liquid - to - solid phase transition or perturbed clearance of sgs . although not observed in a living system so far , in vitro droplets of fus and hnrnps have been shown to mature to a solid state over the course of hours ( molliex et al . , 2015 , patel et al . , 2015 ) . this likely corresponds to a transition of their prlds from a disordered fuzzy state to a rigid sheet structure ( burke et al . , 2015 , molliex et al . , 2015 , patel et al . , 2015 ) . since we found that ( 1 ) pr repeats could engage in interactions with tyrosines , ( 2 ) pr induced the precipitation of several of these proteins in vitro , and ( 3 ) such proteins were enriched in pr100 sgs , we wondered whether pr could affect the dynamics and maturation of prld droplets . addition of pr30 , but not gp30 , to fus lcd droplets interfered with their spontaneous fusion dynamics ( figure 7a ) . additionally , fus lcd phase separation was also enhanced by addition of pr30 ( figures 7a and 7b ) . in agreement with the reduced dynamics of the droplets , kinetic thioflavin - t fluorescence analysis of fus lcd droplets supplemented with pr30 , indicated an increase in sheet content over time ( figures 7c and s7 ) . in this study , we have investigated whether highly repetitive and disordered arginine - rich peptides generated from expanded c9orf72 repeats in als / ftld could play a role in llps . this phenomenon is of intense interest since it could explain the biogenesis and physical underpinnings of sgs , which are the prime suspect of seeding for pathological protein aggregation in als and ftld ( king et al . , 2012 , ramaswami et al . , 2013 ) . until now , llps of prld containing rbps has been mostly attributed to hydrophobic interactions . however , arginine - rich domains are highly enriched in cellular liquid - like compartments , such as stress granules and the nucleolus , and both protein disorder and arginine - content are highly conserved in rbps ( varadi et al . , 2015 ) . this conservation suggests that these arginine - rich domains must be under selective pressure , highlighting their functionality and suggesting that a similar gain - of - function may underlie the pathological role of c9orf72 dprs in als . in fact , rgg boxes have been shown to affect prld aggregation and llps by nucleating on rna or poly(adp - ribose ) ( altmeyer et al . , 2015 , lin et al . , 2015 , , 2015 , patel et al . , 2015 , schwartz et al . , 2013 ) . we hence hypothesized that arginine - rich dprs could play a more direct role in phase separation through their disorder ( zhang et al . , indeed , we found that arginine - rich dprs derived from the c9orf72 repeat expansion undergo llps , a process mimicked by short synthetic peptides corresponding to the rgg boxes of three sg proteins , which also spontaneously phase separated in vitro . this phase transition was directly correlated with the arginine content of the peptides . as shown previously for inorganic polymers , such like - charged phase separation was dependent on counterions ( tiraferri et al . , 2015 ) . recently , two other studies have also reported a similar behavior for artificial arginine - rich peptides ( aumiller and keating , 2016 ) or peptides derived from nucleolar proteins ( mitrea et al . , 2016 ) . intriguingly , when we added cellular proteins to pr , this resulted in the formation of an insoluble pellet , rather than liquid droplets . however , crosslinking experiments showed that specific weak protein - protein interactions did occur in our test tube experiment . this is consistent with recent reports indicating that cellular sgs likely consist of a stable core surrounded by a liquid shell , rather than a homogeneous liquid droplet ( jain et al . the pr30 interactome using ms showed that pr was able to interact with a large , yet specific , set of proteins enriched for rna - binding domains , arginine - rich motifs and protein disorder . numerous hits were known constituents of endogenous liquid organelles , and , interestingly , several have been directly implicated in als / ftld . hence , we suggest that dprs may play a role in disease by upsetting the internal functional balance of membraneless organelles ( brangwynne et al . , 2015 ) . the data of our in vitro precipitation experiment are in agreement with three recent studies showing that intracellular pr and gr peptides can bind with a large set of proteins involved in rna- and sg metabolism ( lee et al . , 2016 , lin et al . , 2016 , lopez - gonzalez et al . , 2016 when overexpressed in cells , pr was indeed able to seed sg assembly , and this sg induction was dependent on phosphorylation of eif2 and the presence of g3bp proteins . although sg induction by pr was shown recently by others ( lee et al . , 2016 , yamakawa et al . , 2015 ) , the mechanism through which this occurred remained unknown and of intense interest from a therapeutic perspective . in this light , eif2 modulation has already been shown to ameliorate toxicity in different tdp-43 als models ( kim et al . , 2014 ) . g3bp proteins have been shown to be essential in eif2-controlled sg assembly ( kedersha et al . , 2016 ) , and the g3bp rgg domain plays a crucial role in this process , once more highlighting the importance of arginine - rich domains in sg metabolism . pr - induced sgs displayed reduced dynamics and were enriched for als - related proteins . moreover , pr was also able to accelerate the liquid - to - solid maturation of prld droplets , a process reminiscent of pathological aggregation ( conicella et al . , 2016 , molliex et al . in line with our findings , two other reports also recently found that arginine - rich dprs can affect sgs and other higher order assemblies in similar ways ( lee et al . sgs are generally considered to be the stepping stone toward pathological aggregation of rbps in disease . identifying the mechanisms behind altered sg dynamics and a deeper appreciation of their biology , hence , will provide us with invaluable clues to better understand the etiology of disease . disease mutations are known to target several sg proteins or proteins involved in sg dynamics ( ramaswami et al . , 2013 ) . however , the reason why wild - type proteins mislocalize to the cytoplasm and aggregate in c9orf72 cases remained elusive . recently , we and others have shown that nucleocytoplasmic transport defects caused by arginine - rich dprs could explain the mislocalization of these rbps ( boeynaems et al . , 2016a , boeynaems et al . , 2016b , freibaum et al . , 2015 , jovii et al . , 2015 ) . by focusing now on the biophysical behavior of arginine - rich dprs , we and others have found that they could also be directly implicated in the cytoplasmic aggregation of rbps . these data suggest that dprs could initiate a pathogenic cascade in c9orf72 als / ftld , by targeting the nuclear transport - stress granule axis ( boeynaems et al . reagent or resourcesourceidentifierantibodiesanti - flag , mousesigma - aldrichf3165 ; rrid : ab_259529anti - flag , rabbitcell signaling#2368santi - g3bp1 , mouseabcamab56574 ; rrid : ab_941699anti - ddx6 , rabbitabcamab40684 ; rrid : ab_941268anti - yb1 , rabbitabcamab76149 ; rrid : ab_2219276anti - tdp-43 , rabbitproteintech12892 - 1-ap ; rrid : ab_2200505anti - ataxin-2 , mousebd biosciences611378 ; rrid : ab_398900anti - tia1 , goatsanta cruzsc-1751 ; rrid : ab_2201433bacterial and virus strainse . coli , bl21 star ( de3)thermo scientificc601003chemicals , peptides , and recombinant proteinspr20this papern / apr30this papern / agr30this papern / agp30this papern / afus rggthis papern / ahnrnpa1 rggthis papern / afmrp rggthis papern / afus lcdr . , 20151,6-hexanediolsigma - aldrich240117polyu rnasigma - aldrichp9528poly - tyrosinesigma - aldrichp1800peg300sigma - aldrich81160anssigma - aldricha1028complete protease inhibitor cocktailsigma - aldrich11697498001arsenitesigma - aldrichs7400lipofectamine 3000thermo scientificl3000015nucbluethermo scientificr37605prolong gold antifadethermo scientificp36934critical commercial assaysalexa fluor 488 protein labeling kitthermo scientifica10235alexa fluor 568 protein labeling kitthermo scientifica10238deposited datamas spec datathis paperpride : pxd005509experimental models : cell lineshelaatccccl-2mef eif2a ssdr . , 2015plasmid : pr100-flag - mcherrythis studyn / a further information and requests for reagents may be directed to and will be fulfilled by the lead contact , ludo van den bosch ( [email protected] ) . paul taylor ( st . jude , usa ) ( figley et al . , 2014 ) . u2os g3bp1/2 ko cell line , and eif2 ss and aa mefs were a kind gift of dr . paul anderson ( harvard , usa ) ( emara et al . , 2012 , kedersha et al . , u2os and hela cells ( atcc ) were cultured in high glucose dmem ( invitrogen ) supplemented with 10% fetal bovine serum ( greiner ) , 4 mm glutamax ( invitrogen ) , penicillin ( 100 u / ml ) , streptomycin ( 100 g / ml ) and non - essencial amino acids ( 1% ) . flag - tagged dpr50 expression constructs were synthesized by genscript ( piscataway , usa ) . fus lc domain was expressed in e. coli and purified as described previously ( burke et al . , 2015 ) . hnrnpa2 lc domain ( residues 190 - 341 ) was expressed with a tev - cleavable n - terminal hexahistidine tag , purified from the inclusion body via histrap ( ge healthcare ) in urea containing buffers , concentrated , diluted into native buffer for tev cleavage of hexahistidine tag , resolublized by addition of solid urea , separated from his - tagged tev protease and cleaved his - tag by histrap , and concentrated to 1 - 2 mm in 8 m urea 20 mm mes ph 5.5 . peptides were diluted to the indicated concentrations in 100 mm k2hpo4/kh2po4 buffer at ph 7 , unless otherwise indicated . peg300 , polyu rna or poly - l - tyrosine ( sigma - aldrich ) were added at the indicated concentrations . for fluorescence microscopy and frap analyses , pr - alexa 568 and polyu30-alexa 488 were spiked in at 200 nm and 100 nm respectively . fus lc droplets were generated by diluting the stock solution to the desired concentration in 50 mm mes buffer at ph 5 , with the indicated nacl concentrations . hnrnpa2 lc droplets were generated by diluting the stock solution to the desired concentration in 20 mm napi buffer with 50 mm nacl at ph 7.5 . od600 of 60l samples was measured using truview microcuvettes in a smartspec plus spectrophotometer ( bio - rad ) . turbidity of 100l of fus and hnrnpa2 lc droplets was measured on a spectra max m5 microplate reader ( molecular devices ) . fus lc droplets were generated by diluting the stock solution to 250m in 50m mes buffer at ph 5 , 200m nacl . fluorescent droplets were incubated in plastic cell counter slides ( bio - rad ) at room temperature . every two hours three arbitrary fields were imaged on a zeiss lsm 510 meta nlo confocal microscope . tht was added to 100l pr30 + lc droplet mixtures at a concentration of 20m . fluorescence intensity over time was followed using a spectra max m5 microplate reader ( molecular devices ) . lag time was estimated as the time point of a local minimum in the curve . cd spectra of the peptides analyzed in this study were recorded in a jasco 715 spectropolarimeter equipped with a ptc 423s peltier element . peptides were loaded into a 0.2 mm quartz cuvette at 30m concentration ( 50 mm k2hpo4/kh2po4 buffer at ph 7 ) , and spectra were recorded with 50nm / min scan speed at 25c with 9 acquisitions averaged on the fly . spectra were background corrected with the buffer spectrum ( 50 mm k2hpo4/kh2po4 buffer at ph 7 ) , and converted into molar ellipticity . pr droplet ( 250m pr20 + 0.04g/l polyu rna ) sample ( 3.5 l ) was applied to a 300 mesh lacey quantifoil grid and incubated for 30 s. next , excess buffer was removed by blotting the grids for 3 s using a whatman 1 filter paper and the sample was snap frozen by plunging in liquid ethane at a temperature of 180c and stored in liquid nitrogen until visualization . next , the samples were transferred to a gatan 914 cryoholder and imaged at low dose conditions at 177c , using a jeol jem1400 tem equipped with an 11 mpxl olympus sis quemesa camera . fluorescent droplets were incubated in plastic cell counter slides ( bio - rad ) at room temperature . fluorescence recovery after bleaching was monitored using zen software on a zeiss lsm 510 meta nlo confocal microscope . for intradroplet frap , a circular are of 1m radius was bleached in droplets with a radius between 5m and 10m . raw data was background substracted and normalized using excell , and plotted using prism software . measurements were carried out in a ls55 luminescence spectrometer ( perkinelmer ) , with 200 nm pr - alexa568 n - terminally labeled protein in 50 mm k2hpo4/kh2po4 buffer at ph 7 . after the addition of 10m of bulk pr30 , fluorescence anisotropy was measured at increasing rna concentration at 578 nm excitation and 603 nm of emission wavelengths . 20m pr30 was titrated with increasing concentration of polyu rna starting at a 0.04g/l concentration in a dynapro nanostar ( wyatt technologies ) in 50 mm k2hpo4/kh2po4 buffer at ph 7 . the size of particles was followed for one hour after every titration step ( each titration step represented a 0.5% dilution ) . to control sedimentation and droplet fusion sample was mixed thoroughly with a pipet randomly between time points . ans ( 8-anilinonaphthalene-1-sulfonic acid ) fluorescence was measured for 40m pr20 or 40m gr20 peptide in the presence and absence of 0.05g/l polyu rna in a biotek mx synergy plate reader in 50 mm k2hpo4/kh2po4 buffer at ph 7 . emission spectra were recorded between 400 nm and 600 nm at 380 nm excitation wavelength . pr30 and polyu rna were dissolved in miliq grade water and added together to make a final concentration of 400 m pr and 4.8 mg / ml polyu rna to induce llps . for experiments on the free peptide , pr the samples were then , either immediately or after an incubation time of 1h for the llps samples , diluted with a 20-fold excess of d2o . after the indicated time points the hdx reaction was quenched by bringing the ph of the solution to 1 using formic acid , followed by snap freezing in liquid nitrogen . mass spectrometry analysis of deuterium incorporation was performed on a synapt g2 hdms mass spectrometer ( waters , wilmslow , uk ) by direct infusion of the undigested sample using nano - electrospray ionization with in - house prepared gold - coated borosilicate needles . hela cells were trypsinized , pelleted and washed three times with 1x pbs ( thermo scientific ) . cells were resuspended in k2hpo4/kh2po4 buffer with edta - free complete protease inhibitor cocktail ( roche ) and sonicated on ice . the lysate was cleared from the insoluble fraction by centrifugation for 15min at 10,000 rpm at 4c . the supernatans was retrieved and protein concentration was measured using micro bca assay ( thermo scientific ) . 1 mg of protein was added to 0.05 mole of pr30 to a final concentration of 100m , and left incubating for 10 min at rt . paraformaldehyde ( sigma - aldrich ) was added to a final concentration of 0.5% , and crosslinking was left for 5 min . 500l of 2 m glycine ( sigma - aldrich ) was added to quench paraformaldehyde , and samples were left for 5 min . the volume of the samples was increased to 1.5ml with k2hpo4/kh2po4 buffer , before gently spinning down the pr droplets at 4,000 rpm for 5 min . pellets were subsequently washed with 1ml pbs with 0.4% triton x-100 ( sigma - aldrich ) and vortexed before spinning down . samples for sds - page were generated identically , but procedure was downscaled four times . pr llps pellets were resuspended in 1x reducing laemmli containing sds ( thermo scientific ) . 1/8 of pellet or input lysate were loaded onto nupage novex 4%12% bis - tris precast gels ( thermo scientific ) . after running gels were stained using coomassie brilliant blue r-250 according to manufacturer s instructions ( bio - rad ) . uncrosslinked ( 2 replicates ) and crosslinked pellets ( 3 replicates ) were redissolved in 300 mm nacl tris buffer , sonicated and boiled to remove crosslinks . proteins in each sample were reduced with 5 mm dtt and incubation for 30 min at 55c and then alkylated by addition of 10 mm iodoacetamide for 15 min at room temperature in the dark . samples were further diluted to a final urea concentration of 2 m and proteins were digested with trypsin ( promega ) ( 1/100 , w / w ) overnight at 37c . peptides were then purified on omix c18 tips ( agilent ) , dried and re - dissolved in solvent a ( 25 l 0.1% tfa in water / acetonitrile ( 98:2 , v / v ) ) of which 10 l was injected for lc - ms / ms analysis on an ultimate 3000 rslcnano system ( dionex , thermo fisher scientific ) in line connected to a q exactive hf mass spectrometer with a nanospray flex ion source ( thermo fisher scientific ) . trapping was performed at 10 l / min for 4 min in solvent a ( on a reverse - phase column produced in - house , 100 m i.d . x 20 mm , 5 m beads c18 reprosil - pur , dr . maisch ) followed by loading the sample on a 40 cm column packed in the needle ( produced in - house , 75 m i.d . 400 mm , 1.9 m beads c18 reprosil - hd , dr . maisch ) . peptides were eluted by an increase in solvent b ( 0.1% formic acid in water / acetonitrile ( 2:8 , v / v ) ) in linear gradients from 2% to 30% in 100 min , then from 30% to 56% in 40 min and finally from 56% to 99% in 5 min , all at a constant flow rate of 250 nl / min . the mass spectrometer was operated in data - dependent mode , automatically switching between ms and ms / ms acquisition for the 16 most abundant ion peaks per ms spectrum . full - scan ms spectra ( 375 - 1500 m / z ) were acquired at a resolution of 60,000 after accumulation to a target value of 3,000,000 with a maximum fill time of 60 ms . the 16 most intense ions above a threshold value of 22,000 were isolated ( window of 1.5 th ) for fragmentation at a normalized collision energy of 32% after filling the trap at a target value of 100,000 for maximum 45 ms . the s - lens rf level was set at 55 and we excluded precursor ions with single and unassigned charge states . data analysis was performed with maxquant ( version 1.5.3.30 ) ( cox and mann , 2008 ) using the andromeda search engine with default search settings including a false discovery rate set at 1% on both the peptide and protein level . spectra were searched against the human proteins in the uniprot / swiss - prot database ( database release version of april 2016 containing 20,103 human protein sequences , www.uniprot.org ) . the mass tolerance for precursor and fragment ions were set to 4.5 and 20 ppm , respectively , during the main search . enzyme specificity was set as c - terminal to arginine and lysine , also allowing cleavage at proline bonds with a maximum of three missed cleavages . variable modifications were set to oxidation of methionine residues and acetylation of protein n - termini . only proteins with at least one unique or razor peptide proteins were quantified by the maxlfq algorithm integrated in the maxquant software ( cox et al . , 2014 ) . further data analysis was performed with the perseus software ( version 1.5.3.0 ) after loading the protein groups file from maxquant . proteins only identified by site and reverse database hits were removed and replicate samples of uncrosslinked and crosslinked were grouped . proteins with less than three valid values in at least one group were removed and missing values were imputed from a normal distribution around the detection limit . then , a t test was performed ( fdr = 0.01 and s0 = 1 ) to compare samples uncrosslinked and crosslinked and calculate the fold change for each protein between both samples . after removal of potential contaminants , 874 proteins were quantified in total ( table s1 ) . cells were transiently transfected using lipofectamine 3000 ( invitrogen ) according to manufacturer s instructions . cells were fixed 24h after transfection in 4% formaldehyde in pbs and stained according standard protocols ( including methanol fixation and permeabilization by pbs - t 0.04% ) . following antibodies were used : anti - flag ( f3165 , sigma ) , rabbit anti - flag ( # 2368s , cell signaling ) , mouse anti - g3bp1 ( ab56574 , abcam ) , rabbit anti - ddx6 ( ab40684 , abcam ) , rabbit anti - yb1 ( ab76149 , abcam ) , rabbit anti - tdp-43 ( 12892 - 1-ap , proteintech ) , mouse anti - ataxin-2 ( 611378 , bd biosciences ) , goat anti - tia1 ( sc-1751 , santa cruz ) . alexafluor 555 and alexafluor 488 secondary antibodies ( life technologies ) were used . control stress granules were induced by incubating the cells for 1h with 0.5 mm naaso2 ( sigma ) . percentage of cells carrying stress granules was assessed by g3bp1 staining in all tested cell lines . stress granule enrichment was quantified as the ratio of fluorescence intensity in the stress granule over the intensity of the surrounding cytoplasm . two stress granules per cell were quantified , of at least 30 cells from 3 independent experiments . for stress granule enrichment of pr100 , one stress granule per cell was counted , of 40 cells from 2 independent experiments . pr100 stress granules were visualized for quantification with flag staining , arsenite stress granules were visualized by tia1 staining . for analysis of dose- and length dependency of pr cytoplasmic structures , cells were categorized as having diffuse cytoplasmic pr , small stress granules , large stress granules or coarse / granular / heterogeneous cytoplasmic pr staining . data was subsequently also plotted as the percentage of cells displaying non - diffuse cytoplasmic pr staining after manual binning according to cytoplasmic pr100-flag intensity . percentages were fitted by a one - phase exponential curve , illustrating a strong dose - dependency of cytoplasmic structure formation . the mass spectrometry proteomics data have been deposited to the proteomexchange consortium via the pride partner repository with the dataset identifier pride : pxd005509 . , w.r . , and p.v.d . provided ideas for the project and participated in writing the paper . s.b .

summaryliquid - liquid phase separation ( llps ) of rna - binding proteins plays an important role in the formation of multiple membrane - less organelles involved in rna metabolism , including stress granules . defects in stress granule homeostasis constitute a cornerstone of als / ftld pathogenesis . polar residues ( tyrosine and glutamine ) have been previously demonstrated to be critical for phase separation of als - linked stress granule proteins . we now identify an active role for arginine - rich domains in these phase separations . moreover , arginine - rich dipeptide repeats ( dprs ) derived from c9orf72 hexanucleotide repeat expansions similarly undergo llps and induce phase separation of a large set of proteins involved in rna and stress granule metabolism . expression of arginine - rich dprs in cells induced spontaneous stress granule assembly that required both eif2 phosphorylation and g3bp . together with recent reports showing that dprs affect nucleocytoplasmic transport , our results point to an important role for arginine - rich dprs in the pathogenesis of c9orf72 als / ftld .

Introduction Results Discussion STARMethods Author Contributions

PMC3224444

cardio - pulmonary fitness is related to the ability to perform large muscle , dynamic , moderate - to - high exercises for a prolonged period . the performance of such exercises depends on the functional state of the cardiovascular , respiratory , and skeletal muscle systems . a variety of tests have been used to check cardio - pulmonary fitness and some of these tests are quite popular , such as , the cooper 12-minute test . the procedure of this test involves the participants running with maximal effort for 12 minutes exactly . the advantages of cooper test are that the whole team can participate together , and compete against each other . the test is relatively easy to carry out , and does not involve too much time or equipment . it can also be used easily for training ; and , any improvements can be easily appreciated . the disadvantages include that it is an indirect test , the results of which are not very accurate . also , it is suitable only for well trained individuals due to the fact it requires maximal effort for the entire length of the test . another disadvantage is that the test does not give actual maximum oxygen consumption values ; it only gives a classification of poor to excellent . hence , it has been suggested that this test is best for comparing athlete 's individual scores against each other to monitor improvements , rather than comparing the scores of the whole team . in the laboratory , maximum oxygen consumption is the gold standard in the assessment of cardio - pulmonary fitness . normally , heart rate is controlled by autonomic nervous system activity . heart rate variability ( hrv ) hrv demonstrates a relationship between autonomic nervous system function ( sympathetic or vagal activity ) and cardiovascular system . on the other hand , hrv has been recognized as a predictor of cardiovascular events , both in symptomatic and asymptomatic population.[36 ] also , it was found that reduced hrv predicts hypertension and hyperglycemia in the future . the simplest variable is the standard deviation of normal - to - normal qrs intervals ( sdnn ) in a continuous electrogram ( ecg ) recording . traditionally , hrv is recorded over a longer period ( 24 hour ) ; however , some investigators have reported that ultra - short hrv ( 2 - 15 minutes ) is strongly correlated with the 24-hour hrv . the aim of this study was to assess the cardio - pulmonary fitness by ultra - short hrv . study population was divided into 3 groups : group-1 ( n = 40 ) consisted of military sports man who volunteered to participate in this study . group-2 ( n = 40 ) were healthy age - matched sedentary male subjects who had a normal bmi [ bmi = 19 - 25 kg / m ) . group-3 ( n = 40 ) consisted of healthy age - matched obese male subjects [ bmi > 29 the subjects of the research were all free of cardiovascular and pulmonary disease , alcohol use , and diabetes mellitus ; and , were not taking any kind of medication , and did not present with any abnormal electrocardiographic patterns . , all the subjects rested in a supine position for a minimum of 15 minutes in a room with a constant temperature of 25 degree c. for the holter recording , standard digital devices were used . after holter recording , bruce protocol treadmill test was used . at timed stages during the run , the slope of the treadmill is increased as detailed in table 1 . maximum oxygen consumption ( vo2max ) , which has been shown to be an objective criterion of cardiopulmonary fitness , was calculated by foster equation : bruce protocol treadmill test vo2max ( ml / kg / min ) = 14.8 [ 1.379 t ] + [ 0.451 t ] [ 0.012 t ] t is the total time of the test expressed in minutes and fractions of a minute . comparisons between groups were made using student 's t - test and mann - whitney u test as appropriate . study population was divided into 3 groups : group-1 ( n = 40 ) consisted of military sports man who volunteered to participate in this study . group-2 ( n = 40 ) were healthy age - matched sedentary male subjects who had a normal bmi [ bmi = 19 - 25 kg / m ) . group-3 ( n = 40 ) consisted of healthy age - matched obese male subjects [ bmi > 29 the subjects of the research were all free of cardiovascular and pulmonary disease , alcohol use , and diabetes mellitus ; and , were not taking any kind of medication , and did not present with any abnormal electrocardiographic patterns . before the test , all the subjects rested in a supine position for a minimum of 15 minutes in a room with a constant temperature of 25 degree c. for the holter recording , standard digital devices were used . after holter recording , bruce protocol treadmill test was used . at timed stages during the run , the slope of the treadmill is increased as detailed in table 1 . maximum oxygen consumption ( vo2max ) , which has been shown to be an objective criterion of cardiopulmonary fitness , was calculated by foster equation : bruce protocol treadmill test vo2max ( ml / kg / min ) = 14.8 [ 1.379 t ] + [ 0.451 t ] [ 0.012 t ] t is the total time of the test expressed in minutes and fractions of a minute . comparisons between groups were made using student 's t - test and mann - whitney u test as appropriate . the mean age was identical between 3 groups as might be expected from the matching . baseline characteristics of the 3 groups involved in the study relationship between sdnn and estimated vo2 max are demonstrated in table 3 . relation between standard deviation of normal - to - normal qrs intervals ( sdnn ) and maximum oxygen consumption ( vo2max ) of the three groups involved in the study when the study population was divided into quartiles of sdnn ( first quartile : < 60 msec ; second quartile : > 60 and < 100 msec ; third quartile : > 100 and < 140 msec ; and fourth quartile : > 140 msec ) , progressive increase was found in vo2max ; and , sdnn was significantly linked with estimated vo2max values [ figure 1 ] . maximum oxygen consumption ( vo2max ) of study population compared by quartiles of standard deviation of normal - to - normal qrs intervals ( sdnn ) . when the study population was divided into quartiles of sdnn ( first quartile : < 60 msec ; second quartile : > 60 and < 100 msec ; third quartile : > 100 and < 140 msec ; and fourth quartile : > 140 msec ) , progressive increase was found in vo2max . the mean age was identical between 3 groups as might be expected from the matching . relation between standard deviation of normal - to - normal qrs intervals ( sdnn ) and maximum oxygen consumption ( vo2max ) of the three groups involved in the study when the study population was divided into quartiles of sdnn ( first quartile : < 60 msec ; second quartile : > 60 and < 100 msec ; third quartile : > 100 and < 140 msec ; and fourth quartile : > 140 msec ) , progressive increase was found in vo2max ; and , sdnn was significantly linked with estimated vo2max values [ figure 1 ] . maximum oxygen consumption ( vo2max ) of study population compared by quartiles of standard deviation of normal - to - normal qrs intervals ( sdnn ) . when the study population was divided into quartiles of sdnn ( first quartile : < 60 msec ; second quartile : > 60 and < 100 msec ; third quartile : > 100 and < 140 msec ; and fourth quartile : > 140 msec ) , progressive increase was found in vo2max . previously , 24 hour was determined as an optimal cut point for hrv recording and analysis . bigger et al . found that measures of rr variability calculated from short ( 2 to 15 minutes ) ecg recordings are remarkably similar to those calculated over 24 hours . hrv has been recognized as a predictor of cardiovascular events both in symptomatic and asymptomatic population.[36 ] it was also used in risk stratification for sudden cardiac death and diabetic autonomic neuropathy . they used hrv as a tool for noninvasive testing of autonomic nervous system activity with exercise training . they showed that regular exercises result in a significant improvement of all hrv indices . in the present study , we have analyzed cardio - pulmonary fitness ( vo2max ) and ultra - short hrv ( sdnn ) . we showed that sdnn is a simple cardio - pulmonary fitness test which just requires 15 minutes , and involves no exercise such as in the treadmill or cycle test . previously , 24 hour was determined as an optimal cut point for hrv recording and analysis . bigger et al . found that measures of rr variability calculated from short ( 2 to 15 minutes ) ecg recordings are remarkably similar to those calculated over 24 hours . hrv has been recognized as a predictor of cardiovascular events both in symptomatic and asymptomatic population.[36 ] it was also used in risk stratification for sudden cardiac death and diabetic autonomic neuropathy . they used hrv as a tool for noninvasive testing of autonomic nervous system activity with exercise training . they showed that regular exercises result in a significant improvement of all hrv indices . in the present study , we have analyzed cardio - pulmonary fitness ( vo2max ) and ultra - short hrv ( sdnn ) . we showed that sdnn is a simple cardio - pulmonary fitness test which just requires 15 minutes , and involves no exercise such as in the treadmill or cycle test . in conclusion , the results of this study demonstrate that exercise training improves cardio - respiratory autonomic function ( and increase heart rate variability ) . improvement in cardio - respiratory autonomic function seems to translate into a lower rate of long term mortality . ultra - short hrv is a simple cardio - pulmonary fitness test which just requires 15 minutes , and involves no exercise such as in the treadmill or cycle test .

objectives : it is known that exercise induces cardio - respiratory autonomic modulation . the aim of this study was to assess the cardio - pulmonary fitness by ultra - short heart rate variability.materials and methods : study population was divided into 3 groups : group-1 ( n = 40 ) consisted of military sports man . group-2 ( n = 40 ) were healthy age - matched sedentary male subjects with normal body mass index [ bmi = 19 - 25 kg / m2 ) . group-3 ( n = 40 ) were healthy age - matched obese male subjects [ bmi > 29 kg / m2 ) . standard deviation of normal - to - normal qrs intervals ( sdnn ) was recorded over 15 minutes . bruce protocol treadmill test was used ; and , maximum oxygen consumption ( vo2max ) was calculated.results:when the study population was divided into quartiles of sdnn ( first quartile : < 60 msec ; second quartile : > 60 and < 100 msec ; third quartile : > 100 and < 140 msec ; and fourth quartile : > 140 msec ) , progressive increase was found in vo2max ; and , sdnn was significantly linked with estimated vo2max.conclusion:in conclusion , the results of this study demonstrate that exercise training improves cardio - respiratory autonomic function ( and increases heart rate variability ) . improvement in cardio - respiratory autonomic function seems to translate into a lower rate of long term mortality . ultra - short heart rate variability is a simple cardio - pulmonary fitness test which just requires 15 minutes , and involves no exercise such as in the treadmill or cycle test .

INTRODUCTION MATERIALS AND METHODS Study population Ultra-short heart rate variability recording and analysis Cardio-pulmonary fitness test Statistical analysis RESULTS Baseline characteristics Relation between standard deviation of normal-to-normal QRS intervals (SDNN) and maximum oxygen consumption (VO DISCUSSION Heart rate variability versus ultra-short heart rate variability recording Ultra-short heart rate variability as a novel tool for cardio-pulmonary fitness test CONCLUSION

PMC4578552

human performance is affected by both the genetic makeup of the individual and the environmental factors . current research and interest in sports genetics focus on genetic variants that may make a significant contribution to the individual s performance . it is known that personal traits such as endurance , strength , power , muscular coordination , and psychological willingness and motivation , all have a genetic background.1 the most frequently investigated genetic polymorphisms in terms of athletic performance or predisposition to athletic capacity are angiotensin-1 converting enzyme ( ace ) gene and -actinin-3 ( actn3 ) gene.2 variants in these genes have been reported to be associated with elite athletic performance and with quantitative physical performance traits in the general population.35 actn3 was the first structural gene specific to skeletal muscle that has been associated with athletic performance.6 the actinins , major and important z line functional and structural proteins in sarcomers , are members of the actin - binding protein family.7 actn3 codes for actn3 in humans , and its expression is restricted to fast , type 2 fibers.8 north et al9 reported that a deficiency of -actinin protein because of a premature stop codon in actn3 alters the designation for the amino acid arginine to a stop codon at position 577 ( r577x ; dbsnp rs1815739 ) in exon 16 and may contribute to the possibility of differential fitness . the actn3 577r allele and 577rr genotype are associated with top - level , power - orientated athletic performance in a wide array of ethnic groups.10 the actn3 r577x polymorphism has been reported to be associated with elite athletic status,6 endurance athletes,11 and many other groups.1216 ace is located on chromosome 17q23 and comprises 26 exons and 25 introns . it contains a polymorphism due to an insertion ( i ) or a deletion ( d ) of a 287 bp alu sequence in intron 16 , resulting in the three genotypes of insertion / insertion ( ii ) , insertion / deletion ( i d ) , and deletion / deletion ( dd).17 the i / d polymorphism is associated with circulating and tissue ace levels . individuals homozygous for the d allele had higher tissue and plasma ace concentrations than heterozygotes and ii homozygotes.18 many of the case control studies reported that success in speed - strength disciplines , such as short - distance running , long jump , high jump , and short - distance swimmers , is associated with ace dd genotype.19,20 on the other hand , individuals with the ii genotype have a lower ace serum concentration and have more success in endurance - related disciplines such as medium- and long - distance running , race walking , and rowing.21,22 two different studies involving spanish and lithuanian football players showed that players had a significantly higher percentage of the ace i d genotype when compared to the nonathletic population.23,24 previous studies , including related genetic polymorphisms and football players , are very limited , especially in turkish subjects . the aim of this study was to determine the genotype and allele distribution of actn3 and ace in turkish male football players and assess the impact on predisposition to football . all players enrolled for the study had turkish ancestry and were members of a turkish football team . the study was conducted in accordance with the principles of the declaration of helsinki ii . skdar university ethical committee approved the study protocol , and written informed consent was obtained from each player once prospective participants understood and accepted the aim and protocol of the study . dna isolation was carried out by using high pure polymerase chain reaction template preparation kit ( roche diagnostics ) using peripheral blood . the region of interest , actn3 , was amplified using the following primers : forward 5-ctg ttg cct gtg gta agt ggg-3 and reverse 5-tgg tca cag tat gca gga ggg-3 , as described previously.5 polymerase chain reaction ( pcr ) was performed by initial denaturation at 95c for five minutes , followed by 35 cycles of denaturation at 95c for 30 seconds , annealing and extension at 72c for one minute , and a final extension for seven minutes at 72c . the 290-bp amplicons were digested by ddei ( new england biolabs ) as recommended by the manufacturer . digested fragments were separated on 10.0% polyacrylamide gel electrophoresis and visualized under uv light by ethidium bromide staining . the wild - type allele , 577r , showed fragments of 205 and 85 bp , whereas the variant allele , 577x , showed fragments of 108 , 97 , and 85 bp ( fig . the final volume of the pcr mixture was 50 l and contained 50100 ng genomic dna , 1 mm of each primer , 50 mm kcl , 1 mm deoxynucleotide triphosphate ( dntp ) , 1.5 mm mgcl2 , 10 mm tris hcl , an initial denaturation at 94c for five minutes was followed by annealing at 58c for one minute and extension at 72c for two minutes . amplification was finalized with 30 cycles : denaturation at 94c for one minute , annealing at 58c for one minute , and extension at 72c for two minutes , followed by a final elongation at 94c for one minute , annealing at 58c for one minute , and extension at 72c for seven minutes . amplicons were separated by electrophoresis on a 2% agarose gel and visualized under uv light after ethidium bromide staining . electrophoresis gave rise to three possible patterns : a 490-bp band ( ii genotype ) , a 190-bp band ( dd genotype ) , or both 490- and 190-bp bands ( i / d genotype ) ( fig . amplicons had 190 bp in the presence of the d allele and 490-bp fragment in the presence of the i allele . all players enrolled for the study had turkish ancestry and were members of a turkish football team . the study was conducted in accordance with the principles of the declaration of helsinki ii . skdar university ethical committee approved the study protocol , and written informed consent was obtained from each player once prospective participants understood and accepted the aim and protocol of the study . dna isolation was carried out by using high pure polymerase chain reaction template preparation kit ( roche diagnostics ) using peripheral blood . the region of interest , actn3 , was amplified using the following primers : forward 5-ctg ttg cct gtg gta agt ggg-3 and reverse 5-tgg tca cag tat gca gga ggg-3 , as described previously.5 polymerase chain reaction ( pcr ) was performed by initial denaturation at 95c for five minutes , followed by 35 cycles of denaturation at 95c for 30 seconds , annealing and extension at 72c for one minute , and a final extension for seven minutes at 72c . the 290-bp amplicons were digested by ddei ( new england biolabs ) as recommended by the manufacturer . digested fragments were separated on 10.0% polyacrylamide gel electrophoresis and visualized under uv light by ethidium bromide staining . the wild - type allele , 577r , showed fragments of 205 and 85 bp , whereas the variant allele , 577x , showed fragments of 108 , 97 , and 85 bp ( fig . the final volume of the pcr mixture was 50 l and contained 50100 ng genomic dna , 1 mm of each primer , 50 mm kcl , 1 mm deoxynucleotide triphosphate ( dntp ) , 1.5 mm mgcl2 , 10 mm tris an initial denaturation at 94c for five minutes was followed by annealing at 58c for one minute and extension at 72c for two minutes . amplification was finalized with 30 cycles : denaturation at 94c for one minute , annealing at 58c for one minute , and extension at 72c for two minutes , followed by a final elongation at 94c for one minute , annealing at 58c for one minute , and extension at 72c for seven minutes . amplicons were separated by electrophoresis on a 2% agarose gel and visualized under uv light after ethidium bromide staining . electrophoresis gave rise to three possible patterns : a 490-bp band ( ii genotype ) , a 190-bp band ( dd genotype ) , or both 490- and 190-bp bands ( i / d genotype ) ( fig . amplicons had 190 bp in the presence of the d allele and 490-bp fragment in the presence of the i allele . the percentage of the ace genotype in the examined players was 16 , 44 , and 40 for ii , i d , and dd genotypes , respectively . for the actn3 genotype , the respective frequencies were 20 , 36 , and 44 for xx , rx , and rr . an allelic count gave rise to 19 ( 38% ) i and 31 ( 62% ) d alleles for ace and 31 ( 62% ) r and 19 ( 38% ) x alleles for actn3 . table 1 lists the genotype and allelic frequencies of ace and actn3 . according to the genotypes , nine different combinations were found , five of the players had dd + rr , the same number of the players had i d + rr , four players had i d + rx , three players had dd + rx , two players had dd + xx , the same number of the players had i d + xx , two players had ii + rx , one player had ii + xx , and one player had ii + rr ( data not shown in table ) . recent studies have indicated that several genes are involved in determining the performance of the players , both physiologically and psychologically.25 genetic models could be developed and used to find the optimal genetic endowment of a player to help scientists establish which genetic polymorphisms are advantageous for proper performance in different sports types . therefore , the creation of genomic databases will be very useful for sport scientists . in this study , we solely examined 25 senior football players in terms of ace and actn3 polymorphisms and aimed to associate these polymorphisms with predisposition to football . in our study cohort , the r allele , considered to be the wild - type allele of the gene and associated with the rapid contraction of the sarcomers , was also high when compared to x allele . egorova et al26 reported similar results in russian football players ; they examined 240 football players , and 46.25% had the rr genotype . santiago et al16 analyzed 60 brazilian football players , and in their cohort , 48.3% of the players had rr genotype . pimenta et al27 aimed to compare acute inflammatory responses , muscle damage , and hormonal variations with eccentric training in soccer athletes and examined 37 professional soccer players of which 40.5% were rr . to the best of our knowledge , this is the first report in turkish male professional soccer players , and our results were in agreement with previous studies , indicating r allele and rr genotype superiority for soccer players . in our cohort , 44% of the players had i d genotype , 40% of the players had dd genotypes for ace , and 84% of the players had at least one d allele . d allele is responsible for high ace concentration and is associated with success in speed - strength disciplines.17 gineviciene et al24 examined 199 lithuanian football players and reported similar results to ours . they showed that i d was the highest genotype ( 46.7% ) , and together with dd genotype , the percentage of the players having at least one d allele was 76.3% . juffer et al23 analyzed 54 male professional soccer players , and i d was the most detected genotype . unlike our results , egorova et al26 examined 213 russian football players and found the frequency of i d and dd genotypes as 28.6% and 50.7% , respectively , giving both genotypes an overall frequency of 79.3% , which is similar to our results . the cumulative effect of genotypes in human metabolism had always received great attention . in an attempt to find the optimal genotypes , several researchers have examined candidate genes separately , in different populations , to create a pool of genomic data . genotype combinations are very useful for identifying a certain metabolism , or a part of a metabolism , if they are part of the same reaction . for example , actn3 and ace have crucial roles for their metabolisms , but they do not have roles in the same biologic pathway . but it is important to understand performance - enhancing polymorphisms to create an optimal genomic score for being an elite athlete . in our cohort , five of the players had the dd + rr genotype combinations and the same number had i d + rr genotypes . the d and r alleles were found together in these genotypes , the most important similarity between these genotypes . on the other hand , one player had the ii + xx genotype , thought to be associated with endurance capacity . we hope this preliminary study will guide new studies , with extended numbers of players in different kinds of sports to evaluate the effect of the combination of these polymorphisms more accurately . in this study , we examined the distribution of ace and actn3 polymorphisms in male turkish professional soccer players for the first time . actn3 rr genotype and r allele and ace i d genotype and d allele dominated our cohort . therefore , we did not compare the genomic results with sedentary people ; rather , we examined the combined effect of these polymorphisms . according to our previous and current results , we hypothesize that elite soccer players tend to have a power / strength - oriented genotype . these polymorphisms , alone or in combination with the additional polymorphisms , should be taken into account when deciding a genomic score profile for success in sports .

angiotensin-1 converting enzyme ( ace ) gene and -actinin-3 ( actn3 ) gene polymorphisms are considered to be the most important candidate genes for genetic predisposition to human athletic performance . in the present study , we aimed to analyze the distribution of ace and actn3 polymorphisms for the first time in male turkish soccer players . in this prospective study , our cohort consisted of 25 professional players , all with turkish ancestry . polymerase chain reaction ( pcr)-restriction length polymorphism was used for the characterization of the genotype of actn3 and single pcr for ace . for ace genotype , 16% , 44% , and 40% of the players had insertion / insertion ( ii ) , insertion / deletion ( i d ) , and deletion / deletion ( dd ) genotypes , respectively , whereas 20% had xx , 36% had rx , and 44% had rr genotypes for actn3 . when we examined the allelic percentages , for ace , d allele was recorded as 62 and i as 38 , and for actn3 , r allele was 62 and x was 38 . our results were in agreement with the previous reports , indicating the presence of actn3 d and ace x allele in soccer players . we suggest that ace and actn3 genotypes are important biomarkers for genetic counseling for the individuals who are prone to be successful soccer players .

Introduction Materials and Methods Study subjects Genotyping Results Discussion Conclusion

PMC5088128

negative change within the carotid artery is reportedly an independent risk factor for cardiovascular disease ( cvd ) and stroke1 . several studies report an association of cvd risk with increased of carotid intima - media thickness ( cimt)2,3,4 and luminal diameter ( cld)5 , 6 . recently , a followed - up study reported that low carotid artery flow velocity ( i.e. peak - systolic velocity [ psv ] and end - diastolic velocity [ edv ] ) is associated with cvd risk in hypertensive patients7 . another study focusing on risk factor evolution and cvd development demonstrated that low psv and edv were independently associated with future cvd8 . a cross - sectional study comparing patients with ischemic stroke patients and subjects without stroke found an association between lower carotid flow velocity ( fv ) and increased stroke risk9 . several cross - sectional studies reported that improved of oxygen uptake and walking speed is associated with preventing to the negative change of carotid arteries ( in cimt or cld ) in elderly3 , 10 . resistance exercise is effectively improves of physical performance such as muscle strength , agility and dynamic balance , flexibility , walking ability , and aerobic endurance in the elderly11,12,13 . additionally , resistance exercise can improve body composition , including decreasing body fat mass or increasing of lean body mass in elderly populations11 , 12 . in contrast , studies on the effect of resistance exercise on arterial function demonstrated that resistance exercise improves vascular endothelial function and increases blood flow velocity in the brachial artery in healthy overweight women and young adults14 , 15 . also , resistance exercise is reported to decrease the cimt and increase the cld in the carotid artery in patients with chronic heart failure patients16 . however , the effects of resistance exercise on carotid artery fv parameters are not yet known in healthy elderly men . therefore , we examined the effect of a 24 weeks resistance exercise program on carotid artery flow velocity in this population . in this study initially enrolled 42 healthy elderly men ( age 65 years ) without hypertension , hyperlipidemia , diabetes , history of cvd , or other diagnosed illness . twelve subjects were excluded for smoking ( n=7 ) , and regular participation in physical activity within the past 6 months ( n=5 ) . the remaining 30 elderly men were randomly divided into a control group ( n=15 ) and a resistance exercise group ( n=15 ) . table 1table 1.the anthropometric characteristics , body composition and blood pressure of subjectsvariablescontrol group(n=15)exercise group(n=15)age ( years)70.9 3.973.1 3.0height ( cm)164.9 7.5164.8 5.4body mass ( kg)64.5 8.066.0 6.1body mass index ( kg / m)23.8 2.924.3 2.1percent body fat mass ( % ) 23.7 7.125.6 6.2skeletal muscle mass ( kg)27.3 3.027.4 3.7systolic blood pressure ( mmhg)127.7 6.7129.1 7.4diastolic blood pressure ( mmhg)74.4 8.075.1 8.2values are means sd shows the participants physical characteristics . in accordance with the ethical standards of the declaration of helsinki , informed consent was obtained from all participants after they were provided with a detailed description of the study . values are means sd after measuring the subjects height and body mass , their body fat mass ( fm ) percent and skeletal muscle mass ( sm ) were measured by bioelectrical impedance analysis , using a body composition analyzer ( inbody 370 , biospace , seoul , south korea ) . body mass index ( bmi ) was calculated as weight ( kg ) divided by height squared ( m ) . for the 2 days prior to measurement of body composition , subjects were asked to not engage in physical activities other than the activities they usually performed and to refrain from drinking or eating excessively . systolic blood pressure ( sbp ) and diastolic blood pressure ( dbp ) were measured using a mercury sphygmomanometer ( hico , tokyo , japan ) after 10 minutes of rest . upper and lower body muscle strength , agility / dynamic balance , flexibility , walking ability , and aerobic endurance were measured after a 10-minute warm up . muscle strength was measured by grip strength using a grip strength dynamometer ( tkk-5401 , japan ) for the upper body and chair stand up for the lower body . chair stand up were scored based on the number of repetitions completed within 30 seconds . agility and dynamic balance was measured by the time up and go ( tug , 2.44 m ) . participants were informed that this was a timed test and that the objective was to walk as quickly as possible ( without running ) around a cone and back to the chair . participants were instructed to take a deep breath and then breathe out while leaning forward as far as possible , without bending the knees . in addition , participants started walking two meters before the start line so that walking speed did not include the acceleration time . and maximum walking speed was defined as the ratio between distance and time . two - minute steps was measured as an alternative to the aerobic endurance test . participants score was the number of full steps completed in 2 minutes , raising each knee to a point midway between the patella and iliac crest . resistance exercises used in this study conformed to the guidelines of physical activity recommended for the elderly17 . the resistance exercise was performed with green elastic exercises bands ( thera - band , ohio , usa ) for 3050 minutes per session , 3 days per week , with 1015 repetitions per set ( weeks 112 : 1012 repetitions per set ; weeks 1324 : 1315 repetitions per set ) , 35 sets ( weeks 112 : 3 sets , weeks 1324 : 5 sets , 1 minute rest between sets ) for 14 items ( elbow flexion , lateral raise , shoulder flexion and extension , biceps curl , chest press and flies , abdominal crunch , trunk extension , side bend , mini squat , leg press , calf raise , and ankle inversion)17 , 18 . participants performed a 10-minute warm and cool down before and after the exercise program , respectively . carotid artery variables were measured using b - mode ultrasound and a 10 mhz probe ( logiq 3 , ge healthcare , wauwatosa , wi , usa ) . cimt and systolic cdl were measured from the far wall of the distal common carotid 1 cm proximal to the carotid bifurcation . the cimt was defined as the distance from the lumen - intima interface to the intima - adventitia interface , and systolic cld was defined as the distance between the near and far wall intima - media interfaces19 . for doppler spectral analysis , peak systolic flow velocity ( psv ) and end diastolic flow velocity ( edv ) were measured by continuous - wave doppler examination in the common carotid 13 cm proximal to the bifurcation8 . wall shear rate ( wsr ) was calculated using to the following formula20:wsrpeak=4vpeak / cldwhere wsr is wall shear rate ( s ) , vpeak is the peak systolic flow velocity and cld is systolic luminal diameter . statistical package for the social sciences ( spss ) ver . 17.0 chicago , il , usa ) was used for statistical analysis , and results reported as mean and standard deviation . the student s t - test was used to assess differences in baseline variables . two - way factorial analysis of variance was used to compare parameters at baseline and after the 24 weeks of the study participation . when significant group time interactions were identified , paired t - tests were performed to detect differences between time points . table 2table 2.changed of body composition , blood pressure , physical function , and carotid artery variables in baseline and after 24 weeksvariablescontrol group ( n=15)changeexercise group ( n=15)changebaseline24 weekbaseline24 weekbody fat mass percent ( % ) 23.7 7.123.8 6.60.125.6 4.325.0 3.61.2*skeletal muscle mass ( kg)27.3 3.027.0 3.00.327.4 3.728.2 3.70.8**systolic blood pressure ( mmhg)127.7 6.7127.9 6.30.2129.1 7.4128.3 6.10.8*diastolic blood pressure ( mmhg)74.4 8.074.5 6.90.175.1 8.274.9 7.40.4left grip strength ( kg)33.3 5.532.9 5.40.431.0 6.633.0 5.52.04**right grip strength ( kg)33.2 5.232.9 5.00.331.2 7.134.3 5.83.1**chair stand up ( repetitions/30 sec)16.3 3.616.1 3.00.216.3 3.418.2 3.11.9**sit and reach ( cm)10.0 6.19.9 6.00.18.9 7.89.8 7.50.9**maximum walking speed ( m / sec)1.67 0.31.64 0.30.031.58 0.21.67 0.30.09**time up and go ( sec)5.84 1.05.86 1.00.025.87 1.15.65 0.80.22*two - minute step test ( repetitions)101.4 13.1101.3 12.10.199.3 15.6103.1 12.93.9**carotid intima media thickness ( mm)0.71 0.110.71 0.100.00.71 0.080.71 0.070.0systolic carotid luminal diameter ( cm)0.62 0.10.62 0.10.00.63 0.10.64 0.10.01peak systolic flow velocity ( cm / sec)63.4 9.962.6 11.10.862.2 13.971.4 13.09.2**end diastolic flow velocity ( cm / sec)20.4 3.820.5 3.80.119.2 3.721.0 4.01.8**wall shear rate ( s)409.6 65.3403.7 74.65.9403.1 108.1453.9 116.750.8*values are means sd . significantly different within group : * p<0.05 , * * p<0.01 ; significant group time interaction : p<0.05 , p<0.01 , p<0.001 shows changes of body composition , blood pressure , physical function , and carotid variables in baseline and after 24 weeks . fm percent ( p<0.05 ) , sm ( p<0.01 ) , and sbp ( p<0.05 ) showed significant interactions . grip strength on the left ( p<0.01 ) and right ( p<0.001 ) , chair stand up ( p<0.01 ) , sit and reach ( p<0.05 ) , maximal walking speed ( p<0.01 ) , tug ( p<0.01 ) , and two - minute step ( p<0.01 ) had significant interactions . psv ( p<0.001 ) , edv ( p<0.05 ) , and wsr ( p<0.05 ) showed significant interactions . however , in the dbp , cimt , and cld , interactions were no identified . values are means sd . significantly different within group : * p<0.05 , * * p<0.01 ; significant group time interaction : p<0.05 , p<0.01 , p<0.001 in the paired t - test results of the exercise group , fm percent , sbp , and tug were significantly decreased ( p<0.05 , each ) , and sm , left and right grip strength , chair stand up , sit and reach , maximal walking speed , and two - minutes step were significantly increased ( p<0.01 , each ) , also psv ( p<0.01 ) , edv ( p<0.01 ) , and wsr ( p<0.05 ) were significantly increased after 24 weeks . body composition , blood pressure , and physical function variables showed no change in the control group . but in exercise group , dbp , cimt , and cld were no change , also in control group , all variables were no change . this study examined the effects of a 24-week resistance exercise program on carotid parameters in elderly men . the primary findings of this study are that the resistance exercise program increased pfv , edv , and wsr in this study population . decreased of carotid artery fv is associated with early progress of atherosclerosis , and stoke in elderly patients9 . in addition , two recently cross - sectional studies reported that low psv and edv were independently associated with future dvd7 , 8 . the previous studies suggest that increase of psv and edv is associated with a potential prevention for cvd . studies on the effects of exercise training on arterial parameters demonstrated that resistance exercise increases blood flow in the femoral artery and increases flow mediated dilation of the brachial artery in adults14 , 21 , 22 . resistance exercise is also reported to increase the flow velocity of the brachial and femoral artery in adults and older subjects15 , 22 . changes in brachial and femoral parameters due to resistance exercise are considered to significantly improve of peripheral artery function14 , 15 . however , effects of long - term resistance exercise on carotid artery fv have not been confirmed . several studies reported that carotid fv is increased during dynamic exercise or after acute high intensity exercise in young adults23 , 24 . a study of obese adults reported that resistance exercise alone could improve brachial artery endothelial function14 . in the present study , this study demonstrated that long - term resistance exercise improves to age - related carotid fv in health elderly men . aging is reported as a major risk factor for decreased of carotid artery flow25 . in the elderly , improvement of carotid fv by exercise low wsr is associated with increased cimt , and is directly associated with changes in carotid fv , viscosity , and cld26 . in the current study , wsr was used as a surrogate marker of wall shear stress , and was calculated by systolic cld and psv26 . we found that exercise resulted in increased psv , but there was no change in cld . two intervention studies in overweight or obese adults demonstrated that short- or long - term resistance training is not effective in improving cimt and cld14 , 21 . in this study , this result suggests that resistance exercise does not effectively improve cimt and cld in health elderly . two intervention studies demonstrated that resistance exercise using weightlifting machine is effectively improves of body composition and physical function in the elderly11 , 12 . other intervention study demonstrated that resistance exercise using elastic band is effectively improves physical fitness such as muscle function and balance ability in elderly women13 . and resistance exercise using elastic band are simple and economical , and have safety advantages13 . in this study , resistance exercise was to use elastic bands , and health - related indicators such as blood pressure as well as body composition and physical fitness variables were examined . this study showed that resistance exercise using elastic bands for 24 weeks is effectively improves of blood pressure as well as body composition and physical fitness variables in elderly . therefore , effects of resistance exercise on the carotid artery may be limited to elderly men . in conclusion , 24-week resistance exercise program , using elastic bands , effectively improves carotid flow velocity and wall shear rate in healthy elderly men .

[ purpose ] the aim of this study was to examine the effect of resistance exercise on carotid intima - media thickness , luminal diameter , peak systolic flow velocity , end diastolic flow velocity , and wall shear rate in healthy elderly men . [ subjects and methods ] thirty healthy elderly men ( age 65 years ) were randomly divided into a control ( n=15 ) and resistance exercise ( n=15 ) groups . the 24-week exercise intervention consisted of 3 days of resistance exercise per week using an elastic band per week . body composition , physical function , blood pressure , and carotid variables were measured at baseline and after 24 weeks . [ results ] body fat percent , skeletal muscle mass , systolic blood pressure , grip strength , arm curl , chair stand up , sit and reach , maximum walking speed , time up and go , and two - minute step test showed significant interaction . peak systolic flow velocity , end diastolic flow velocity , and wall shear rate also showed significant interaction . [ conclusion ] a 24-week resistance exercise program , using elastic bands , effectively improves carotid flow velocity and wall shear rate in healthy elderly men .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC4075902

postinfectious obliterative bronchiolitis ( ob ) is an uncommon and severe form of chronic obstructive lung disease that occurs in children after lower respiratory tract injury . ( ) in terms of diagnosis , there are no specific signs or symptoms of postinfectious ob . clinical and imaging findings are used in combination with laboratory test results in order to identify the cause and rule out other forms of chronic lung disease . the diagnostic criteria for postinfectious ob are as follows : severe acute bronchiolitis / viral pneumonia during the first 3 years of life in previously healthy children ; evidence of persistent airway obstruction after the acute event , identified by physical examination , pulmonary function testing , or both ; chest x - ray findings of obstructive lung disease ; a mosaic pattern and air trapping ( at ) on chest ct scans ; and exclusion of other chronic lung diseases progressing to permanent respiratory symptoms . ( , ) histologically , ob is characterized by the presence of granulation tissue plugs in the small airway lumen , complete small airway destruction , or both . ( ) showed that ob is histologically characterized by a constrictive pattern with varying degrees of inflammation and airway obliteration , ranging from minimal bronchiolar inflammation to complete obliteration of the bronchioles and bronchi by fibrotic tissue . indirect signs of obstruction , such as macrophage accumulation , bronchiectasis , mucus accumulation , and hyperinflation , are always present . although there have been several reviews of studies examining long - term sequelae of adenovirus pneumonia , ( , ) few studies have examined ct findings in children with ob . ( , ) some of these studies have reported that abnormal ct findings in such children can predict abnormal lung function later in life . ( ) it has been reported that hrct is an important diagnostic tool for the evaluation of pulmonary damage in patients with ob . ( ) the volume of an organ ( or the volume of abnormal parts of an organ ) can be determined by the use of helical ct images and attenuation coefficient values or densities on the hounsfield scale , expressed in hounsfield units ( hu ) . this method is known as volumetric ct densitometry or ct densitovolumetry , ( , ) and it can be used in order to measure the volume of areas of lung with low attenuation ( i.e. , at ) , as well as total lung volume ( tlv ) , in children with ob . in addition , it can be used in order to measure the percentage of at relative to tlv ( at% ) with the use of thresholds to differentiate normal lung areas from ob areas . it has been shown that at% is a major contributing factor to chronic persistent airflow obstruction in asthma . ( , ) the clinical course following the onset of ob is variable and depends on the volume of affected lung tissue . ( ) measures of clinical status and of the ability to perform physically demanding activities - such as the number of hospital admissions or missed school days , walk test distance , nutritional status , and oxygen saturation ( so2 ) at rest or during exercise - facilitate the selection of appropriate clinical interventions . ( , ) because the prognosis of ob patients depends not only on patient behavior but also on functional impairment and the extent of anatomical damage , clinicians should gather information regarding the extent and type of anatomical abnormalities . bronchiectasis , atelectasis , lobar collapse , and areas of low density are known consequences of postinfectious ob , and hrct is the best method of examining such lesions , ( , , ) all of which can influence the clinical status of patients and their pulmonary function test results . ( , , ) previous studies have shown that low attenuation areas correlate well with pulmonary function test results in patients with at of various causes.(- ) however , to our knowledge , no studies have shown a correlation of ct densitovolumetry findings with pulmonary function in children with ob . ( , , ) our objective was to describe the correlation between the volume of areas of at and pulmonary function test results in children with ob . in addition , we compared at% values with so2 values , pulmonary function test results , and clinical scores in those children in an attempt to obtain objective measurement criteria for ob . this was a prospective study including all of the children treated at our postinfectious ob clinic for more than 5 years and having clinically stable ob and symptom onset before the age of 2 years . the study protocol was approved by the local research ethics committee , and the parents or legal guardians of all participants gave written informed consent . the diagnosis of ob was based on the identification of chronic obstructive respiratory symptoms appearing after an episode of lower respiratory tract infection before the age of 2 years in previously healthy children . ( , ) all clinical diagnoses were confirmed by characteristic findings on contemporary hrct scans . the inclusion criteria were as follows ( ) : acute bronchiolitis / viral pneumonia before the age of 2 years in previously healthy children ; evidence of persistent airway obstruction after the acute event , identified by physical examination , pulmonary function testing , or both , the obstruction being unresponsive to at least 2 weeks of treatment with systemic corticosteroids and bronchodilators ; radiological findings of obstructive lung disease , including hyperinflation , atelectasis , bronchial wall thickening , and bronchiectasis , as well as a mosaic perfusion pattern and at on ct scans ; and absence of other chronic lung diseases progressing to persistent respiratory symptoms , including tuberculosis , cystic fibrosis , bronchopulmonary dysplasia , immunodeficiency , asthma , and severe alpha-1 antitrypsin deficiency . the exclusion criteria were as follows : being unable to undergo pulmonary function testing or ct without sedation ; being unable to hold breath for the duration of ct scanning ; having disease exacerbation < 30 days before ct or pulmonary function testing ; having other lung diseases ; and requiring continuous oxygen therapy . of those 25 patients , 6 were excluded ( because they were unable to perform the required respiratory maneuvers ) . the final sample consisted of 19 children ( 14 males and 5 females ) in the 7 - 15 year age bracket ( mean age , 10 2.5 years ) . all patients performed spirometry and six - minute walk tests ( 6mwts ) in accordance with previous reports . ( , ) spirometry was performed with a vitalograph alpha spirometer ( vitalograph , buckingham , uk ) before and 10 min after the administration of an inhaled dose of albuterol ( 300 g ; aerolin ; glaxosmithkline plc , ware , uk ) with a valve spacer ( fisonair ; glaxosmithkline plc ) . all children performed a 6mwt with so2 control . because the 6mwt was performed after spirometry ( and on the same day as the latter ) , all patients used albuterol before the 6mwt . clinical scores included the following : 1 ) nutritional status ( ) ( z score : 0 = good nutritional status ; 1 = mild malnutrition ; 2 = moderate malnutrition ; and 3 = severe malnutrition ) ; 2 ) cough during remission ( 0 = absent and 1 = present ) ; 3 ) cough within 2 weeks before the examination ( 0 = absent and 1 = present ) ; 4 ) wheezing during remission ( 0 = absent and 1 = present ) ; 5 ) wheezing on most days of the week ( 0 = absent and 1 = present ) ; 6 ) wheezing in the last 2 weeks ( 0 = absent and 1 = present ) ; 7 ) difficulty breathing in the 2 weeks preceding the examination ( 0 = absent and 1 = present ) ; 8) frequency of exacerbations in the last 6 months ( 0 = no exacerbations ; 1 = sporadic exacerbations ; 2 = exacerbations every 2 months ; 3 = exacerbations every month ; and 4 = exacerbations every week ) ; 9 ) increased anteroposterior chest diameter ( 0 = absent and 1 = present ) ; 10 ) so2 at rest ( 95% = 0 ; 90 - 94% = 1 ; and < 90% = 2 ) ; 11 ) minimum so2 during the 6mwt ( 6mwt_so2 ; 95% = 0 ; 90 - 94% = 1 ; and < 90% = 2 ) , mean desaturation during exercise being measured by calculating the difference between so2 at rest and 6mwt_so2 ; 12 ) desaturation > 4% during the 6mwt ( 0 = negative and 1 = positive ) ; 13 ) the fev1/fvc ratio ; and 14 ) percent predicted fev1 ( fev1% ; > 80% = 0 ; 61 - 80% = 1 ; 41 - 60% = 2 ; < 41% = 3 ) . all ct images were acquired with the use of the lowest possible radiation dose and a commercially available helical ct scanner ( xvision ex ; toshiba medical systems corporation , otawara , japan ) , being post - processed on a workstation ( o2 ; sgi , fremont , ca , usa ) running three - dimensional ( 3d ) rendering software ( alatoview ; toshiba medical systems corporation ) . an initial set of nine axial hrct scans ( with 1-mm collimation at increments of 20 mm ) were acquired with the use of a high - frequency algorithm . those hrct images were evaluated by two thoracic radiologists with more than 10 years of experience in chest ct . two additional sets of images were acquired by the helical ct scanner during single breath - hold maneuvers ( during inhalation and exhalation ) . to minimize respiratory motion artifacts , on the basis of previous studies,(- ) the following parameters were used : collimation , 10 mm ; table speed , 14 mm / rotation ( pitch , 1.4 ) ; and low radiation dose ( 120 kv and 50 mas ) . the mean total radiation dose was 5 1.3 msv . we used helical ct scans of 10 mm , low mas , and high pitch in order to reduce radiation exposure . a standard reconstruction algorithm was used in order to avoid the effects of edge - enhancing filters on tissue density . two segmentation steps were applied to each set of helical ct images before the calculation of tlv . the lungs were isolated by eliminating from the image data any structure with a density exceeding 250 hu . subsequently , we eliminated the air within the abdomen and outside the patient ( nonpulmonary air ) by selecting regions of interest with the 3d rendering software . the regions of interest were selected by drawing a line between the lung and the nonpulmonary air on each slice . different 3d software , unavailable to us during the study period , might include different tools for lung segmentation . in order to minimize operator - dependent variability , we considered the air within the trachea or main bronchi to be pulmonary air rather than excluding it . the operator assessed segmentation accuracy by reviewing the 3d lung image generated by volume rendering ( figure 1 ) . figure 1 in a , coronal reformatted ct image ( minimum intensity projection ) showing air trapping . in b , three - dimensional ct reconstruction showing low attenuation volumes , which represent air trapping volumes . for measuring the volume of lung parenchyma with abnormally low attenuation values , areas of extremely low attenuation or density were considered to be abnormal , because of the disproportion between the volume of lung parenchyma ( including interstitial tissue , vessels , blood , lymph , interstitial fluid , and airway walls ) and the air in those regions . in order to simplify measurement , we defined " at volume " as the total volume of lung zones showing extremely low density values . we first calculated the lung volume affected by at using a fixed threshold of 950 hu ( at%950 ) . ( ) and has been used by many others in order to quantify emphysema . ( ) we considered any portion of the lungs with a density below 950 hu to be affected by at . we calculated at%950 by dividing the at volume for that threshold by the tlv . no validated threshold is available for determining the volume of hyperinflation , at , or emphysema in children . because the 950 hu threshold can underestimate the extent of disease in this population , we also calculated at volume using a threshold selected for each patient with the aid of a density mask . the threshold level was adjusted until the mask corresponded to our subjective visual impression of affected lung portions ( figure 2 ) . any portions of the lungs with densities below the selected hu were considered abnormal . figure 2 post - processing tools . after the use of a threshold , the air inside and outside the lungs is isolated . in a , axial ct scan showing air inside and outside the lungs . in b , three - dimensional ( 3d ) volume rendering of the same data . in c , 3d volume rendering of total lung volume after the exclusion of air outside the lungs . the shrink ( deflation ) volume of the lungs was calculated by subtracting the tlv as measured on images acquired during exhalation from the tlv as measured on images acquired during inhalation . the percentage of shrink volume was considered excellent if it was > 50% , reasonable / good if it was 30 - 50% , and poor if it was < 30% . for the statistical analysis , test results were entered into a microsoft excel database and processed by means of excel tools , the analysis being performed with the statistical package for the social sciences , version 11 ( spss inc . , chicago , il , usa ) . we assumed a power of 90% for a sample size of 19 patients and statistical significance of p < 0.05 , on the basis of a previous study . ( ) scatter plots ( figure 3 ) were used in order to classify patients according to the severity of at% . for at%950 and at%dm , in addition , at%950 values of 1 - 5% were considered to indicate moderate disease expression , and at%950 values > 5% were considered to indicate severe disease expression . moreover , at%dm values of 1 - 10% were considered to indicate moderate disease expression , and at%dm values > 10% were considered to indicate severe disease expression . the magnitude of variability in at%950 and at%dm guided the selection of values to differentiate between moderate and severe disease expression ( figure 3 ) . figure 3 scatter plot illustrating the distribution of patients according to the percentage of air trapping relative to total lung volume ( at% ) , which was calculated for a fixed threshold of 950 hu ( at%950 ) and for thresholds set by subjective analysis based on density masks ( at%dm ) . note that at%dm allows better discrimination among the grades of disease severity ( i.e. , grade 1 , normal / mild ; grade 2 , moderate ; and grade 3 , severe ) , especially for moderate disease ( at% > 1% ) and severe disease ( at% > 10% ) . note also that the stratification of disease severity changed from normal / mild to moderate in 1 patient ( case 10 ) and from moderate to severe in 1 patient ( case 14 ) depending on the method for selectin g the threshold . figure 4 distribution of the observations of the percentage of air trapping relative to total lung volume ( at% ) , calculated for a fixed threshold of 950 hu - at%950-(red squares ) and for thresholds selected with the aid of density masks - at%dm-(blue squares ) , including the corresponding linear regression lines . the distribution suggests that patients with fev1% above 70% had only mild anatomical damage as measured by quantification of areas of air trapping or hyperinflation . some patients with fev1% in the range of 50% or less had only mild disease as measured by at%950 , a finding that highlights the limitations of a threshold of 950 hu for the assessment of patients with obliterative bronchiolitis . the correlation between fev1% and at%dm was much better , as shown by the regression line . of the 19 patients , 2 had normal clinical scores , 7 had clinical scores < 5 ( including the two patients with clinical scores = 0 ) , 6 had scores of 5 - 10 , and 6 had scores > 10 . in the evaluation of the nutritional status , 8 patients had z scores < 0 . regarding 6mwt parameters , mean so2 at rest was 96 2% ( range , 92 - 99% ) , and mean 6mwt_so2 was 92 4% ( range , 83 - 99% ) . we found no correlation between so2 at rest and so2 during exercise ( r = 0.00 ) . mean desaturation during exercise ( measured by calculating the difference between so2 at rest and 6mwt_so2 ) was 4 4% ( range , 2% to 15% ) . the mean six - minute walk distance ( 6mwd ) was 552 131 m ( range , 90 - 705 m ) . the 6mwd correlated significantly with 6mwt_so2 ( r = 0.52 ; p < 0.05 ) and desaturation during exercise ( r = 0.58 ; p < 0.05 ) but not with so2 at rest ( r = 0.26 ; p > 0.05 ) . regarding pulmonary function parameters , mean fvc was 75 20% ( range , 43 - 106% ) , mean fev1% was 58 20% ( range , 36 - 100% ) , and mean fev1/fvc was 72 16% ( range , 49 - 107% ) . regarding ct densitovolumetry parameters , mean tlv was 3,009 1,184 ml ( range , 1,252 - 6,673 ml ) . the 6mwd was longer in those with larger normal lung volumes ( r = 0.53 ) . mean shrink volume was 1,174 789 ml ( range , 182 - 3,471 ml ) , and mean percentage of shrink volume was 36 13% ( range , 9 - 63% ) . mean at%dm was 7.28 9% ( range , 0.03 - 24.67% ) , and mean at%950 was 2.4 3% ( range , in 9 , 5 , and 5 of the 19 patients , at%dm was classified as normal or mild , moderate , and severe , respectively ( points above the gray line in figure 3 ) . in 10 , 5 , and 4 of the 19 patients , at%950 was classified as normal or mild , moderate , and severe , respectively ( points above the blue line in figure 3 ) . we found a correlation between at%dm and at%950 , as evidenced by r = 0.83 ( or r = 0.93 after the exclusion of one outlier ) . no significant correlation was found between the percentage of shrink volume and at%dm or between the percentage of shrink volume and at%950 . the correlations of ct densitovolumetry parameters with clinical scores , pulmonary function test results , and 6mwt parameters are summarized in table 1 . table 1 shows the correlation values for each parameter before and after the exclusion of outliers . an uncommon and severe form of chronic obstructive lung disease in children and adults , ob results from lower respiratory tract injury . ( ) the diagnosis of postinfectious ob in children is based on a history of lower respiratory tract infection ( usually an acute viral infection ) , followed by persistent chronic obstructive lung disease . ( , ) hrct is an excellent method for the identification of anatomical damage following the onset of the disease , such damage including areas of low attenuation , areas of consolidation / atelectasis , bronchial wall thickening , bronchiectasis , and mosaic perfusion . in addition , expiratory hrct scans can assist in confirming the presence of at . however , hrct allows only a subjective assessment of the extent of the disease and is dependent on the experience and skill of radiologists . ( , ) our study demonstrated that the 6mwd was longer in patients with larger normal lung ( r = 0.53 ) . in addition , we found that at%950 showed significant correlations ( before and after the exclusion of outliers , respectively ) with the clinical score ( r = 0.72 ; 0.80 ) , fvc ( r = 0.24 ; 0.59 ) , fev1 ( r = 0.58 ; 0.67 ) , and fev1/fvc ( r = 0.53 ; r = 0.62 ) , as did at%dm with the clinical score ( r = 0.58 ; r = 0.63 ) , so2 at rest ( r = 0.40 ; r = 0.61 ) , 6mwt_so2 ( r = 0.24 ; r = 0.55 ) , fvc ( r = 0.44 ; r = 0.80 ) , fev1 ( r = 0.65 ; r = 0.71 ) , and fev1/fvc ( r = 0.41 ; r = 0.52 ) . these data suggest that objective ct measurements adequately represent clinical scores and functional impairment in ob . ct densitovolumetry has been proven to overcome this limitation and is a standard recommendation for the quantification of other lung diseases in which the proportion between pulmonary air and the lung parenchyma is increased , therefore decreasing lung density . ( , ) areas of decreased attenuation can also result from decreased perfusion of hypoventilated alveoli distal to obstructed bronchioles . the main finding on expiratory ct scans is a geographic heterogeneity of lung attenuation ( mosaic attenuation pattern ) , which is seen in 40 - 80% of patients . ( , ) the abnormalities can be subtle on inspiratory ct scans , being usually easier to detect on expiratory ct scans . ( ) in a previous study of the correlation between pulmonary function abnormalities and the extent of hrct features of ob , significant relationships were found only between fev1 and the number of bronchopulmonary segments affected by bronchiectasis . ( ) confirmed that the extent of decreased attenuation was independently associated with a reduction in fev1 . in contrast , bronchial wall thickening was independently associated with the presence of at ( as measured by rv / tlc ) . in patients with sauropus androgynus - associated ob , pulmonary function test results were more closely correlated with at than with any other ct parameter . ( ) to our knowledge , the present study is the first to demonstrate significant correlations of at% with clinical scores and pulmonary function test results . the quantification of areas of abnormally low attenuation is an important diagnostic tool for ob , and the technique has substantial advantages over the traditional subjective assessment of hrct images.(- ) the quantification of anatomical damage is important in patients with ob ; ct densitovolumetry can measure lung volumes directly and therefore aid clinicians in making decisions regarding patient quarantining and the aggressiveness of treatment . in the present study , clinical scores were moderately correlated with fvc and fev1/fvc ( r ~ 0.5 ) . although there was a stronger correlation between clinical scores and fev1 ( r = 0.8 ) , these findings are possibly biased because pulmonary function test results were included in the clinical score parameters . additionally , both measures of at% were significantly correlated with clinical scores ( r 0.6 ) ; the strength of those correlations increased when outliers were eliminated ( censored at%950 , r = 0.8 ) , which suggests that it might increase further in larger series . we found that inspiratory ct scans were much more informative than expiratory ct scans regarding the presence of at . the extent of at areas might have been underestimated on the expiratory ct scans because of the higher expiratory lung density . the 950 hu threshold was validated for emphysema quantification in adult patients , and we used it in the present study despite our conviction that segmentation at this level would result in an underestimation of areas of low attenuation in children , especially in the absence of hyperinflation / emphysema . ( ) although histopathological findings were unavailable for our patients , previous studies ( , ) have examined correlations between imaging findings and histopathological findings in patients with ob . in addition , our clinical score has yet to be validated , its clinical application requiring further investigation . in conclusion , anatomical damage to the lungs as measured by ct ( i.e. , at% ) correlated significantly with clinical scores and pulmonary function test results . after our censoring of extreme values , at%dm showed stronger correlations than did at%950 . determinar as correlaes entre o volume de aprisionamento areo em relao ao volume pulmonar total ( aa% ) e parmetros clnicos e funcionais em crianas com bronquiolite obliterante ( bo ) . tcnicas de ps - processamento de imagem foram usadas em imagens de tc de 19 crianas com bo para quantificar aa% por meio de um limiar fixo de 950 uh ( aa%950 ) e de limiares selecionados por meio de mscaras de densidade ( aa%md ) . os pacientes foram divididos em trs grupos , de acordo com a gravidade de aa% . foram examinadas as correlaes entre aa% e a saturao de oxignio ( so2 ) em repouso , a distncia percorrida no teste de caminhada de seis minutos ( dtc6 ) , a so2 mnima durante o teste de caminhada de seis minutos ( so2_tc6 ) , a cvf , o vef1 , a relao vef1/cvf e parmetros clnicos . a dtc6 foi maior nos pacientes com maiores volumes pulmonares normais ( r = 0,53 ) . na amostra como um todo , encontramos ( antes e depois da excluso de valores extremos , respectivamente ) , correlaes estatisticamente significativas entre aa%950 e o escore clnico ( r = 0,72 ; 0,80 ) , a cvf ( r = 0,24 ; 0,59 ) , o vef1 ( r = 0,58 ; 0,67 ) e a relao vef1/cvf ( r = 0,53 ; r = 0,62 ) , bem como entre aa%md e o escore clnico ( r = 0,58 ; r = 0,63 ) , a so2 em repouso ( r = 0,40 ; r = 0,61 ) , a so2_tc6 ( r = 0,24 ; r = 0,55 ) , a cvf ( r = 0,44 ; r = 0,80 ) , o vef1 ( r = 0,65 ; r = 0,71 ) e a relao vef1/cvf ( r = 0,41 ; r = 0,52 ) . os resultados deste estudo mostram que aa% correlaciona - se significativamente com escores clnicos e testes de funo pulmonar em crianas com bo . a bronquiolite obliterante ( bo ) ps - infecciosa uma forma incomum e grave de doena pulmonar obstrutiva crnica que ocorre em crianas aps uma agresso ao trato respiratrio inferior . ( ) no tocante ao diagnstico , no h sinais ou sintomas especficos de bo ps - infecciosa . achados clnicos e de imagem so usados em conjunto com resultados de testes laboratoriais para identificar a causa e descartar outras formas de doena pulmonar crnica . os critrios diagnsticos para bo ps - infecciosa so os seguintes : bronquiolite aguda grave / pneumonia viral durante os 3 primeiros anos de vida em crianas previamente saudveis ; evidncia de obstruo persistente das vias areas aps o evento agudo , identificada por meio de exame fsico , teste de funo pulmonar ou ambos ; achados de doena pulmonar obstrutiva na radiografia de trax ; padro em mosaico e aprisionamento areo ( aa ) na tc de trax e excluso de outras doenas pulmonares crnicas que progridem para sintomas respiratrios permanentes . ( , ) histologicamente , a bo caracterizada pela presena de tampes de tecido de granulao no lmen das pequenas vias areas , destruio completa das pequenas vias areas ou ambas . ( ) mauad et al . ( ) mostraram que a bo histologicamente caracterizada por um padro constritivo com diversos graus de inflamao e obliterao das vias areas , desde inflamao bronquiolar mnima at obliterao completa dos bronquolos e brnquios por tecido fibrtico . sinais indiretos de obstruo , tais como acmulo de macrfagos , bronquiectasia , acmulo de muco e hiperinsuflao , esto sempre presentes . embora vrias revises tenham examinado as sequelas em longo prazo da pneumonia por adenovrus , ( , ) poucos estudos examinaram os achados tomogrficos em crianas com bo . ( , ) alguns desses estudos relataram que achados tomogrficos anormais nessas crianas podem predizer a presena de funo pulmonar anormal anos mais tarde . ( ) relatou - se que a tcar uma importante ferramenta diagnstica para avaliar o dano pulmonar em pacientes com bo . ( ) o volume de um rgo ( ou o volume de partes anormais de um rgo ) pode ser determinado por meio de imagens de tc helicoidal e valores do coeficiente de atenuao ou densidades na escala de hounsfield , em unidades hounsfield ( uh ) . esse mtodo conhecido como densitometria e anlise volumtrica pulmonar por tc ou densitovolumetria pulmonar por tc ( , ) e pode ser usado para medir o volume de reas pulmonares com baixa atenuao ( ou seja , aa ) e o volume pulmonar total ( vpt ) em crianas com bo . alm disso , pode ser usado para medir a porcentagem de aa em relao ao vpt ( aa% ) por meio de limiares para diferenciar reas pulmonares normais de reas de bo . demonstrou - se que aa% contribui sobremaneira para a obstruo ao fluxo areo persistente e crnica na asma . ( ) a funo pulmonar permanece anormal durante longos perodos aps um episdio de bo . ( , ) o curso clnico aps o aparecimento de bo varivel e depende do volume do tecido pulmonar afetado . ( ) medidas do estado clnico e da capacidade de realizar atividades fsicas intensas - medidas como o nmero de internaes hospitalares ou de dias de aula perdidos , a distncia percorrida em testes de caminhada , o estado nutricional e a saturao de oxignio ( so2 ) em repouso ou durante o exerccio - facilitam a seleo de intervenes clnicas apropriadas . ( , ) como o prognstico dos pacientes com bo depende no s de seu comportamento , mas tambm do comprometimento funcional e da extenso do dano anatmico , os clnicos devem reunir informaes sobre o tipo de anormalidade anatmica e sua extenso . bronquiectasia , atelectasia , colapso lobar e reas de baixa densidade so conhecidas consequncias da bo ps - infecciosa , e a tcar o melhor mtodo para examinar tais leses , ( , , ) as quais podem influenciar o estado clnico dos pacientes e seu desempenho nos testes de funo pulmonar . ( , , ) estudos anteriores demonstraram que reas de baixa atenuao correlacionam - se bem com os resultados de testes de funo pulmonar em pacientes com aa de causas variadas . ( - ) no entanto , at onde sabemos , nenhum estudo mostrou uma correlao entre achados de densitovolumetria pulmonar por tc e funo pulmonar em crianas com bo . ( , , ) nosso objetivo foi descrever a correlao entre o volume de reas de aa e os resultados de testes de funo pulmonar em crianas com bo . alm disso , comparamos os valores de aa% aos valores de so2 , aos resultados dos testes de funo pulmonar , e aos escores clnicos observados nessas crianas , na tentativa de obter critrios objetivos de medio de bo . trata - se de um estudo prospectivo incluindo todas as crianas que foram atendidas em nossa clnica de bo ps - infecciosa durante mais de 5 anos e que apresentavam bo clinicamente estvel e incio dos sintomas antes da idade de 2 anos . o protocolo do estudo foi aprovado pelo comit de tica em pesquisa da instituio , e todos os pais ou responsveis assinaram um termo de consentimento livre e esclarecido . o diagnstico de bo baseou - se na identificao de sintomas respiratrios obstrutivos crnicos aps um episdio de infeco do trato respiratrio inferior antes da idade de 2 anos em crianas anteriormente saudveis . ( , ) todos os diagnsticos clnicos foram confirmados por achados caractersticos em tcar contempornea . os critrios de incluso foram os seguintes ( ) : bronquiolite aguda / pneumonia viral antes dos 2 anos de idade em crianas previamente saudveis ; evidncia de obstruo persistente das vias areas aps o evento agudo , identificada por meio de exame fsico , teste de funo pulmonar ou ambos e sem resposta a pelo menos 2 semanas de tratamento com corticosteroides sistmicos e broncodilatadores ; achados radiolgicos de doena pulmonar obstrutiva , incluindo hiperinsuflao , atelectasia , espessamento das paredes brnquicas e bronquiectasia , alm de padro de perfuso em mosaico e aa na tc ; ausncia de outras doenas pulmonares crnicas que progridem para sintomas respiratrios persistentes , incluindo tuberculose , fibrose cstica , displasia broncopulmonar , imunodeficincias , asma e deficincia grave de alfa-1 antitripsina . os critrios de excluso foram os seguintes : incapacidade de se submeter aos testes de funo pulmonar ou tc sem sedao ; incapacidade de segurar a respirao durante todo o exame de tc ; exacerbao da doena < 30 dias antes da tc ou dos testes de funo pulmonar ; presena de outras doenas pulmonares e necessidade de oxigenoterapia contnua . um total de 25 pacientes com bo preencheram os critrios de incluso . desses 25 pacientes , 6 foram excludos ( porque no foram capazes de realizar as manobras respiratrias necessrias ) . a amostra final foi composta por 19 crianas ( 14 do sexo masculino e 5 do feminino ) de 7 - 15 anos de idade ( mdia : 10 2,5 anos ) . todos os pacientes realizaram espirometria e teste de caminhada de seis minutos ( tc6 ) em conformidade com relatos anteriores . ( , ) a espirometria foi realizada com um espirmetro vitalograph alpha ( vitalograph , buckingham , reino unido ) antes e 10 min depois da administrao de 300 g de salbutamol inalatrio ( aerolin ; glaxosmithkline plc , ware , reino unido ) com um espaador valvulado ( fisonair ; glaxosmithkline plc ) . como o tc6 foi realizado aps a espirometria ( e no mesmo dia que esta ) , todos os pacientes usaram salbutamol antes do tc6 . o escore clnico incluiu os seguintes itens : 1 ) estado nutricional ( ) ( escore z : 0 = bom estado nutricional ; 1 = desnutrio leve ; 2 = desnutrio moderada e 3 = desnutrio grave ) ; 2 ) tosse durante a remisso ( 0 = ausente e 1 = presente ) ; 3 ) tosse 2 semanas antes do exame ( 0 = ausente e 1 = presente ) ; 4 ) sibilncia durante a remisso ( 0 = ausente e 1 = presente ) ; 5 ) sibilncia na maioria dos dias da semana ( 0 = ausente e 1 = presente ) ; 6 ) sibilncia nas ltimas 2 semanas ( 0 = ausente e 1 = presente ) ; 7 ) dificuldade para respirar nas 2 semanas anteriores ao exame ( 0 = ausente e 1 = presente ) ; 8) frequncia de exacerbaes nos ltimos 6 meses ( 0 = sem exacerbaes ; 1 = exacerbaes espordicas ; 2 = exacerbaes a cada 2 meses ; 3 = exacerbaes todos os meses e 4 = exacerbaes todas as semanas ) ; 9 ) aumento do dimetro anteroposterior do trax ( 0 = ausente e 1 = presente ) ; 10 ) so2 em repouso ( > 95% = 0 ; 90 - 94% = 1 e < 90% = 2 ) ; 11 ) so2 mnima durante o tc6 ( so2_tc6 ; > 95% = 0 ; 90 - 94% = 1 e < 90% = 2 ) , a dessaturao mdia durante o exerccio sendo medida por meio do clculo da diferena entre so2 em repouso e so2_tc6 ; 12 ) dessaturao > 4% durante o tc6 ( 0 = negativo e 1 = positivo ) ; 13 ) relao vef1/cvf e 14 ) vef1 em porcentagem do previsto ( vef1% ; > 80% = 0 ; 61 - 80% = 1 ; 41 - 60% = 2 ; < 41% = 3 ) . todos as imagens de tc foram adquiridas com a dose de radiao mais baixa possvel e um tomgrafo helicoidal disponvel comercialmente ( xvision ex ; toshiba medical systems corporation , otawara , japo ) e foram ps - processadas em uma estao de trabalho ( o2 ; sgi , fremont , ca , eua ) com software para reconstruo tridimensional ( alatoview ; toshiba medical systems corporation ) . o tomgrafo foi calibrado periodicamente , conforme recomendado pelo fabricante . um conjunto inicial de nove imagens de tcar axial ( com 1 mm de colimao em incrementos de 20 mm ) foi adquirido por meio de um algoritmo de alta frequncia . essas imagens de tcar foram avaliadas por dois radiologistas torcicos com mais de 10 anos de experincia em tc de trax . a deciso final foi tomada por consenso . dois conjuntos adicionais de imagens foram adquiridos pelo tomgrafo helicoidal durante manobras de pausa respiratria nica ( durante a inspirao e a expirao ) . para reduzir o nmero de artefatos causados pelo movimento respiratrio , a tc helicoidal foi realizada no sentido caudocranial . anteriores,(- ) foram usados os seguintes parmetros : colimao = 10 mm ; velocidade da mesa = 14 mm / rotao ( pitch = 1.4 ) e baixa dose de radiao ( 120 kv e 50 mas ) . a mdia da dose total de radiao foi de 5 1,3 msv . usamos cortes tomogrficos helicoidais de 10 mm , mas baixa e pitch elevado para reduzir a exposio radiao . um algoritmo de reconstruo convencional foi usado para evitar os efeitos de filtros de realce de bordas sobre a densidade do tecido . o primeiro passo da densitovolumetria pulmonar por tc foi a segmentao pulmonar para medir o vpt . dois passos de segmentao foram aplicados a cada conjunto de imagens tomogrficas helicoidais antes do clculo do vpt . os pulmes foram isolados por meio da eliminao de qualquer estrutura com densidade superior a 250 uh dos dados de imagem . posteriormente , eliminamos o ar dentro do abdome e fora do paciente ( ar no pulmonar ) selecionando regies de interesse com o software de reconstruo tridimensional . as regies de interesse foram selecionadas por meio de uma linha desenhada entre o pulmo e o ar no pulmonar em cada corte . diferentes programas de reconstruo tridimensional , indisponveis para ns durante o perodo de estudo , podem incluir ferramentas diferentes para a segmentao pulmonar . a fim de reduzir a variabilidade atribuda ao operador , o ar dentro da traqueia ou dos brnquios principais foi considerado ar pulmonar em vez de ser excludo . o operador avaliou a preciso da segmentao por meio da reviso da imagem tridimensional do pulmo gerada pela reconstruo volumtrica ( figura 1 ) . o software ento calculou o vpt automaticamente . figura 1 em a , imagem tomogrfica coronal reformatada ( projeo de intensidade mnima ) mostrando aprisionamento areo . em b , tc com reconstruo tridimensional mostrando volumes de baixa atenuao , que representam volumes de aprisionamento areo . para medir o volume do parnquima pulmonar com valores de atenuao anormalmente baixos , reas de atenuao ou densidade extremamente baixa foram consideradas anormais , em virtude da desproporo entre o volume do parnquima pulmonar ( incluindo o tecido intersticial , vasos , sangue , linfa , lquido intersticial e paredes das vias areas ) e o ar nessas regies . a fim de simplificar a medio , consideramos " volume de aa " o volume total de zonas pulmonares com valores de densidade extremamente baixos . primeiro calculamos o volume pulmonar afetado por aa usando um limiar fixo de 950 uh ( aa%950 ) . ( ) qualquer poro dos pulmes com densidade inferior a 950 uh foi considerada afetada por aa . para calcular aa%950 , o volume de aa calculado com esse limiar foi dividido pelo vpt . o prximo passo foi calcular aa% com limiares selecionados por meio de mscaras de densidade . no h limiares validados para a determinao do volume de hiperinsuflao , aa ou enfisema em crianas . como o limiar de 950 uh pode subestimar a extenso da doena nessa populao , tambm calculamos o volume de aa com um limiar selecionado para cada paciente com o auxlio de uma mscara de densidade . o limiar foi ajustado at que a mscara correspondesse a nossa impresso visual subjetiva a respeito de pores pulmonares afetadas ( figura 2 ) aps o uso de um limiar , o ar dentro e fora dos pulmes isolado . em a , imagem de tc axial mostrando o ar dentro e fora dos pulmes . em c , reconstruo tridimensional do volume pulmonar total aps a excluso do ar fora dos pulmes . para calcular o volume de encolhimento ( desinsuflao ) pulmonar , o vpt medido em imagens adquiridas durante a expirao foi subtrado do vpt medido em imagens adquiridas durante a inspirao . a porcentagem de volume de encolhimento era considerada excelente se > 50% , razovel / boa se = 30 - 50% e ruim se < 30% . para a anlise estatstica , os resultados dos testes foram inseridos em um banco de dados a anlise foi feita com o programa statistical package for the social sciences , verso 11 ( spss inc . , assumiu - se um poder de 90% para uma amostra de 19 pacientes e significncia estatstica de p < 0,05 , com base em um estudo anterior . ( ) grficos de disperso ( figura 3 ) foram usados para classificar os pacientes de acordo com a gravidade de aa% . valores de aa%950 de 1 - 5% foram considerados indicativos de moderada expresso da doena , e valores > 5% foram considerados indicativos de grave expresso da doena . alm disso , valores de aa%md de 1 - 10 % foram considerados indicativos de moderada expresso da doena , e valores > 10% foram considerados indicativos de grave expresso da doena . a magnitude da variabilidade de aa%950 e aa%md orientou a seleo de valores para distinguir doena moderada de doena grave ( figura 3 ) . figura 3 grfico de disperso ilustrando a distribuio dos pacientes de acordo com a porcentagem de aprisionamento areo em relao ao volume pulmonar total ( aa% ) , calculada com um limiar fixo de 950 uh ( aa%950 ) e com limiares estabelecidos por anlise subjetiva baseada em mscaras de densidade ( aa%md ) . nota - se que aa%md permite melhor discriminao dos graus de gravidade da doena ( ou seja , grau 1 , normal / leve ; grau 2 , moderada e grau 3 , grave ) , especialmente doena moderada ( aa% > 1% ) e grave ( aa% > 10% ) . de normal / leve para moderada em 1 paciente ( caso 10 ) e de moderada para grave em 1 paciente ( caso 14 ) dependendo do mtodo usado para selecionar o limiar . todas as variveis foram analisadas por meio do coeficiente de correlao produto - momento de pearson . as correlaes foram determinadas antes e depois da excluso de valores extremos ( figura 4 ) . os valores de r e p foram calculados separadamente para dados censurados e no censurados . foram tambm calculadas as correlaes com os resultados dos testes de funo pulmonar e escores clnicos . figura 4 distribuio das observaes da porcentagem de aprisionamento areo em relao ao volume pulmonar total ( aa% ) , calculada com um limiar fixo de 950 uh - at%950 - ( quadrados vermelhos ) e com limiares selecionados com o auxlio de mscaras de densidade - aa%md - ( quadrados azuis ) , incluindo as linhas de regresso linear correspondentes . a distribuio sugere que os pacientes com vef1% acima de 70% apresentavam dano anatmico leve conforme medido pela quantificao das reas de aprisionamento areo ou hiperinsuflao . alguns pacientes com vef1% na faixa de 50% ou menos tinham doena leve conforme medida por aa%950 , um achado que evidencia as limitaes do limiar de 950 uh para avaliar pacientes com bronquiolite obliterante . a correlao entre vef1% e aa%md foi muito melhor , como mostra a linha de regresso . dos 19 pacientes , 2 apresentaram escores clnicos normais , 7 apresentaram escores clnicos < 5 ( incluindo os dois pacientes com escore clnico = 0 ) , 6 apresentaram escores de 5 - 10 e 6 apresentaram escores > 10 . na avaliao do estado nutricional , 8 pacientes apresentaram escore z < 0 . no tocante aos parmetros do tc6 , a mdia da so2 em repouso foi de 96 2% ( variao : 92 - 99% ) , e a mdia da so2_tc6 foi de 92 4% ( variao : 83 - 99% ) . no encontramos nenhuma correlao entre so2 em repouso e so2 durante o exerccio ( r = 0,00 ) . a dessaturao mdia durante o exerccio ( medida por meio do clculo da diferena entre so2 em repouso e so2_tc6 ) foi de 4 4% ( variao : 2% a 15% ) . a mdia da distncia percorrida no tc6 ( dtc6 ) foi de 552 131 m ( variao : 90 - 705 m ) . a dtc6 correlacionou - se significativamente com so2_tc6 ( r = 0,52 ; p < 0,05 ) e dessaturao durante o exerccio ( r = 0,58 ; p < 0,05 ) , mas no com so2 em repouso ( r = 0,26 ; p > 0,05 ) . no tocante aos parmetros de funo pulmonar , a mdia da cvf foi de 75 20% ( variao : 43 - 106% ) , a mdia do vef1% foi de 58 20% ( variao : 36 - 100% ) e a mdia da relao vef1/cvf foi de 72 16% ( variao : 49 - 107% ) . no tocante aos parmetros da densitovolumetria pulmonar por tc , a mdia do vpt foi de 3.009 1.184 ml ( variao : 1.252 - 6.673 ml ) . a dtc6 foi maior nos pacientes com maiores volumes pulmonares normais ( r = 0,53 ) . a mdia do volume de encolhimento foi de 1.174 789 ml ( variao : 182 - 3.471 ml ) , e a mdia da porcentagem do volume de encolhimento foi de 36 13% ( variao : 9 - 63% ) . a mdia de aa%md foi de 7,28 9% ( variao : 0,03 - 24,67% ) , e a mdia de aa%950 foi de 2,4 3% ( variao : 0,03 - 8,67% ) . em 9 , 5 e 5 dos 19 pacientes , aa%md foi classificada em normal ou leve , moderada e grave , respectivamente ( pontos acima da linha cinza na figura 3 ) . em 10 , 5 e 4 dos 19 pacientes , aa%950 foi classificada em normal ou leve , moderada e grave , respectivamente ( pontos acima da linha azul na figura 3 ) . encontramos uma correlao entre aa%md e aa%950 , evidenciada por r = 0,83 ( ou r = 0,93 , aps a excluso de um valor extremo ) . no encontramos correlao significativa entre a porcentagem do volume de encolhimento e aa%md ou entre a porcentagem do volume de encolhimento e aa%950 . as correlaes dos parmetros de densitovolumetria pulmonar por tc com os escores clnicos , resultados dos testes de funo pulmonar e parmetros do tc6 esto resumidas na tabela 1 . a tabela 1 mostra os valores de correlao para cada parmetro , antes e depois da excluso de valores extremos . a figura 4 ilustra as correlaes de aa%md e aa%950 com os parmetros no densitomtricos . a bo uma forma incomum e grave de doena pulmonar obstrutiva crnica em crianas e adultos que resulta de uma agresso ao trato respiratrio inferior . ( ) o diagnstico de bo ps - infecciosa em crianas baseia - se em uma histria de infeco do trato respiratrio inferior ( geralmente uma infeco viral aguda ) seguida de doena pulmonar obstrutiva crnica persistente . ( , ) a tcar um excelente mtodo para a identificao de dano anatmico aps o incio da doena , incluindo reas de baixa atenuao , reas de consolidao / atelectasia , espessamento das paredes brnquicas , bronquiectasia e perfuso em mosaico . alm disso , imagens de tcar expiratria podem ajudar a confirmar a presena de aa . no entanto , a tcar permite apenas uma avaliao subjetiva da extenso da doena e depende da experincia e habilidade dos radiologistas . ( , ) nosso estudo demonstrou que a dtc6 foi maior em pacientes com maior pulmo normal ( r = 0,53 ) . alm disso , encontramos correlaes significativas ( antes e aps a excluso de valores extremos , respectivamente ) entre aa%950 e o escore clnico ( r = 0,72 ; 0,80 ) , a cvf ( r = 0,24 ; 0,59 ) , o vef1 ( r = 0.58 ; 0.67 ) e a relao vef1/cvf ( r = 0,53 ; r = 0,62 ) , bem como entre aa%md e o escore clnico ( r = 0,58 ; r = 0,63 ) , a so2 em repouso ( r = 0,40 ; r = 0,61 ) , a so2_tc6 ( r = 0,24 ; r = 0,55 ) , a cvf ( r = 0,44 ; r = 0,80 ) , o vef1 ( r = 0,65 ; r = 0,71 ) e a relao vef1/cvf ( r = 0,41 ; r = 0,52 ) . esses dados sugerem que medidas tomogrficas objetivas representam adequadamente escores clnicos e comprometimento funcional na bo . provou - se que a densitovolumetria pulmonar por tc supera essa limitao e uma recomendao - padro para a quantificao de outras doenas pulmonares nas quais a proporo entre o ar pulmonar e o parnquima pulmonar maior , diminuindo , portanto , a densidade pulmonar . ( , ) reas de atenuao reduzida tambm podem resultar da diminuio da perfuso de alvolos hipoventilados distais aos bronquolos obstrudos . o principal achado da tomografia expiratria uma heterogeneidade geogrfica da atenuao pulmonar ( padro de atenuao em mosaico ) , observado em 40 - 80% dos pacientes . ( , ) as alteraes podem ser sutis nas imagens de tc inspiratria ; so geralmente mais fceis de detectar em imagens de tc expiratria . ( ) em um estudo anterior da correlao entre alteraes da funo pulmonar e a extenso das caractersticas de bo na tcar , foram encontradas relaes significativas somente entre vef1 e o nmero de segmentos broncopulmonares afetados por bronquiectasia . ( ) confirmaram que a extenso da diminuio da atenuao associou - se independentemente a uma reduo do vef1 . em contraste , o espessamento das paredes brnquicas associou - se independentemente presena de aa ( medida por vr / cpt ) . em pacientes com bo associada a sauropus androgynus , os resultados dos testes de funo pulmonar correlacionaram - se mais com aa do que com qualquer outro parmetro tomogrfico . ( ) at onde sabemos , o presente estudo o primeiro a demonstrar correlaes significativas entre aa% e escores clnicos e resultados de testes de funo pulmonar . a quantificao das reas de atenuao anormalmente baixa uma importante ferramenta para o diagnstico de bo , e a tcnica tem vantagens substanciais sobre a tradicional avaliao subjetiva de imagens de tcar.(- ) a quantificao do dano anatmico importante em pacientes com bo ; a densitovolumetria pulmonar por tc capaz de medir volumes pulmonares diretamente e , portanto , ajudar os clnicos a tomar decises sobre a quarentena de pacientes e a agressividade do tratamento . no presente estudo , os escores clnicos correlacionaram - se moderadamente com a cvf e a relao vef1/cvf ( r ~ 0,5 ) . embora a correlao entre os escores clnicos e o vef1 tenha sido mais forte ( r = 0,8 ) , esses resultados so possivelmente tendenciosos , pois os resultados dos testes de funo pulmonar foram includos nos parmetros do escore clnico . alm disso , ambas as medidas de aa% correlacionaram - se significativamente com os escores clnicos ( r 0,6 ) ; a fora dessas correlaes aumentou quando foram eliminados os valores extremos ( aa%950 censurada : r = 0,8 ) , o que sugere que pode aumentar ainda mais em sries maiores . as imagens de tc inspiratria foram muito mais informativas que as de tc expiratria no tocante presena de aa . a extenso das reas de aa pode ter sido subestimada nas imagens de tc expiratria por causa da densidade pulmonar expiratria maior . o limiar de 950 uh foi validado para a quantificao do enfisema em pacientes adultos , e ns o usamos no presente estudo no obstante nossa convico de que a segmentao nesse nvel resultaria em subestimao das reas de baixa atenuao em crianas , especialmente na ausncia de hiperinsuflao / enfisema . ( ) embora no tivssemos os achados histopatolgicos em nossos pacientes , estudos anteriores ( , ) examinaram as correlaes entre achados de imagem e achados histopatolgicos em pacientes com bo . alm disso , nosso escore clnico ainda no foi validado ; so necessrios mais estudos para sua aplicao clnica . em suma , o dano anatmico aos pulmes medido por meio de tc ( ou seja , aa% ) correlacionou - se significativamente com escores clnicos e resultados de testes de funo pulmonar . aps a censura de valores extremos , aa%md apresentou correlaes mais fortes do que aa%950 .

objective : to determine whether air trapping ( expressed as the percentage of air trapping relative to total lung volume [ at% ] ) correlates with clinical and functional parameters in children with obliterative bronchiolitis ( ob ) . methods : ct scans of 19 children with ob were post - processed for at% quantification with the use of a fixed threshold of 950 hu ( at%950 ) and of thresholds selected with the aid of density masks ( at%dm ) . patients were divided into three groups by at% severity . we examined at% correlations with oxygen saturation ( so2 ) at rest , six - minute walk distance ( 6mwd ) , minimum so2 during the six - minute walk test ( 6mwt_so2 ) , fvc , fev1 , fev1/fvc , and clinical parameters . results : the 6mwd was longer in the patients with larger normal lung volumes ( r = 0.53 ) . we found that at%950 showed significant correlations ( before and after the exclusion of outliers , respectively ) with the clinical score ( r = 0.72 ; 0.80 ) , fvc ( r = 0.24 ; 0.59 ) , fev1 ( r = 0.58 ; 0.67 ) , and fev1/fvc ( r = 0.53 ; r = 0.62 ) , as did at%dm with the clinical score ( r = 0.58 ; r = 0.63 ) , so2 at rest ( r = 0.40 ; r = 0.61 ) , 6mwt_so2 ( r = 0.24 ; r = 0.55 ) , fvc ( r = 0.44 ; r = 0.80 ) , fev1 ( r = 0.65 ; r = 0.71 ) , and fev1/fvc ( r = 0.41 ; r = 0.52 ) . conclusions : our results show that at% correlates significantly with clinical scores and pulmonary function test results in children with ob .

Introduction Methods Results Discussion OBJETIVO: MTODOS: RESULTADOS: CONCLUSES: Introduo Mtodos Resultados Discusso

PMC3185260

to date , one of the most understudied etiologies of older adults ' survival and longevity has been the role of their cognitive beliefs and worldviews that possibly interact with their functional and mental capacities to endure , challenge , overcome , and survive in the face of the numerous struggles and obstacles in advanced old age . in earlier research on predictors of mortality , the focus has been exclusively on variations in physical health and sociodemographic variables to explain and predict differences in longevity and mortality rates across a wide age range . more recently , studies have explored the relationship between the 5-factor personality traits [ 14 ] and other stress - inducing traits of perfectionism and dysfunctional dependency traits to predict greater longevity or increased risk of mortality . the present study presents a clear departure from earlier studies that have focused on sociodemographic and personality factors to explain and predict differences in all - cause mortality rates in later life . the goal of the present research is to move outside the personality and traits model to other second - order cognitive - behavioral factors to predict differences in all - cause mortality rates in later life . cognitive - behavioral theorists argue on both theoretical and empirical grounds that individuals ' cognitive beliefs exert a great deal of influence on their health , resilience , and longevity and may logically be assumed to be robust predictors of impending mortality or conversely of longevity . however , the ability of cognitive belief systems to predict important health outcomes of survival and longevity has traditionally been questioned because of the putative effects of individuals ' earlier life experiences such as parental loss and divorce . more recent explorations into individuals ' cognitive beliefs have been drawing attention to a cluster of beliefs systems that may counter the effects of earlier negative experiences and may serve as strengthening factors toward enhancing longevity . recent research using more modern concepts of evaluating dominant cognitive beliefs and cognitive perspectives of individuals ( e.g. , beliefs about a just world ( bjw ) for self and others , beliefs about one 's future time , beliefs about one 's self - continuity with the future , and beliefs about social , political , and interpersonal trust ) has provided growing evidence that individuals ' cognitive beliefs and perspectives are indeed related to health - related processes [ 7 , 8 ] leading to longer survival and longevity as a final health outcome . also , in recent years , the availability of reliable and valid measures of cognitive beliefs of justice and fairness [ 9 , 10 ] , future time perspectives , and future self - continuity perspectives [ 12 , 13 ] has increased our understanding of the predictive value of individuals ' cognitive beliefs as strengthening or debilitating factors in health - related outcomes of resilience and longevity . the present study addresses the relationship between newly emerging sets of important cognitive belief systems and the potential for increased longevity . for example , a number of researchers [ 10 , 14 ] have demonstrated empirically that individuals ' tenacious cognitive beliefs in a just world ( bjw ) society are not only predictive of their subjective well being and resilience , but more importantly drive them toward investment in long - term goals and a commitment to better self - care of health and a longer life . as a result , life - span scholars are now more keenly exploring the proactive role that individuals ' beliefs about a just world ( bjw ) may play in their future well being and healthy physical survival ( see tomaka and blascovitch , 1994 ) , low levels of depression , and less loneliness . other dominant cognitive beliefs which have been seen to be related to healthy survival processes or which present increased risks of mortality include beliefs about interpersonal trust and trust in key institutions [ 18 , 19 ] . individuals who have strong positive beliefs of trust in the interpersonal and institutional domains are commonly expected to live lives that are more organized and planned , as distinguished from lives of instability , anxiety , and caution . other perspectives and belief systems that are predictive of planned healthy survival include future time perspective ( ftp ) and self - continuity with the future perspective ( fsc ) . the ftp perspective is a measure of individuals ' perceived belief about how much time participants had left in life . according to carstensen , the subjective sense of remaining time has profound effects on basic human processes , including motivation , cognition , and behaviors . with increasing age , constraints on time left shift individuals ' priorities about how remaining time can be protected . along somewhat similar lines , the future self - continuity perspective ( fsc ) indicates that participants ' beliefs about their similarity and connection as well as caring and liking for their future self 10- , 15- , or 20-years from now determine and shift their motivation to protect the potential future person . for purposes of the present study , our underlying conceptual assumption in both these futuristic perspectives is that individuals who perceive their time horizons and their self - continuity with the future as more limited would be more likely to discount the future , and thus more unlikely to plan for self - care and self - management of the future , whereas those who feel the future horizons are more open - ended and expansive are more likely to plan and organize for a secure future . implicit in these perspectives is the prediction that individual differences in the experience of self - continuity could have positive pragmatic consequences for future health care and healthy survival . one logical assumption is that people who experience little or no continuity with the future self may not aspire to control future health - related processes whereas people who experience much similarity or self - continuity with the future self are likely more motivated to work toward shaping a better survival . while previous research has demonstrated empirically the predictive value of the preceding sets of cognitive beliefs and perspectives in regard to well - being and physical and mental health - related processes and outcomes , findings have been drawn from the study of a wide range of ages . thus , there is the question of whether the prognostic value of these belief systems continues into advanced old age . to date , the number of longitudinal studies of cognitive beliefs as predictors of longevity or as risk to mortality in old age is limited . to address this issue , accordingly , the purpose of our current research was to examine longitudinally the extent to which specific and select sets of cognitive beliefs are enabling , strengthening , or disabling with respect to long - term health , resilience , and longevity of older adults and have predictive value for all - cause mortality in advanced old age . the question is of increasing interest to health professionals and gerontologists for both practical and conceptual reasons . in the section which follows , we review briefly the research literature that both explains and extends the assumptions underlying the theory and goals of concepts of bjw , ftp , and fsc and their potential for predicting longer survival / or reduced risk for mortality . the just world hypothesis and how it may relate to health and longevity is easily stated . individuals have a strong need to believe that they live in a world where people generally get what they deserve . the belief that the world is just enables the individual to confront his / her physical and social environment as though they were stable and orderly . without such a belief , it would be difficult for the individual to commit himself / herself to the pursuit of long - range goals or even to the socially regulated behavior of day - to - day life . since the belief that the world is just serves such an important adaptive function for the individual , people are very reluctant to give up this belief , and they can be greatly troubled if they encounter evidence that suggests that the world is not really just or orderly after all . according to lerner and miller 's just - world theory , people who believe that the world treats them fairly may plan confidently for their future , expecting their lives to be orderly , meaningful , and controllable , foreseeing a positive future or viewing one 's living situation as justly deserved and hence fair . in turn , this expectation promotes mental health , meaning that the belief in a just world ( bjw ) can be seen as a positive illusion . indeed research links bjw to many indices of subjective well being including a greater purpose in life and commitment to planned healthy survival . there is empirical evidence showing that individuals who strongly believe in a just world have been seen to experience less stress and more positive affect than individuals with a weaker bjw ( e.g. , [ 16 , 24 ] ) . the preceding conceptual underpinnings and the recent theory and research related to cognitive beliefs of a just world ( bjw ) lend weight to the proposed hypothesis of our present study that strong bjw beliefs about justice for the self portend positive social consequences and health - related benefits for the future . as such , individuals ' strong cognitive beliefs about a just world ( bjw ) may be early predictors of their continued physical and mental well being at later stages of life and would serve to protect them against the stress associated with the challenges of later life . in the current study , we reason that the predictive power of bjw to enhance longevity derives uniquely from perceived justice and bjw beliefs . in light of the preceding discussion , our leading hypotheses for the current study were ( 1 ) that individuals ' beliefs that the world is just to themselves ( bjw - self ) are particularly predictive of their longer term survival and longevity , and ( 2 ) that the associations between cognitive beliefs of a just world would be observed more powerfully among measures of bjw for self only ( as distinguished from bjw for others ) . in essence , we reason that it is the perception of one 's own , more so than other individuals ' , outcomes that would most powerfully predict longevity or possible risks of mortality ; ( 3 ) that individuals ' stronger levels of interpersonal trust and trust in the major communal institutions ( as associated with their perceptions of justice in the bjw beliefs ) are early predictors of their longevity , or conversely stronger levels of distrust in the major communal institutions would be associated with increased risk of mortality . a related second goal of our current study was to examine the predictive value of other related cognitive perspectives such as future time perspectives not previously studied as predictors of mortality and longevity . on the basis of postulates derived from carstensen 's theory on the influence of a sense of time on human development ( also see [ 13 , 25 ] ) theory on individual differences in future self - continuity , we reason that while time eventually runs out for all individuals , individuals who hold more expansive and open - ended future time horizons or who foresee stronger self - continuity with the future self ( compared to those who hold more limited future time horizons and less self - continuity with the future self ) are less likely to discount the significance of future time and are more invested in self - preservation for the future . accordingly , we hypothesize ( 4 ) that individuals ' varying beliefs about future time left ( ftp ) and their beliefs about their ability to maintain self - continuity with the future ( fsc ) are critical markers or early predictors of longevity and of the risks of mortality . while we acknowledge that the preceding associations between mortality and cognitive beliefs may not have been apparent in early and middle - age adulthood , our expectation is that the associations will be especially observable and relevant in late life functioning and will emerge as early markers or predictors of mortality or longevity . participants for the study were randomly recruited from the registry listings of four branch offices of community services and community organizations for seniors ( ministry of health services and health policy 1992 ) , a governmental organization responsible at the time for social services and health policy in southern alberta . a sampling strategy with proportional stratification in function of geographical zone ( metropolitan , urban , or rural ) was used to ensure that the sample was representative of the general population of older adults living in three big cities and various rural areas in southern alberta . participants came from three mid - sized cities ( populations ranging from 170,000 to 300,000 individuals ) and surrounding suburban and rural areas in southern alberta ( canada ) . it should be noted that various levels of community dwellings ranging from upper middle class private houses to low income apartment housing , and assisted living homes were included in the final recruitment . initially 760 brochures briefly describing the research were mailed on a staggered basis to seniors ' households requesting individuals ' participation with a one - time offer of a $ 30 gift certificate to compensate for their time . the purpose of the study was explained as an attempt to understand older individuals ' beliefs , hopes , expectancies , and planned goals for the foreseeable future . by the end of eight weeks the remaining 628 individuals were subsequently recontacted by mail , and of these 137 individuals who wanted additional information about the research were contacted by phone . another 333 individuals agreed to participate in response to further advertising and a to a second and third call for participants , made 3 and 4 months later , bringing the total number of willing participants to 470 . eligibility criteria for the selection of participants included ( 1 ) being 65 years or older ; ( 2 ) being able to understand , speak , and write english ; ( 3 ) not having a diagnosis of cognitive dysfunction registered in the medical files ; ( 4 ) being available for baseline assessments ; ( 5 ) able to specify by name , address , phone number , and other relevant details , one or more family members or care - givers willing to serve as informants to the research team ; ( 6 ) willing to sign a consent form . the initial interview with each of the 470 participants and their family members took approximately one - and - a - half hours , with interviews staggered over a period of 16 weeks . the majority of participants were interviewed in their homes in order to obtain baseline information about health status in relation to the chronic condition presented , to obtain participants ' formal consent to participate and to arrange for their family member or primary care giver to contact us periodically concerning the participant 's general progress . we provided informants with postage - paid envelopes to contact us at regular intervals , as stated in the contact form , concerning the participant 's health status ( improving , stable , declining somewhat , seriously declining ) , and in the event of the participant 's death , to provide the exact date of death . within the first four months of the completion of baseline assessments for the study , a total of 30 participants withdrew their participation for a number of reasons , mostly because of the care - givers ' reluctance to cooperate . data are presented for the remaining 440 participants who , following baseline assessments , stayed the entire 6.5 years ' course of the study . we wrote thank you notes and sent gift coupons to care givers at every wave of the study , as a token of appreciation of their continuing participation . there was no further attrition of participants during the course of the next 6.5 years . ascertainment of mortality ( date of death ) was done solely on the basis of report of the informants . family members preferred that we used this follow - up and contact procedure because it was more personal and private . however , as far as possible , we double checked dates of death against easily accessible local / provincial mortuary listings . the study comprised 11 waves of data collection . following a small pilot study , we conducted in 1995 on study procedures and assessment scales , baseline measures ( wave 1 ) were obtained between september and december 2000 in a staggered way , followed by 10 subsequent waves of contacts with informants and/or participants to obtain summary progress data on participants . contacts were made at approximately eight - month intervals ( 240 days apart ) till early december 2007 , approximately 6.5 years after baseline . participants agreed to complete paper - and - pencil tests at their own pace , at home or in their place of study , and approximately 10 percent sought the help of research assistants to record their responses . at baseline , participants completed the following . measure of beliefs of a just world ( bjw)bjw were assessed with a scale originally developed by lipkus and siegler but further improved by bgue and bastounis , in order to separate items of bjw pertaining to self from items of bjw pertaining to others . participants rated on a scale of 1 ( strongly disagree ) to 6 ( strongly agree ) 8 items of bjw beliefs pertaining to the self . sample items are i feel that the world treats me fairly in life , i feel that i get what i deserve , i feel that when people meet with misfortune , they have brought it upon themselves . scores for bjw ( self ) and scores for the bjw ( others ) ranged from 8 to 48 . we used the option of scoring the bjw - self and bwj - others as continuous scales . cronbach 's alpha were .84 and .74 for the bjw - self and bwj - others , respectively . bjw were assessed with a scale originally developed by lipkus and siegler but further improved by bgue and bastounis , in order to separate items of bjw pertaining to self from items of bjw pertaining to others . participants rated on a scale of 1 ( strongly disagree ) to 6 ( strongly agree ) 8 items of bjw beliefs pertaining to the self . sample items are i feel that the world treats me fairly in life , i feel that i get what i deserve , i feel that when people meet with misfortune , they have brought it upon themselves . scores for bjw ( self ) and scores for the bjw ( others ) ranged from 8 to 48 . we used the option of scoring the bjw - self and bwj - others as continuous scales . cronbach 's alpha were .84 and .74 for the bjw - self and bwj - others , respectively . measure of future time perspective ( ftp)ftp was assessed with the future time perspective scale developed by carstensen . participants rated on a scale from 1 ( very untrue for me ) to 7 ( very true for me ) the degree with which they agreed with each of 10 items . i have the sense that time is running out , as i get older , i begin to experience time as limited . we used the option of scoring the ftp as a continuous scale in the first instance . participants rated on a scale from 1 ( very untrue for me ) to 7 ( very true for me ) the degree with which they agreed with each of 10 items . sample items are many opportunities await me in the future , most of my life still lies ahead of me , i have the sense that time is running out , as i get older , i begin to experience time as limited . we used the option of scoring the ftp as a continuous scale in the first instance . measure of future self - continuity ( fsc)fsc was assessed by means of an adapted psychometric measure of future self - continuity originally devised by frederick that used a single - item measure ( i.e. , how similar / connected are you to your past and future self for 5- , 10- , 15- , and 20-year intervals on a 1100 scale ? ) . to facilitate the comprehension of the concept of continuity , the individual 's endorsement of similarity between present and future selves was presented pictorially by a range of five circles with no overlap to circles with complete overlap . the index of future self - continuity featured two questions on 7-point scale marked at each point by two circles that ranged from showing no overlap at one end of the scale to depicting almost complete overlap at the other end ( see for circles depicting no overlap to complete overlap ) , thus , making the measuring devise more concretely comprehensible to older adults . participants first selected the pair of circles that best described how similar / connected they felt to the future self 15 years from now , and how much they cared about the future self . subsequently , their responses were invited to two questions : how connected do you feel to your future self 15 years from now ? ( not at all = 0 to very much = 7 ) . how much do you care for your future self ? ( not at all = 0 to very much = 7 ) . we used the option of scoring the fsc scale ( similarity / caring ) as a continuous scale in the first instance . the index score of future self - continuity ranged from 7 to 35 , and caring for the future self ranged from 7 to 35 , assessed in terms of one continuous total index score ranging from 14 to 70 . the high scores represented higher levels of future self - continuity / caring for future self . fsc was assessed by means of an adapted psychometric measure of future self - continuity originally devised by frederick that used a single - item measure ( i.e. , how similar / connected are you to your past and future self for 5- , 10- , 15- , and 20-year intervals on a 1100 scale ? ) . to facilitate the comprehension of the concept of continuity , the individual 's endorsement of similarity between present and future selves was presented pictorially by a range of five circles with no overlap to circles with complete overlap . the index of future self - continuity featured two questions on 7-point scale marked at each point by two circles that ranged from showing no overlap at one end of the scale to depicting almost complete overlap at the other end ( see for circles depicting no overlap to complete overlap ) , thus , making the measuring devise more concretely comprehensible to older adults . participants first selected the pair of circles that best described how similar / connected they felt to the future self 15 years from now , and how much they cared about the future self . subsequently , their responses were invited to two questions : how connected do you feel to your future self 15 years from now ? ( not at all = 0 to very much = 7 ) . how much do you care for your future self ? ( not at all = 0 to very much = 7 ) . we used the option of scoring the fsc scale ( similarity / caring ) as a continuous scale in the first instance . the index score of future self - continuity ranged from 7 to 35 , and caring for the future self ranged from 7 to 35 , assessed in terms of one continuous total index score ranging from 14 to 70 . the high scores represented higher levels of future self - continuity / caring for future self . interpersonal and society trust measureindividuals ' degree of interpersonal trust and trust in the surrounding public institutions that are perceived to represent justice and fairness were assessed by means of scale items adapted from the the rotter trust scale . as a first step in adapting the scale , a number of items were written using a 5-point likert format , ( 1 ) strongly agree to ( 5 ) strongly disagree . an attempt was made to sample a wide variety of social objects so that a subject would be called upon to express his / her trust of outside agents , friends , and family members . sample items include in dealing with strangers one is better off to be cautious until they have provided evidence that they are trustworthy , most elected public officials are not really sincere in their campaign promises ; most friends can be trusted to support you for life ; i am able to share my innermost thoughts and feelings with family because of my trust in them ; i do not like to reveal personal information to outside agents even when they claim to be helping me ; i am a private person and find it hard to trust people i do not know well . in the final form of this scale , the 24 items selected were similarly balanced . twelve items indicated trust for agreeing , and 12 items indicated distrust for agreeing ( with the range of scores for both the trust items and distrust items being 12 to 60 ) . a few filler items were included to disguise the true purpose of the scale . cronbach 's alpha for the trust scale and distrust scale were .77 and .72 , respectively . individuals ' degree of interpersonal trust and trust in the surrounding public institutions that are perceived to represent justice and fairness were assessed by means of scale items adapted from the the rotter trust scale . as a first step in adapting the scale , a number of items were written using a 5-point likert format , ( 1 ) strongly agree to ( 5 ) strongly disagree . an attempt was made to sample a wide variety of social objects so that a subject would be called upon to express his / her trust of outside agents , friends , and family members . sample items include in dealing with strangers one is better off to be cautious until they have provided evidence that they are trustworthy , most elected public officials are not really sincere in their campaign promises ; most friends can be trusted to support you for life ; i am able to share my innermost thoughts and feelings with family because of my trust in them ; i do not like to reveal personal information to outside agents even when they claim to be helping me ; i am a private person and find it hard to trust people i do not know well . in the final form of this scale , the 24 items selected were similarly balanced . twelve items indicated trust for agreeing , and 12 items indicated distrust for agreeing ( with the range of scores for both the trust items and distrust items being 12 to 60 ) . a few filler items were included to disguise the true purpose of the scale . cronbach 's alpha for the trust scale and distrust scale were .77 and .72 , respectively . spheres of control the scale has little or no conceptual overlap with the bjw scale and , hence , was selected to provide an independent measure of control . the scale is comprised of 30 items with the three spheres of control ( personal efficacy , interpersonal control , and sociopolitical control ) each represented by 10 items each rated on a 5-point likert scale ranging from disagree to agree . in the present research , we collapsed the scores across these three spheres of control because the results of our hypothesis tests were not affected by distinguishing between them . specimen items for the personal efficacy scale , interpersonal scale , and sociopolitical scale , respectively , include it 's pointless to keep working on something that is too difficult for me ; i find it easy to play an important part in most group or individual situations . in the long run , we as voters are responsible for bad government on a local or national level ; it is difficult for people to have much control over things politicians do in offices . a total index score for the 30 items was obtained with scores ranging from 30 to 150 . the scale has little or no conceptual overlap with the bjw scale and , hence , was selected to provide an independent measure of control . the scale is comprised of 30 items with the three spheres of control ( personal efficacy , interpersonal control , and sociopolitical control ) each represented by 10 items each rated on a 5-point likert scale ranging from disagree to agree . in the present research , we collapsed the scores across these three spheres of control because the results of our hypothesis tests were not affected by distinguishing between them . specimen items for the personal efficacy scale , interpersonal scale , and sociopolitical scale , respectively , include it 's pointless to keep working on something that is too difficult for me ; i find it easy to play an important part in most group or individual situations . in the long run , we as voters are responsible for bad government on a local or national level ; it is difficult for people to have much control over things politicians do in offices . a total index score for the 30 items was obtained with scores ranging from 30 to 150 . measure of self - esteem ( sei : ) this inventory was used to measure self - esteem as a global and stable disposition . this inventory was used to measure self - esteem as a global and stable disposition . physical functionphysical function was assessed by means of a single item taken from the physical function mobility index inquiring about one 's ability ( yes / no ) to climb one flight of stairs without help . this one - item question was intended to seek information on physical fitness.it should be noted that all measures administered to the participants were formatted in terms of language and structure appropriate for adults having a ninth - grade education . all paper - and - pencil tests and self - report measures used in the study were previously piloted on a volunteer group of 20 men and women aged 60 to 80 years . subsequent modifications were made in the instructions and illustrations given for responding to the five - point ratings of test items . this procedure was undertaken to ensure that even those participants who were elderly and had relatively limited education could validly complete the measures . physical function was assessed by means of a single item taken from the physical function mobility index inquiring about one 's ability ( yes / no ) to climb one flight of stairs without help . it should be noted that all measures administered to the participants were formatted in terms of language and structure appropriate for adults having a ninth - grade education . all paper - and - pencil tests and self - report measures used in the study were previously piloted on a volunteer group of 20 men and women aged 60 to 80 years . subsequent modifications were made in the instructions and illustrations given for responding to the five - point ratings of test items . this procedure was undertaken to ensure that even those participants who were elderly and had relatively limited education could validly complete the measures . advanced old age is commonly associated with an increase in disabilities , higher rates of health care use , and higher need for social support . thus , we felt the need to examine further fluctuations in risk of mortality after controlling for health - related covariates , which were assessed as follows : survey of number of visits to health providersas a part of a separate survey , respondents listed the number of visits that they had paid in the previous year to health providers , including visits to family physicians , community health clinics , and emergency health units , including hospital visits . as a part of a separate survey , respondents listed the number of visits that they had paid in the previous year to health providers , including visits to family physicians , community health clinics , and emergency health units , including hospital visits . iadl index of disabilitywe based this index on the self - reported ability for 12 daily living activities included in the iadl . respondents were asked to indicate whether they had experienced limitations in 12 areas of functioning with responses coded ( 1 ) for yes and ( 0 ) for no , for example , able to use a telephone ; boarding a bus without assistance ; lifting or carrying groceries ; personal grooming and personal hygiene care ; doing light house work ; taking medications ; walking one block . all the items were summed to form one index of iadl , with scores ranging from 0 to 12 . we based this index on the self - reported ability for 12 daily living activities included in the iadl . respondents were asked to indicate whether they had experienced limitations in 12 areas of functioning with responses coded ( 1 ) for yes and ( 0 ) for no , for example , able to use a telephone ; boarding a bus without assistance ; lifting or carrying groceries ; personal grooming and personal hygiene care ; doing light house work ; taking medications ; walking one block . all the items were summed to form one index of iadl , with scores ranging from 0 to 12 . measure of satisfaction with family and social supportsocial satisfaction was assessed with 10 items adapted from zimet et al . . participants rated how satisfied they were with their social partners in general , and how satisfied they were with their family and relatives on a rating scale ranging from 1 ( very dissatisfied ) to 3 ( very satisfied ) . participants rated how satisfied they were with their social partners in general , and how satisfied they were with their family and relatives on a rating scale ranging from 1 ( very dissatisfied ) to 3 ( very satisfied ) . we assessed the internal consistency of each trait scale with cronbach 's coefficient alpha and the association of the scales with each other and with other covariates by means of pearson correlation coefficients . for all analyses , differences between survivors and decedents on the date of final censoring were assessed with t - tests and chi - square tests of association . cox proportional hazard models were fitted to estimate the importance of each predictor of mortality . it is important to note that cox regression , or the proportional hazards model , is a well - recognized statistical technique for survival analysis which is concerned with studying the time between entry to a study and a subsequent event such as death , as is the case in the present research . cox regression has the advantage that it allows for the simultaneous exploration of the relationship between survival of persons and several explanatory variables ( in our case variables such as cognitive beliefs , future time perspectives , control and trust , etc . ) . of particular relevance and interest to our research is that the technique allows for age adjustment of the calculated hazard ratios across a wide range of ages , as was the case in our study . , by stephen j. walters , a haywood group plc publication , may 2003 , accessible online at http://www.whatisseries.co.uk/whatis/pdfs/what_is_cox_model.pdf ) ; see also g.d . garson , statnotes , north carolina state university , online at http://faculty.chass.ncsu.edu/garson/pa765/cox.htm ) for details of statistical procedures for achieving adjustment for the effect of age as a covariate . the cox regression model assumes that the death rate of the population depends on a continuous time variable , which in the present study was the interval between the wave 1 assessment and date of death , calculated in terms of total number of days . in the present study , predictor variables included beliefs in a just society ( bjw : self and others ) , future time perspective ( ftp ) , future self - continuity ( similarity / caring ) perspective ( fsc ) , demographic variables , and health and physical functioning variables . the initial risk ratios ( irrs ) ( frequently referred to as hazard ratios ) are calculated by exponentiating the beta weight for the predictor , and regressions are expressed in risk ratios and 95 percent confidence intervals . all cox regression hazard ratios ( irrs ) presented in the present analyses were first adjusted for age . in subsequent analyses , we controlled for three health - related covariates ( see [ 3 , 5 , 32 , 33 ] for significance of studying health variables ) . results are reported in two sections corresponding to our two main research questions . in the first section , we examine key differences between survivors and decedents in baseline characteristics of participants . in specific , we conducted cox regression analysis of risk rations of mortality to estimate the relative risk of death and the importance of each predictor of mortality for the whole sample . all variables entered in the cox regression analysis were entered as continuous variables for these analyses . in the second section , we present additional analysis of data where we were unable to derive easily interpretable results from using cox regressions of continuous variable data . subsequently , we analyzed the data using categorical variables that we considered were appropriate for the otherwise continuous variables . four hundred and forty participants were followed starting in december 2000 when baseline assessments were completed . as of december 2007 , after an average of 6.5 years of observation one hundred and forty ( 32% ) deaths had occurred , and 300 ( 68% ) survived . table 1 provides crude data at baseline on the two subgroups of decedents and survivors . those who died during the study period were somewhat older . among health - related variables , the respondents in the two groups were quite different with respect to physical function scores , but not with respect to number of medical visits reported at time of baseline assessment , or in respect of number of disabilities reported on the iadl index ( see table 1 ) . survivors compared to decedents had significantly higher scores on measures of beliefs in just world ( bjw - self ) , on the future time perspective ( ftp ) and future self - continuity / caring for the future self ( fsc ) perspective scale . by contrast , decedents compared to survivors had significantly higher scores on the measure of distrust on the interpersonal and society scale and on the control scale . first , we examined the intercorrelations of measures of beliefs in a just world ( bjw ; self and others ) , future time perspective ( ftp ) , future self - continuity perspective ( continuity / caring for future self ) , trust ( agreement with trust items and agreement with distrust items ) and control with one another , and with demographic variables ( age and education ) , and baseline indicators of number of medical visits , iadl disability , and satisfaction with social support . as expected , the two scales of bjw were positively related to each other , with correlations ranging from .41 to .64 ; p < .001 . overall , the bjw - self scale , the ftp scale , and fsc scale were positively correlated with one another , with correlations ranging from .34 to .68 ; p < .001 . in general , the bjw , ftp , fsc , and trust measures were not correlated significantly with demographic variables of age and education . the one notable exception , however , was that the distrust measure was significantly correlated with age and education , with correlations ranging from .34 to .39 ; p < .001 ( note : the detailed table of correlations is available from the authors on request ) . table 2 provides psychometric information on the predictor variables for mortality risks selected for the research . examination of the mean scores for the various scales for which baseline data were obtained shows that the mean score of the control scale ( 80.1 ) was quite elevated when considered within the context of validity norms outlined in paulhus . however , the mean scores for the bjw , ftp , fsc . trust and distrust the internal consistencies of the various trait measures as reported in table 2 are consistent with and converge appropriately with internal consistencies reported for the various scales elsewhere ( see [ 10 , 12 , 19 , 25 ] ) . when cox proportional hazard models adjusted for age were fitted to estimate the relative risk of death and the importance of each predictor of mortality for the whole sample of 440 participants , the findings showed that bjw ( self ) , ftp , and fsc were positively and significantly related to reduced risk of mortality . in other words , scores on these measures were inversely related to risk of mortality , and associated with a significantly reduced risk of mortality . as seen in table 3 , bjw ( others ) , trust , self - esteem , and social support satisfaction were associated with a rather weak or marginal reduction in risk of mortality . by contrast we conducted a hierarchical regression analysis ( n = 440 ) in order to examine further the relative contribution of the variables to risk of mortality ( table 4 ) . this analysis revealed the ability of bjw - self to predict risks of mortality over and above other predictor variables . as seen in table 4 ( model 1 model 2 ) , results clearly show that adding the cognitive beliefs of bjw ( self ) variable separately contributed to the explanation of variance in mortality risks significantly and uniquely , over and above that offered by the three variables of ftp , fsc , and distrust . in other words , the results in table 4 confirm that bjw ( self ) predicts an association with significant reduction in mortality risks independent of the other predictor variables . this rules out the sceptical view that the ability of bjw - self to predict outcomes is an artifact of correlations with other factors previously associated with outcomes of bjw , for example , control or trust . additionally , we conducted a preliminary examination of how deaths were distributed across low , average , and high trait standard scores . the proportionality assumption is violated when the relative risk of the outcome does not change in the same manner for equivalent changes in the levels of a risk factor or covariate . categorical variables may then be appropriate for an otherwise continuous variable and predictors may be trichotomized based on broad cut - off points . although trichotomization may reduce statistical power , it has the possible advantage that trichotomized domain scores will be more readily interpretable . accordingly , following scores on each of the predictor variables ( converted to standard scores ) , tertiles were computed for each of the predictor variable subscale measures . we plotted survival curves showing specifically and with greater focus on the relationship of predictor variables to mortality , with survival rates plotted for extreme high scorers ( at 67th percentile and above ) and extreme low scorers ( at 33rd percentile and below ) . four survival curves of particular interest and relevance to our research hypotheses are portrayed in figures 1to 3 . figure 1 showing the survival curve for beliefs in a just world ( bjw - self ) suggests that individuals with extreme high scores ( at 67th percentile and above ) on this dimension are at significantly reduced risk for mortality compared to individuals with extreme low scores ( at 33rd percentile and below ) . the percentages for those who survived in the two groups are 79.6% and 70.7% percent , respectively , indicating a 9% difference in mortality . the ratio of fractions of decedents to survivors ( 1 0.707)/(1 0.796 ) = 1.433 ( see figure 1 ) suggests a 43.3% lower risk of death for extreme high scorers compared to extreme low scorers . the findings from the survival curve presented in figure 1 further confirm the predictive ability of this measure seen earlier in table 3 where bjw - self was used as continuous variable in the cox regression analyses . figure 2(a ) showing the survival curve for future time perspectives ( ftps ) suggests that individuals with extreme high scores on this dimension ( at 67th percentile and above ) are at a significantly reduced risk for mortality compared to individuals with extreme low scores ( at 33rd percentile and below ) . the percentages for those who survived in the two groups are 81.6% and 74.8% percent , respectively , indicating a 7% difference in mortality . the ratio of fractions of decedents to survivors ( 1 0.748)/(1 0.816 ) = 1.370 ; ( see figure 2(a ) ) suggests a 37.0% reduced risk of death for extreme high scorers compared to extreme low scorers . the findings from the survival curve presented in figure 2(a ) further confirm the predictive ability of this measure seen earlier in table 3 where future time perspective was used as continuous variable in the cox regression analyses . figure 2(b ) showing the survival curve for future self - continuity perspectives ( fscs ) suggests that individuals with extreme high scores ( at 67th percentile and above ) on this dimension are at a significantly reduced risk for mortality compared to individuals with extreme low scores ( at 33rd percentile and below ) . the percentages for those who survived in the two groups are 79.6 and 74.1 percent , respectively , indicating a 5.5% difference in mortality . the ratio of fractions of decedents to survivors ( 1 0.741)/(1 - 0.796 ) = 1.267 ( see figure 2(b ) ) suggests a 26.7% reduced risk of death for extreme high scorers compared to extreme low scorers . the findings from the survival curve presented in figure 2(b ) further confirm the predictive ability of this measure seen earlier in table 3 where the future self - continuity perspective measure was used as continuous variable in the cox regression analyses . conversely , the survival curve for distrust ( figure 3 ) suggests that individuals with extreme high scores ( at 67th percentile and above ) on this dimension , compared with those with extreme low scores ( at 33rd percentile and below ) , are at a significantly increased risk for mortality . the percentages for those who survived in the two groups are 80.3% and 74.8% , respectively , indicating a 5.5% difference in mortality . the ratio of fractions of decedents to survivors ( see figure 3 ) ( 1 0.748)/(1 0.803 ) = 1.276 suggests a 27.6% higher risk of death for extreme high scorers on distrust , compared to extreme low scorers . the findings from the survival curve presented in figure 3 further confirm the predictive ability of this measure seen earlier in table 3 where the distrust measure was used as continuous variable in the cox regression analyses . to sum up the results , when the tertile split analysis was done , bjw ( self ) , future time perspectives ( ftps ) and future self - continuity perspectives ( fscs ) were found to be differentially associated with mortality risks . when bjw ( self ) scores , future time perspective scores , and self - continuity or similarity with the future self scores were extremely low ( 33rd percentile and below ) , mortality risk was significantly greater , compared to extremely high scores on these measures ( 67th percentile and above ) . however , the variable of distrust revealed a somewhat contrasting pattern in the tertile analysis . when distrust scores were extremely low ( 33rd percentile and below ) , mortality risk was significantly lower , compared to extremely high distrust scores ( 67th percentile and above ) which were associated with an increase in mortality risk . in a separate set of regression analyses , we explored the influence of three health - related covariates ( table 5 ) . these analyses were intended mainly to see whether differences in cognitive predictors of mortality would be maintained after controlling for three health - related covariates assessed at baseline : ( 1 ) number of visits in the previous year to medical practitioners and health providers , ( 2 ) total index score for the measure of disability ( iadl ) in daily life activities , and ( 3 ) total score for self - rated satisfaction with family and social support . initially , these health - related covariates were controlled for one at a time , using a cox regression analysis procedure . we then re - ran the analysis using multiple regression , adjusting for the covariates ( table 5 ) . as seen in table 5 , the negative beta values for beliefs in a just world , future time perspective , future self - continuity , and social support satisfaction , and the significant f values observed in the multiple regression analysis are consistent with the direction of reduced risk ratios shown in the relative risk ( rr ) and 95% confidence interval ( ci ) model ( table 3 ) . similarly , after adjusting for the covariates , the positive direction of the beta values and the significant f values for distrust in the multiple regression analyses is consistent with the direction of increased risk ratios for this variable in the relative risk ( rr ) and 95% ci model . thus , overall , the relationship between cognitive beliefs about a just world , future time perspectives , and risks for mortality were maintained after adjusting for the health - related covariates . in other words , the direction of the association we saw in the earlier cox regression analyses ( table 3 ) between scores on cognitive beliefs , future perspectives , distrust , and risks for mortality remained unchanged after adjusting for the health - related covariates . in further analyses , we found no statistically significant interaction effects among the sociodemographic variables and health variables relating to the prediction of mortality . consistent with our hypothesis , our findings from the cox regression analysis of the predictor variable of bjw revealed that bjw is positively related to survival . additionally , our findings based on a hierarchical regression analysis of a two - model design show that the variable of bjw ( self ) had stronger ability than all other cognitive variables explored in this research to predict mortality risk . these findings lend weight to recent theory and research suggesting that the predictive power of bjw - self seems to derive uniquely from perceived justice and that most persons have a strong need to perceive the world to be just , a belief system that conceivably serves to protect their health and survival [ 10 , 14 , 23 ] . our findings that high levels of beliefs about justice and fairness are early predictors of longer survival are consistent with our hypothesis . these findings advance and extend the assumptions of earlier researchers ' work on perceived justice and fairness [ 9 , 10 ] as linked to better health and well being . our findings show that individuals ' concern that the world is just to themselves , as distinguished from others , is particularly predictive of their healthy survival . along with earlier researchers , we speculate that bjw for self contributes to a larger extent to individuals ' psychosocial adjustment , providing them with useful resources in times of stress , and correspondingly becomes linked with reduced risks of mortality . in our study , bjw ( others ) was not differentially linked with mortality risks . however , we can not conclude that bjw - self alone generates the link with reduced risk of mortality . consequently , the mortality correlates with bjw in the spheres of self and others remain to be determined more fully . overall we predicted that while time is finite and limited for all , an increased or more expansive or open - ended future time perspective would reduce the risk of mortality , based on a logical assumption that a more expansive view on time left may promote valuation of future plans to safeguard the future self and protect it against risks of mortality . consistent with our hypothesis , our findings from the cox regression analysis ( adjusted for age ) show that an expansive future time perspective is associated with a reduced risk of mortality for our older sample . our findings lend support to earlier postulates that the subjective sense of time plays an essential role in human motivation , and gradually time left becomes a better predictor than a range of other cognitive variables that contribute to behavioral and psychological processes at work in old age . the present direction of findings advances and extends assumptions of future time perspectives proposed by previous researchers ( e.g. , ) according to whom individuals ' perceptions of remaining time to live determines their potential for evaluating and shaping future time and future prospects concerning identity and health . extrapolating from our findings , we speculate that incorporating an expansive time horizon influences individuals to pay relatively greater attention in the time left to make pragmatic and more practical decisions concerning a healthier future life . additionally , we speculate that individuals in our sample who showed more expansive or open - ended time horizons at the time of baseline assessment would have already maximized attempts to preserve and protect future personal health and survival . however , our findings suggest that older adults ' perception of time left is linked rather nonspecifically with risks of mortality . it remains for future researchers to disentangle the specific psychological processes that link limited horizons of time left with increased risk of mortality . overall , we predicted that increased future self - continuity and caring for future self would reduce the risk of mortality , based on a logical assumption that increased future self - continuity perceptions may promote the valuation of future plans to safeguard the future self and to protect it against risks of mortality . consistent with our hypothesis , our findings from the cox regression analysis ( adjusted for age ) show that future self - continuity as a variable is associated with reduced risk of mortality . extreme high scorers on future self - continuity and caring for the future ( i.e. , individuals who perceived greater similarity between their present and future self ) , compared with extreme low scorers ( i.e. , individuals who perceived minimum similarity between their present and future self ) had a significantly reduced risk of mortality ( 37% ) . although the present results can not specify the causal direction of the association between future self - continuity and reduced risk of mortality nor specify the health - related processes at work to explain this association , the research findings provide initial evidence that high similarity and high self - continuity with future self is a significant predictor of longevity . overall , our findings fit well with the philosophical analysis by parfit that individuals who feel similar to their future selves are more likely to make more prudent decisions for the future , including health - related decisions , because of their perceived close connectedness to the future self . alternatively , we speculate that individuals who perceived their current physical and mental health to be stable at the time of baseline assessments were also able to identify better with their future self as being one of healthy survival and , therefore , worth protecting . of particular interest , in the context of a longitudinal study , is the initial evidence that behavioral differences in longevity are driven differentially by perceived future self - continuity . our findings from the cox regression analysis , showing that high distrust predicted a significantly increased risk of mortality , are consistent with our hypothesis . these findings are not at variance with earlier theoretical proposals suggesting that historically older adults have had difficulty in accepting the status quo as defined by younger authorities in the social system and may tend to be more distrusting of those authorities . our findings suggest that extreme high levels of distrust which typified some individuals in our sample , compared with extreme low levels of trust , increased the risk of mortality by 27.6 percent . overall , the cox regression analysis revealed that distrust is empirically predictive of an increased risk of mortality . we speculate that high levels of distrust may be analogous to a form of pathological social isolation or breakdown in communications that leads to increased risk for physical and psychological disease [ 37 , 38 ] . conceivably distrust becomes linked with a shorter life span . despite the novelty of our data , and thus the importance of their contribution to cognitive etiology of health - related processes such as survival , mortality , or longevity , our study was subject to the limitation that the sample was a volunteer one . our cognitive scale measures of beliefs in a just world , and other measures exploring individuals ' perspectives on future self - continuity and future time , trust and distrust were self - report measures and subject to the biases associated with self - report measures . the preceding sources of unreliability of measurement could potentially both reduce or increase the proportion of variance in the outcome variables for which our models were able to account . however , we are confident that we had a sufficiently large and representative sample of urban and rural older adults , and the measures we used showed high internal consistency . our study provided novel indications that cognitive belief systems and future time perspectives of older adults may have much to contribute to increased or reduced mortality risks in late life . thus , we suggest that basic cognitive beliefs and future time perspectives that were examined in the present research for their association with increased or reduced risk for mortality are an important direction for future research . it remains for future researchers to try to integrate these various cognitive factors into some kind of theoretical frameworks to gain a precise appreciation of how these factors may be causally linked with longevity . extrapolating from the findings of the study concerning the association between beliefs in a just world and the potentials for a longer survival , it may be reasonable to postulate that the findings have possible implications for clinical practice with older adults who may be dealing with traumatic life events such as life threatening illness , loss of loved ones , and other natural disasters . it is conceivable that these individuals may , as a result of adverse and stressful experiences , feel an abrupt decline in their beliefs in a just world or a decline in their faith or trust in interpersonal connectedness . it is suggested that clinicians be particularly mindful of clients who score extremely low on measures of bjw and future time perspectives . . similarly , those individuals scoring extremely high on measures of distrust and control may be at greater risk of mortality . by exploring with older clients their beliefs about a just world and their trust or faith in interpersonal connectedness , geriatric service providers and psychologists may be able to assist clients to develop a realistic self - profile of their sources of life strengths in relation to self - growth , resilience , and control over every day life situations . thus , clinicians may be able to assist elderly clients to draw on internal sources of strengths , such as reflecting on their beliefs about a just world and their beliefs about trusting others , as a means to strengthening and enhancing future self - continuity . based on our findings suggesting that individuals ' strong beliefs in a just world and positive perspectives regarding future time may possibly protect them against risk to mortality , geriatric service providers are encouraged to engage in a dialogue with their clients on these themes . such dialogues may not only serve to act as buffers for clients against stress and anxiety about the future , but may also assist clients in building up greater motivation for living . from a pragmatic standpoint , we postulate that interventions aimed at strengthening individuals ' beliefs in a just world at an earlier age , and encouraging future - self continuity in the earlier adulthood years , might motivate people to care more for their potential person in the later years , and conceivably keep people healthier longer and thereby reduce risk of mortality . similarly , communication programs that foster interpersonal and institutional trust or help older adults combat high levels of distrust ( which in our research were associated with significantly increased risk of mortality ) may be of value to a longer survival .

on the basis of postulates derived from cognitive - behavioral theory , research and therapy , the authors explored the extent to which older adults ' cognitive beliefs of a just world and their perspectives on future time and similarity or self - continuity with the future self are predictors of long - term survival . after baseline assessment of health and cognitive beliefs and future perspectives of time and self - continuity as predictors of mortality , 440 participants ( ages 65 to 87 ) were followed longitudinally for 6.5 years . consistent with our hypotheses , findings demonstrated that a significantly higher percentage of survivors were individuals who showed higher scores on beliefs in a just world and on both the future time perspective and the future self - continuity perspective at the time of baseline assessments . conversely , mortality risk was much higher for individuals who scored low on these predictor variables , and high on distrust . implications for health and longevity are discussed .

1. Introduction 2. Conceptual and Theoretical Framework for the Research 3. Sampling Frame for the Study and Recruitment of Participants 4. Results 5. Discussion and Interpretation of Results

PMC5054075

calculating nonsynonymous ( ka ) and synonymous ( ks ) substitution rates is of great significance in reconstructing phylogeny and understanding evolutionary dynamics of protein - coding sequences across closely related and yet diverged species 1 . , 2 . , 3 .. it is known that ka and ks , or often their ratio ( ka / ks ) , indicate neutral mutation when ka equals to ks , negative ( purifying ) selection when ka is less than ks , and positive ( diversifying ) selection when ka exceeds ks . therefore , statistics of the two variables in genes from different evolutionary lineages provides a powerful tool for quantifying molecular evolution . over the past two decades , several methods have been developed for this purpose , which can generally be classified into two classes : approximate method and maximum likelihood method . the approximate method involves three basic steps : ( 1 ) counting the numbers of synonymous and nonsynonymous sites , ( 2 ) calculating the numbers of synonymous and nonsynonymous substitutions , and ( 3 ) correcting for multiple substitutions . on the other hand , the maximum likelihood method integrates evolutionary features ( reflected in nucleotide models ) into codon - based models and uses the probability theory to finish all the three steps in one go ( 4 ) . however , these methods adopt different substitution or mutation models based on different assumptions that take account of various sequence features , giving rise to varied estimates of evolutionary distance ( 5 ) . in other words , ka and in addition , since the amount and the degree of sequence substitutions vary among datasets from diverse taxa , a single model or method may not be adequate for accurate ka and ks calculations . therefore , a model selection step , that is , to choose a best - fit model when estimating ka and ks , becomes critical for capturing appropriate evolutionary information 6 . , 7 .. toward this end , we have applied model selection and model averaging techniques for ka and ks estimations . we use a maximum likelihood method based on a set of candidate substitution models and adopt the akaike information criterion ( aic ) to measure fitness between models and data . after choosing the best - fit model for calculating ka and ks , we average the parameters across the candidate models to include as many features as needed since the true model is seldom one of the candidate models in practice ( 8) . finally , these considerations are incorporated into a software package , namely kaks_calculator . substitution models play a significant role in phylogenetic and evolutionary analyses of protein - coding sequences by integrating diverse processes of sequence evolution through various assumptions and providing approximations to datasets . , 14 . , 15 . , 16 . as shown in table 1 17 . , 18 . , ranging from the jukes - cantor ( jc ) model , which assumes that all substitutions have equal rates and equal nucleotide frequencies , to the general time - reversible ( gtr ) model that considers six different substitution rates and unequal nucleotide frequencies . subsequently , we incorporated the parameters in each nucleotide model into a codon - based model 19 . , 20 .. as a result , a general formula of the substitution rate qij from any sense codon i to j ( i j ) is given for all candidate models ( 19):qij={0ifiandjdifferbymorethanonedifferencekxyjifiandjdifferbyasynonymoussubstitutionofxforykxyjifiandjdifferbyanonsynonymoussubstitutionofxforywhere j is the frequency of codon j , is the ka / ks ratio , and kxy is the ratio of rxy to rca , x , y {a , c , g , t } ( table 1 ) . for example , in the jc model , kxy and j are equal to 1 owing to equal substitution rates and equal nucleotide frequencies assumed . in the hasegawa - kishino - yano ( hky ) model , ktc and kag become equivalent to the transition / transversion rate ratio and j can be estimated from sequences , similar to the method reported by goldman and yang ( 19 ) . therefore , we could acquire maximum likelihood scores in various values generated from individual candidate model by implementing the codon - based models in a maximum likelihood framework 19 . , 20 .. aic ( 21 ) has been widely used in model selection aside from other methods such as the likelihood ratio test ( lrt ) and the bayesian information criterion ( bic ) ( 8) . aic characterizes the kullback - leibler distance between a true model and an examined model , and this distance can be regarded as quantifying the information lost by approximating the true model . kaks_calculator uses a modification of aic ( aicc ) , which takes account of sampling size ( n ) , maximum likelihood score ( lnli ) , and the number of parameters ( ki ) in model i as follows : aicci = aici+2ki(ki+1)nki1=2lnli+2ki+2ki(ki+1)nki1 aicc is proposed to correct for small sampling size , and it approaches to aic when sampling size comes to infinity . consequently , we could use this equation to compute aicc for each candidate model and then identify a model that possesses the smallest aicc , which is a sign for appropriateness between models and data . model selection is merely an approximate fit to a dataset , whereas a true evolutionary model is seldom one of the candidate models ( 8) . therefore , an alternative way is model averaging , which assigns each candidate model a weight value and engages more than one model to estimate average parameters across models . accordingly , we first need to compute the akaike weight ( wi , where i = 1 , 2 , , m ) for each model in a set of candidate models : wi = exp[12(aicciminaicc)]j=1mexp[12(aiccjminaicc)]where min aicc is the smallest aicc value among candidate models . we can then estimate model - averaged parameters . taking ktc as an example , a model - averaged estimate can be calculated by : ktc=i=1m[wii(ktc , i)ktc , i]i=1m[wii(ktc , i)]where ktc , i is ktc in model i andi(ktc , i)={1ifrtcrcainmodeli0otherwise substitution models play a significant role in phylogenetic and evolutionary analyses of protein - coding sequences by integrating diverse processes of sequence evolution through various assumptions and providing approximations to datasets . , 14 . , 15 . , 16 . as shown in table 1 17 . , 18 . , ranging from the jukes - cantor ( jc ) model , which assumes that all substitutions have equal rates and equal nucleotide frequencies , to the general time - reversible ( gtr ) model that considers six different substitution rates and unequal nucleotide frequencies . subsequently , we incorporated the parameters in each nucleotide model into a codon - based model 19 . , 20 .. as a result , a general formula of the substitution rate qij from any sense codon i to j ( i j ) is given for all candidate models ( 19):qij={0ifiandjdifferbymorethanonedifferencekxyjifiandjdifferbyasynonymoussubstitutionofxforykxyjifiandjdifferbyanonsynonymoussubstitutionofxforywhere j is the frequency of codon j , is the ka / ks ratio , and kxy is the ratio of rxy to rca , x , y {a , c , g , t } ( table 1 ) . for example , in the jc model , kxy and j are equal to 1 owing to equal substitution rates and equal nucleotide frequencies assumed . in the hasegawa - kishino - yano ( hky ) model , ktc and kag become equivalent to the transition / transversion rate ratio and j can be estimated from sequences , similar to the method reported by goldman and yang ( 19 ) . therefore , we could acquire maximum likelihood scores in various values generated from individual candidate model by implementing the codon - based models in a maximum likelihood framework 19 . aic ( 21 ) has been widely used in model selection aside from other methods such as the likelihood ratio test ( lrt ) and the bayesian information criterion ( bic ) ( 8) . aic characterizes the kullback - leibler distance between a true model and an examined model , and this distance can be regarded as quantifying the information lost by approximating the true model . kaks_calculator uses a modification of aic ( aicc ) , which takes account of sampling size ( n ) , maximum likelihood score ( lnli ) , and the number of parameters ( ki ) in model i as follows : aicci = aici+2ki(ki+1)nki1=2lnli+2ki+2ki(ki+1)nki1 aicc is proposed to correct for small sampling size , and it approaches to aic when sampling size comes to infinity . consequently , we could use this equation to compute aicc for each candidate model and then identify a model that possesses the smallest aicc , which is a sign for appropriateness between models and data . model selection is merely an approximate fit to a dataset , whereas a true evolutionary model is seldom one of the candidate models ( 8) . therefore , an alternative way is model averaging , which assigns each candidate model a weight value and engages more than one model to estimate average parameters across models . accordingly , we first need to compute the akaike weight ( wi , where i = 1 , 2 , , m ) for each model in a set of candidate models : wi = exp[12(aicciminaicc)]j=1mexp[12(aiccjminaicc)]where min aicc is the smallest aicc value among candidate models . we can then estimate model - averaged parameters . taking ktc as an example , a model - averaged estimate can be calculated by : ktc=i=1m[wii(ktc , i)ktc , i]i=1m[wii(ktc , i)]where ktc , i is ktc in model i andi(ktc , i)={1ifrtcrcainmodeli0otherwise kaks_calculator is written in standard c++ language . it is readily compiled and run on unix / linux or workstation ( tested on aix / irix / solaris ) . in addition , we use visual c++ 6.0 for graphic user interface and provide its windows version that can run on any ibm compatible computer under windows operating system ( tested on windows 2000/xp ) . compiled executables on aix / irix / solaris and setup application on windows , as well as source codes , example data , instructions for installation and documentation for kaks_calculator is available at http://evolution.genomics.org.cn/software.htm . , kaks_calculator employs model - selected and model - averaged methods based on a set of candidate models to estimate ka and ks . it integrates as many features as needed from sequence data and in most cases gives rise to more reliable evolutionary information ( see the comparative results on simulated sequences at http://evolution.genomics.org.cn/doc/simulatedresults.xls ) ( 24 ) . kaks_calculator also provides comprehensive information estimated from compared sequences , including the numbers of synonymous and nonsynonymous sites and substitutions , gc contents , maximum likelihood scores , and aicc . , 25 . , 26 . , 27 . , 28 . , 29 . , 30 . , 31 . and allows users to choose one or more methods at one running time ( table 2 ) . although there exist 203 time - reversible models of nucleotide substitution ( 8) , model selection in practice is often limited to a subset of them ( 32 ) , and thus model averaging can reduce biases arising from model selection . therefore , model - averaged methods should be preferred for general calculations of ka and ks . some planned improvements include application of model selection and model averaging to detect positive selection at single amino acid sites , which requires high - speed computing for maximum likelihood estimation , especially when an adopted model becomes complex . in conclusion , kaks_calculator incorporates as many features as needed for accurately extracting evolutionary information through model selection and model averaging , therefore it may be useful for in - depth studies on phylogeny and molecular evolution . zz designed and programmed this software , and drafted the manuscript . jl carried out computer simulations to generate sequences .

kaks_calculator is a software package that calculates nonsynonymous ( ka ) and synonymous ( ks ) substitution rates through model selection and model averaging . since existing methods for this estimation adopt their specific mutation ( substitution ) models that consider different evolutionary features , leading to diverse estimates , kaks_calculator implements a set of candidate models in a maximum likelihood framework and adopts the akaike information criterion to measure fitness between models and data , aiming to include as many features as needed for accurately capturing evolutionary information in protein - coding sequences . in addition , several existing methods for calculating ka and ks are also incorporated into this software . kaks_calculator , including source codes , compiled executables , and documentation , is freely available for academic use at http://evolution.genomics.org.cn/software.htm .

Introduction Algorithm Candidate models Model selection Model averaging Application Authors contributions Competing interests

PMC4227978

polycystic ovarian morphology is displayed on ultrasound imaging in approximately 11.92.4 of women ( 1 ) . the most common features of pcos are hyperandrogenism , unovulation , infertility and weight gain . these women are at the risk of diabetes , atherosclerosis , and cardiovascular diseases ( 1 , 3 ) . since obesity is the most common cause of insulin resistance and hyper - androgenism , weight reduction might be the first recommendation for pcos women ( 4 , 5 ) . many researchers introduced hypocaloric diet as an operant treatment for weight loss and improvement of fertility in the overweight / obese pcos women . however , only 5 - 10% weight loss has a useful effect on insulin resistance and cardiovascular hemodynamic function ( 46 ) . weight loss up to 5 - 7% during 6 month can restore ovulation and fertility in more than 75% of women ( 5 ) . weight loss induced by hypocaloric diet was intensified by treatment with metformin ( 7 , 8) . in addition , several studies have shown weight loss reduces the severity of insulin resistance ( 9 , 10 ) . treatment with insulin sensitizer agents may also alter endocrine imbalance , improves ovulation and fertility ( 11 ) . even some researchers recommended 3 months of pretreatment with metformin for obese women ( 12 , 13 ) . pasquali et al . showed hirsutism and irregular menses in pcos patients who received metformin were significantly improved in comparison to placebo group and also hypocaloric diet reduced bmi and waist circumference ( wc ) in pcos patients as well as the controls . in both groups , treatment with metformin reduced weight and bmi significantly ( 14 ) . treatment with metformin reduces lh and androgens level and restores regular menses and ovulation ( 1 , 2 ) . also , treatment with the antiandrogen drugs such as flutamide reduced hirsutism and abdominal fat ( 15 ) . in sahin however , flutamide only reduces androgen levels without improvement of insulin resistance in non - obese patients ( 16 ) . studied 40 obese pcos patients during 6 months and showed that hypocaloric diet with metformin or flutamide alone or in combination had a significant effect on body lipid distribution , androgen levels , hirsutism and menses ( 17 ) . gambineri in another study evaluated 80 women within 12 months and showed the benefits of long term treatment with metformin and flutamide and their compounds in obese pcos patients under hypocaloric diet . in his study , flutamide reduced visceral fat , androgen levels and lipid profile after 12 months and improved glucose tolerance value in comparison to placebo and metformin though there was no synergistic effect in patients who simultaneously received the combination of the two drugs ( 18 ) . however , the other study showed that hypocaloric diet with metformin or flutamide alone or in combination had a better therapeutic effect ( 14 ) . more recently , literature revealed contradictory findings about adding medicine to the diet and especially there has been little agreement on the beneficial effects of polytherapy ; hence , in this study , an attempt was made to address the following question : do metformin and flutamide alone or combination of both improve anthropometric indices and laboratory tests in overweight and obese pcos patients under hypocaloric diet ? first , 156 overweight and obese infertile pcos women entered this single blind clinical trial ( clinical trial registration number : irct13880 7271760n3 , may 21 , 2010 ) . these patients were referred to our pcos clinic ( fatemezahra infertility and reproductive health research center ) in babol ( north of iran ) in 2009 . out of them , 117 women were recruited and randomized to the treatment after considering exclusion and inclusion criteria . this study was approved by the ethical committee of babol university of medical science . at the beginning , all participants signed an informed consent and entered the study . inclusion criteria included all pcos women of 18 - 40 years of age , body mass index ( bmi ) > 19 and < 35 the diagnosis of pcos was determined by presence of two or more following conditions of rotterdam criteria ; oligo - ovulation or anovulation ( manifested as oligomenorrhea or amenorrhea ) , hyperandrogenemia ( elevated levels of circulating androgens ) , hyperandrogenism ( clinical manifestations of androgens excess ) and polycystic ovaries detected by ultrasonography ( 20 ) . exclusion criteria were any drug consumers ( hormonal and metformin ) or cases who tried weight loss in 3 recent months , cases with hyperprolactinemia , cushing syndrome , late onset congenital adrenal hyperplasia , thyroid dysfunction , diabetes , cardiovascular , renal and liver diseases , pregnancy and also athletes . they were advised to use barrier methods of contraception throughout the study . before the initiation of the study , the patients underwent the physical examination focused on anthropometric indices ( weight , bmi , waist and hip circumferences and hirsutism ) . the clinical examination focused on the evaluation of anthropometric and parametric values ( height , weight and waist circumference ) and also hirsutism using the ferriman - gallwey score ( 21 ) . waist circumference was obtained as the minimum value between the iliac crest and the lateral costal margin , whereas hip circumference was determined as the maximum value over the glottal region to cm . waist and hip circumferences were measured to the nearest centimeter with a soft tape ( cm ) according to world health organization ( who ) criteria . before the study hormonal and metabolic determinations included the evaluation of circulating concentrations of free and total testosterone , dehydroepiandroster - one sulfate ( dhea - s ) , sex hormone - bonding globulin ( shbg ) , triglyceride ( tg ) , total and high - density lipoprotein ( hdl ) and low - density ( ldl ) cholesterol and triglycerides ( tg ) in fasting blood samples . also , fasting glucose test , 2-hour oral glucose tolerance test ( ogtt ) with 75 gr glucose ( parsazmoon co , iran ) and fasting insulin test were administered . all blood tests were performed , regardless of the menstrual cycle , at baseline and after 6 months . a qualified laboratory examined the blood samples ( at baseline and the following 6-month reevaluation ) through the same methods and materials . it should be noted that no significant differences were seen in the anthropometric indices and laboratory tests . to eliminate the confounding effect of diet , all patients received a standardized hypocaloric diet containing 1200 - 1400 kcal / day from a research dietitian . the diet consisted of 50% carbohydrates , 30% proteins and 20% fat prescribed by the same dietician attending our division , who calculated the dietary energy composition by subtracting at least 500 kcal from the usual energy intake of an individual . it should be noted that all women were visited monthly for compliance with both dietary and pharmacological treatments . diet was evaluated by the dietician at each monthly visit , according to previously defined criteria providing quantitative information on daily energy intake and composition of the diet consumed during the previous month . there was a strong reason to initiate the hypocaloric diet before treatment with medicine ; all patients equally underwent the similar diet as the only impressive element without any medication intervention . furthermore , patients were checked for their usual physical activity throughout the treatment by the standard physical activity questionnaire ( the persian version with high validity ) before , 3 months following and at the end of the study ( 21 , 22 ) . from total of 156 overweight or obese pcos women , following one month of diet , the participants were visited again . while continuing dietary treatment , the patients who had anthropometric indices and the recording of all preliminary laboratory tests , were randomly divided in 4 groups including , 1 : metformin hydrocloride ( tablet , 500 mg , tehran - shimi co. , tehran , iran ) , orally , 3/day , 2 : flutamide - biosyn ( tablet , 250 mg , sobhan co. , tehran , iran ) , 2/day , 3 : metformin 500 mg , 3/day+ flutamide 250 mg , 2/day and 4 : placebo group . patients were randomly assigned to groups to receive the drugs ( active or placebo ) using a computer generated sequence concealed from the study participants and then allocation sequence was done by a third party . fatemezahra investigation pharmacy transferred metformin , flutamide and placebo capsules with appropriate dose and the same color , then packed and labeled them according to subject number . all subjects were given a 1-month supply of capsules and they returned for monthly visits . also , to evaluate compliance with medications and to assess the side effects , patients were visited monthly . to evaluate the liver and renal toxicity at the beginning which were probably the results of medicines , renal and liver functional tests were requested and repeated every 2 months . moreover , the remaining capsules were counted and a new supply of capsules was given to the patients . patients recruited during 6-month treatment with hypocaloric diet and placebo , metformin , flutamide , or metformin+flutamide finally , at the end of the sixth month of treatment , physical examination focusing on anthropometric indices and all preliminary laboratory tests were done again and hirsutism was reevaluated . all patients underwent anthropometric , hormonal and metabolic evaluation at baseline and 6 months following . the sample size was calculated by g*power software and set error prob to 0.05 , power to 0.80 . the values in anova had normal distribution and the variances of assumed variables were equal . paired t - test was performed to estimate the within - group modification ( before and after treatment at each time point ) . a one - way anova ( post hoc ) was applied to compare values amongst the four groups at each time point ( baseline , 6 months following ) , whereas tukey test was applied to find differences amongst the groups . before the initiation of the study , the patients underwent the physical examination focused on anthropometric indices ( weight , bmi , waist and hip circumferences and hirsutism ) . the clinical examination focused on the evaluation of anthropometric and parametric values ( height , weight and waist circumference ) and also hirsutism using the ferriman - gallwey score ( 21 ) . waist circumference was obtained as the minimum value between the iliac crest and the lateral costal margin , whereas hip circumference was determined as the maximum value over the glottal region to cm . waist and hip circumferences were measured to the nearest centimeter with a soft tape ( cm ) according to world health organization ( who ) criteria . before the study , the baseline values of anthropometric parameters were measured . hormonal and metabolic determinations included the evaluation of circulating concentrations of free and total testosterone , dehydroepiandroster - one sulfate ( dhea - s ) , sex hormone - bonding globulin ( shbg ) , triglyceride ( tg ) , total and high - density lipoprotein ( hdl ) and low - density ( ldl ) cholesterol and triglycerides ( tg ) in fasting blood samples . also , fasting glucose test , 2-hour oral glucose tolerance test ( ogtt ) with 75 gr glucose ( parsazmoon co , iran ) and fasting insulin test were administered . all blood tests were performed , regardless of the menstrual cycle , at baseline and after 6 months . a qualified laboratory examined the blood samples ( at baseline and the following 6-month reevaluation ) through the same methods and materials . it should be noted that no significant differences were seen in the anthropometric indices and laboratory tests . to eliminate the confounding effect of diet , all patients received a standardized hypocaloric diet containing 1200 - 1400 kcal / day from a research dietitian . the diet consisted of 50% carbohydrates , 30% proteins and 20% fat prescribed by the same dietician attending our division , who calculated the dietary energy composition by subtracting at least 500 kcal from the usual energy intake of an individual . it should be noted that all women were visited monthly for compliance with both dietary and pharmacological treatments . diet was evaluated by the dietician at each monthly visit , according to previously defined criteria providing quantitative information on daily energy intake and composition of the diet consumed during the previous month . there was a strong reason to initiate the hypocaloric diet before treatment with medicine ; all patients equally underwent the similar diet as the only impressive element without any medication intervention . furthermore , patients were checked for their usual physical activity throughout the treatment by the standard physical activity questionnaire ( the persian version with high validity ) before , 3 months following and at the end of the study ( 21 , 22 ) . from total of 156 overweight or obese pcos women , 117 women were recruited and randomized to the treatment ( figure 1 ) . following one month of diet , the participants were visited again . while continuing dietary treatment , the patients who had anthropometric indices and the recording of all preliminary laboratory tests , were randomly divided in 4 groups including , 1 : metformin hydrocloride ( tablet , 500 mg , tehran - shimi co. , tehran , iran ) , orally , 3/day , 2 : flutamide - biosyn ( tablet , 250 mg , sobhan co. , tehran , iran ) , 2/day , 3 : metformin 500 mg , 3/day+ flutamide 250 mg , 2/day and 4 : placebo group . patients were randomly assigned to groups to receive the drugs ( active or placebo ) using a computer generated sequence concealed from the study participants and then allocation sequence was done by a third party . fatemezahra investigation pharmacy transferred metformin , flutamide and placebo capsules with appropriate dose and the same color , then packed and labeled them according to subject number . all subjects were given a 1-month supply of capsules and they returned for monthly visits . also , to evaluate compliance with medications and to assess the side effects , patients were visited monthly . to evaluate the liver and renal toxicity at the beginning which were probably the results of medicines , renal and liver functional tests were requested and repeated every 2 months . moreover , the remaining capsules were counted and a new supply of capsules was given to the patients . patients recruited during 6-month treatment with hypocaloric diet and placebo , metformin , flutamide , or metformin+flutamide finally , at the end of the sixth month of treatment , physical examination focusing on anthropometric indices and all preliminary laboratory tests were done again and hirsutism was reevaluated . all patients underwent anthropometric , hormonal and metabolic evaluation at baseline and 6 months following . the sample size was calculated by g*power software and set error prob to 0.05 , power to 0.80 . the values in anova had normal distribution and the variances of assumed variables were equal . paired t - test was performed to estimate the within - group modification ( before and after treatment at each time point ) . a one - way anova ( post hoc ) was applied to compare values amongst the four groups at each time point ( baseline , 6 months following ) , whereas tukey test was applied to find differences amongst the groups . out of 156 pcos patients who were eligible , 39 women were excluded ; 32 women had no inclusion criteria , 6 women declined to participate and one woman met the exclusion criteria . 117 women were randomized in 4 groups ; 28 women were allocated to receive metformin , 28 received flu - tamide ( 1 woman had no contribution ) and 31 women received metformin+flutamide ( 1 woman had no contribution ) and 27 women received placebo . out of the metformin group , 3 women ( 1 woman for high transaminase level and 2 women for refusing to follow up ) , from the flutamide group , one woman for wrong use of medicine , were lost to follow up . also , from the metformin+flutamide group , 4 women ( 2 women for the high transaminase level , 1 for refusing the follow up and one woman for the wrong use of medicine ) and from placebo group , one woman were lost to follow up ( for changing the place of living ) . finally , 25 women in the metformin group , 27 in the flutamide group , 27 in the metformin+flutamide group and 26 in the placebo group were analyzed . no significant difference was seen in the anthropometric indices and laboratory tests in baseline values . table 1 summarizes the anthropometric indices and laboratory test changes ( before and after treatment ) within the four groups . to determine the within - group changes , changes in anthropometric indices and laboratory tests within the 4 groups under study ( before and after treatment ) changes ( post treatment value- pre treatment value ) was significant at p<0.05 changes ( post treatment value- pre treatment value ) was significant at p<0.01 changes ( post treatment value- pre treatment value ) was significant at p<0.001 comparing changes between groups was significant at p<0.05 comparing changes between groups was significant at p<0.01 ferriman - gallway score as well as placebo , the groups treated with metformin , flutamide , and metformin+flutamide showed significant reduction in all antropometric indices following 6 months in comparison to their baseline ( p<0.01 ) except hc in flutamide group which was not significant . in the glucose profile , the fbs initially demonstrated significant reduction within metformin+flutamide group and then within placebo group before and after treatment , respectively ( p<0.001 ) ( p<0.05 ) . however , the fasting insulin showed no further modification after treatment compared to baseline except within metformin+flutamide group ( p<0.001 ) . in the lipid profile , ldl had significant reduction in all groups except metformin+ flutamide following the treatment compared to baseline ( metformin ; p<0.001 ) , ( flutamide ; p<0.01 ) ( placebo ; p<0.05 ) . in sex hormones , all groups demonstrated no significant reduction except dhea ( s ) in which significant reduction was seen in metformin+flutamide group and also placebo after treatment compared to the baseline . the anthropometric indices and laboratory tests of four groups after treatment are summarized in table 2 . parametric indices and metabolic parameters of women among the four groups under study results are expressed as meansd . the values were evaluated with the post hoc test ; no significant differences among groups in baseline values ; difference by group p-0.05 ; ns = non significant > 0.05 ; met = metformin ; flu = flutamide ; p = placebo the difference between groups ( in a row ) is significant ferriman - gallway score there were no significant differences in the baseline values in our study . in anthropometric indices , significantly greater reductions were observed in bmi , wc and hc in the metformin group after treatment in comparison to other groups ( p < 0.001 ) . in sugar profile , the post treatment fasting blood sugar and fasting insulin were diminished significantly in metformin+flutamide group compared to other groups after treatment ( p<0.001 ) , while in the lipid profile , metformin+flutamide revealed reduction in tg and placebo reduced ldl significantly after treatment compared to other groups ( p<0.001 ) . in sex hormone profiles , the flutamide group was superior to others in reduction of total testosterone ( p<0.01 ) and in the placebo , the reduction of dhea(s ) was observed after treatment ( p<0.05 ) ( table 2 ) . it should be mentioned that , throughout the study , markers of liver dysfunction such as transaminases and lactate dehydrogenase were checked monthly and they remained unaltered except among 3 women . table 1 summarizes the anthropometric indices and laboratory test changes ( before and after treatment ) within the four groups . to determine the within - group changes , changes in anthropometric indices and laboratory tests within the 4 groups under study ( before and after treatment ) changes ( post treatment value- pre treatment value ) was significant at p<0.05 changes ( post treatment value- pre treatment value ) was significant at p<0.01 changes ( post treatment value- pre treatment value ) was significant at p<0.001 comparing changes between groups was significant at p<0.05 comparing changes between groups was significant at p<0.01 ferriman - gallway score as well as placebo , the groups treated with metformin , flutamide , and metformin+flutamide showed significant reduction in all antropometric indices following 6 months in comparison to their baseline ( p<0.01 ) except hc in flutamide group which was not significant . in the glucose profile , the fbs initially demonstrated significant reduction within metformin+flutamide group and then within placebo group before and after treatment , respectively ( p<0.001 ) ( p<0.05 ) . however , the fasting insulin showed no further modification after treatment compared to baseline except within metformin+flutamide group ( p<0.001 ) . in the lipid profile , ldl had significant reduction in all groups except metformin+ flutamide following the treatment compared to baseline ( metformin ; p<0.001 ) , ( flutamide ; p<0.01 ) ( placebo ; p<0.05 ) . in sex hormones , all groups demonstrated no significant reduction except dhea ( s ) in which significant reduction was seen in metformin+flutamide group and also placebo after treatment compared to the baseline . the anthropometric indices and laboratory tests of four groups after treatment are summarized in table 2 . parametric indices and metabolic parameters of women among the four groups under study results are expressed as meansd . the values were evaluated with the post hoc test ; no significant differences among groups in baseline values ; difference by group p-0.05 ; ns = non significant > 0.05 ; met = metformin ; flu = flutamide ; p = placebo the difference between groups ( in a row ) is significant ferriman - gallway score there were no significant differences in the baseline values in our study . in anthropometric indices , significantly greater reductions were observed in bmi , wc and hc in the metformin group after treatment in comparison to other groups ( p < 0.001 ) . in sugar profile , the post treatment fasting blood sugar and fasting insulin were diminished significantly in metformin+flutamide group compared to other groups after treatment ( p<0.001 ) , while in the lipid profile , metformin+flutamide revealed reduction in tg and placebo reduced ldl significantly after treatment compared to other groups ( p<0.001 ) . in sex hormone profiles , the flutamide group was superior to others in reduction of total testosterone ( p<0.01 ) and in the placebo , the reduction of dhea(s ) was observed after treatment ( p<0.05 ) ( table 2 ) . it should be mentioned that , throughout the study , markers of liver dysfunction such as transaminases and lactate dehydrogenase were checked monthly and they remained unaltered except among 3 women . the result of our study indicates that the use of metformin and flutamide improves anthropometric indices and laboratory tests in obese / over - weight pcos women under hypocaloric diet . generally , in obese women with anovulatory infertility , weight loss makes spontaneous ovulation facile and improves the chance of spontaneous conception ( 24 ) . those patients who ovulate and conceive while remaining obese will encounter predictable risks during pregnancy and post - pregnancy . since pre - pregnancy weight loss reduces the incidence of gestational diabetes in these women ( 25 ) , most therapists encourage pcos women to reduce weight before pregnancy . the findings of the current study are consistent with that of morin - papunen et al . who found metformin is able to decrease glucose disposal in skeletal muscle ( 26 ) . also , a number of studies have found the reduction in weight and bmi by metformin ( 7 , 27 , 28 ) , however , there are many conflicting ideas about the effect of metformin on weight and bmi . tang et al . concluded that metformin is less effective in very obese anovulatory women and a higher dose instead of routinely prescribed doses and a longer duration of therapy are necessary for these patients ( 29 ) . in addition , nieuwenhuis - ruifrok in his systematic review concluded that addition of metformin treatment does not lead to greater weight loss in patients who are on a diet or who follow a lifestyle program ( 8) . a possible explanation for those might be that regarding our patients mean bmi ( 32 k / m ) , no further doses were needed for them . our unanticipated finding shows that the combination of the two drugs as a part of polytherapy provides beneficial effects on glucose reduction . wadden concluded that more weight loss is achieved by combination of a lifestyle modification program with drug therapy in comparison to a lifestyle program alone ( 30 ) . 's research hypocaloric diet alone has a main role in improving insulin resistance and hyperinsulinaemia . gambineri also conducted another study on pcos patients during 12 months and concluded metformin alone decreased glucose - stimulated insulin but the combination of both drugs had no additive effect . he explained that some therapeutic effects would not be present during the first six months of treatment ( 18 ) though , in this paper this incidence took place within 6 month . our interesting finding was that metformin+ flutamide reduced tg more than other medicines except hypocaloric diet . this finding is not in agreement with gambineri et al.s findings which showed none of the active treatment groups differed in changes of lipid concentrations after 6 months of treatment , whereas they observed a greater reduction in ldl cholesterol after 12 months in the metformin+flutamide group . maybe the initial results of ldl reduction in our study relates to the lesser weights of our patients in comparison to the patients in their study ( 18 ) . one of the limitations of our study was small sample size of the obese women which overlapped with overweight women , so the effect of medicine on them versus overweight women could not be evaluated . in our study , the menstrual pattern of our patients showed no significant differences with taking medicines or diet before and after treatment , whereas metformin further increased frequency of menstruation . reported that flutamide therapy showed a significant enhancement in the percentage of ovulation cycles and regulated the menstrual profile in their pcos patients . also , gambineri reported that metformin further increased frequency of menstruation compared with placebo after 6 months ( 18 , 32 ) . maybe , by dividing our patients menstruation pattern to regular and irregular , further beneficial findings could be offered . well designed clinical trials on the current topic are therefore recommended , especially use of flutamide and metformin and also hypocaloric diet and determining their effect on clinical and biochemical characteristics of pcos versus non pcos patients can be an important area for investigation . it causes improvement in their general health and prevents long term consequences . also , different beneficial treatment options according to our desired outcomes in the diet of overweight / obese pcos women would help the experts to plan and treat patients according to each of their profile disturbance ( any of the 4 groups ) . considering the beneficial effects of hypochaleric diet compared to the medicine , life style and nutrition modification in pcos patients was also effective in our study . of course , considering findings about metformin and flutamide versus diet alone , it is supposed that our conclusions should be considered with caution and judiciously and they require more attention before clinical recommendations . the most obvious finding of this study is that the use of metformin and flutamide improves and accelerates normalizing the anthropometric indices and laboratory tests in obese / overweight pcos women under hypocaloric diet .

backgroundthis study was designed to investigate the effect of metformin and flutamide alone or in combination with anthropometric indices and laboratory tests of obese / overweight pcos women under hypocaloric diet.methodsthis single blind clinical trial was performed on 120 pcos women . at the beginning , hypocaloric diet was recommended for the patients . after one month while they were on the diet , the patients were randomly divided in 4 groups ; metformin ( 500 mg , 3/day ) , flutamide ( 250 mg , 2/day ) , combined , metformin ( 500 mg , 3/day ) with flutamide ( 250 mg , 2/day ) and finally placebo group . the patients were treated for 6 months . anthropometric indices and laboratory tests ( fasting and glucose - stimulated insulin levels , lipid profile and androgens ) were measured . a one - way anova ( post hoc ) and paired t - test were performed to analyze data . a p 0.05 was considered statistically significant.resultsafter treatment , reduction in weight , bmi , hip circumference was significantly greater in the metformin group in comparison to other groups ( p<0.05 ) . in addition , the fasting insulin was significantly greater in metformin group and flutamide group in comparison to metformin+flutamide and placebo groups after treatment ( p<0.05 ) . within groups , insulin level showed significant changes ( before and after treatment ) in metformin+flutamide group and ldl reduction was significant in flutamide group before and after treatment . post hoc tukey and two - tailed with p0.05 were used to define statistical significance.conclusionusing combination of metformin and flutamide improves anthropometric indices and laboratory tests in obese / overweight pcos women under hypocaloric diet .

Introduction Methods Participants Randomization and intervention Statistical Analysis Results Comparing changes within the four groups (before and after treatment) Comparing amongst the four groups Discussion Conclusion Conflict of Interest

PMC5085309

skeletal muscle injury may result from toxin injection , crushing , freezing , and mechanical disruption induced by forceful stretches of muscle fibers . skeletal muscle fiber damage may also result from intense , unaccustomed , extreme ( i.e. , ultra - endurance events such as marathon running , triathlon , etc . ) and/or eccentric types of exercises that are characterized by forceful lengthening contractions during which muscle fibers are overstretched.1 according to the model of proske and allen,2 within stretched fibers , some sarcomeres may be more resilient than others resulting in greater absorption of the stretch by the weaker sarcomeres which , depending on the magnitude of the stretch , become more vulnerable as the overlap between myosin and actin filaments is minimized toward the end of the stretch . following numerous eccentric stretches , more and more of the weaker sarcomeres are gradually overstretched , and as fiber stretching continues , the more resilient sarcomeres become overstretched . since myofilaments of these sarcomeres may not return to their original overlapping state during the relaxation phase of the muscle , a mechanical disruption of these sarcomeres develops which is eventually transmitted to neighboring areas in muscle vicinity resulting in subcellular damage , that is , a collapse of membrane surrounding the sarcoplasmic reticulum , transverse tubules , and the muscle fibers themselves . this series of events compromises the process of excitation contraction coupling in damaged sarcomeres and results in the release of calcium ions from sarcoplasmic reticulum into the cytoplasm where they stimulate proteolytic enzymes that promote further muscle fiber degradation.1,2 during this first phase , aseptic exercise - induced muscle damage ( eimd ) leads to the onset of an inflammatory response associated with the activation of leukocytes , muscle edema , deterioration of muscle function , delayed - onset of muscle soreness ( doms ) , increased release of muscle proteins into the interstitial space , and circulation and a rise in muscle temperature.3 even after an extensive injury , skeletal muscle demonstrates an extraordinary ability for healing . consequently , a regeneration or healing phase follows the first inflammatory phase.4 muscle regeneration is related to the activation of a set of mononucleated cells , known as satellite cells , which subsequently proliferate , differentiate , and enter the damaged myofibers to synthesize new fibers or contribute to the healing of other fibers with a less severe damage.5 this phase is characterized by a marked rise of muscle protein synthesis.4 the inflammatory and the regeneration phases are operationally interconnected , and the disturbance of the former may hamper the later.6 evidence suggests that suppression of the inflammatory phase may lead to an attenuated overcompensation or healing during the regeneration phase.7 the scope of the present article is to review the first inflammatory phase and disclose important implications for exercise training and overall athletic performance . finally , the third part presents the changes in skeletal muscle performance during the inflammatory response and discusses important implications for sports performance . eimd is associated with muscle soreness or discomfort and a marked decline of muscle strength during the first 1272 h postexercise depending on the magnitude of the muscle - damaging exercise , and as stated earlier , it is related to the disruption of subcellular structures.8 although this phenomenon was described as early as the first part of the 20th century,9,10 the mechanisms underlying eimd are not entirely understood . despite that isometric ( static work , length of the muscle remains unchanged ) and concentric ( length of the muscle decreases ) muscle contractions are able to elevate skeletal muscle damage markers , the vast majority of published studies support the notion that eccentric work is primarily associated with eimd.1,3,4,8 eccentric ( lengthening ) work , either in their isolated form on an isokinetic dynamometer or as a part of the stretch - shortening cycle during downhill running , is used experimentally to study eimd . in fact , eccentric eimd is thought to be the basis for the increase of skeletal muscle mass ( hypertrophy ) seen with exercise training , especially resistance exercise training , suggesting that muscle micro - trauma induced by mechanical stress of acute exercise is a prerequisite for its subsequent growth during the adaptation phase.11,12 moreover , givli12 suggested that eimd may also be utilized to develop safer and more effective training and recovery protocols based on a personalized approach . study of eimd aids to understand the mechanisms governing muscle degeneration and regeneration , so that findings may be extrapolated to various clinical conditions that demonstrate deviations in these mechanisms such as in dystrophies , inflammaging , cancer , and so on . for example , muscular dystrophies are characterized by extensive skeletal muscle degeneration due to the lack of dystrophin , calcium toxicity , free radical generation , activation of proteolytic enzymes , degradation of muscle proteins , and finally loss of muscle function and patient s physical independence.13 understanding the molecular mechanisms linking muscle damage with inflammatory cascade and proteolysis using the eimd model to induce mechanical strain to the cytoskeleton of the muscle could help toward the development of potential treatments for the clinical symptoms associated with this disease.14,15 during the last few years , a number of pharmacological agents and/or nutritional supplements have been tested in this direction using the eimd model.1618 rheumatoid arthritis ( ra ) represents another example . ra demonstrates an intense inflammatory response through the production of the inflammatory cytokines at the site of the disease ( i.e. , joints ) affecting distant tissues and play a significant role in skeletal muscle wasting.19 furthermore , oxidative stress is frequently reported in patients with ra since cells present in inflamed joints ( e.g. , macrophages , neutrophils , and lymphocytes ) have the ability to produce free radicals.20 therefore , finding ways to manipulate the inflammatory responses and modulate the redox response following eimd could give insights on cellular mechanisms leading to muscle cachexia and how to confront clinical conditions like ra and potentially others . doms and strength loss during eimd were originally attributed to subcellular disruption of skeletal muscle fibers.10,12 this notion was further supported by the indirect evidence of increased concentration of muscle proteins into the circulation in response to eimd , suggesting a potential leakage of intracellular content due to damage of muscle fiber membrane.12,21,22 this theory was further supported by marked sarcomeric distortions ( e.g. , z - line streaming ) identified by electron microscopy following eimd in both human and animal studies12,23,24 or infiltration of extracellular dyes into the muscle in animal studies.16,25 damage of muscle fiber membrane also causes influx of extracellular substances ( e.g. , albumin and fibronectin).12,26,27 increased membrane permeability has been linked to the stimulation of sodium and calcium channels in response to repetitive stretching of muscle fibers by lengthening contractions.28,29 nevertheless , a large variation exists in the manifestations of the magnitude of eimd induced by eccentric exercise protocols ranging from extensive local or systemic inflammatory responses and myofiber trauma7,12,30 to less pronounced injury in humans31 probably due to large variations in muscles tested as well as exercise protocols used . moreover , protocols using electrical stimulation on animals to simulate eccentric contractions , although useful to study molecular mechanisms underlying eimd , may not be physiologically relevant to that produced by humans.32 electron microscopy revealed that eimd is characterized by extensive fiber necrosis , z - line streaming at sarcomere level for as long as 13 days following eccentric exercise depending on the magnitude of the exercise stimulus,12,23,24,30,33 and deterioration of a significant structural ( e.g. , desmin and dystrophin ) and myofibrillar proteins ( e.g. , myosin ) , suggesting an increased rate of degradation.32,3436 necrotic parts of injured myofibers appear histochemically as inflated and orbicular.37 although others argue that eccentric exercise does not cause necrosis,38 myofiber necrosis may be accompanied by macrophage infiltration , especially at the perimysium and endomesium , through toll - like receptors indicating an abrupt mobilization of the immune system during the first hours postexercise.30,39,40 interestingly , damaged myofibers also demonstrate the aggregation of satellite cell - derived myoblasts.30,38,4143 these findings suggest that doms and strength loss are primarily related to this disruption of contractile and structural proteins located in sarcomeres . yu et al44 suggested that these structural deformations of muscle fibers do not indicate muscle damage but rather a regeneration process suggestive of an adaptation at cellular level . animal models seem to demonstrate a greater rate of subcellular derangement than human models.44,45 muscles of the upper limbs seem to be more susceptible to eimd probably due to their smaller muscle mass and utilization in daily activities.46,47 extracellular matrix , mainly connective tissue , is affected by eimd as well . muscle fibers are mechanically connected with extracellular matrix through a complex organization of membrane - bound proteins ( i.e. , integrins , dystroglycans , and proteoglycans ) that enable mechanical transmission among muscle fibers and fascicles.48 histological data indicate that eimd widens parts of the perimysium and endomysium with connective tissue dissociated from muscle fibers into a more expanded interstitium.27 these findings are coupled with others showing increased and prolonged collagen turnover indicative of a remodeling of extracellular matrix in response to its damage by exercise.49,50 however , extracellular matrix contains not only collagen fibers but also endothelial and pericyte cells , immune cells , stromal cells such as fibroblasts , nerve cells , and satellite cells that all combine to produce a physiological response , that is , muscle contraction . it is uncertain how all these cells are affected by eimd and whether their potential damage or remodeling affects skeletal muscle fiber homeostasis . for example , intramuscular mechanical pressure during eimd is transferred through the extracellular matrix inducing an early release of growth factors from fibroblasts that result in the activation of adjacent satellite cell populations via selective intracellular signaling pathways which may promote muscle healing.51,52 vascular cells such as pericytes are also involved in eimd by activating nuclear factor - kappa b ( nf-b ) signaling which is implicated in immune cell mobilization and satellite cell proliferation.7,53,54 however , it must be mentioned that nf-b pathway mediates the upregulation of the activity of the ubiquitin proteosome system ( ups ) by proinflammatory cytokines ( i.e. , tumor necrosis factor - alpha [ tnf- ] and interleukin-6 [ il-6 ] ) thereby contributing to protein degradation of skeletal muscle proteins and overall eimd.55 it is unclear whether the activation of ups contributes or exacerbates eimd or it represents a protective mechanism that clears damaged protein molecules and as such accelerates the inflammatory cascade and the subsequent healing . the involvement of the nf-b ups pathway in eimd constitutes a valuable experimental model to study skeletal muscle atrophy in conditions such as sepsis and cachexia induced by cancer and other clinical conditions . therefore , it seems that extracellular matrix may be actively involved during eimd and may affect muscle fiber healing , a perspective that needs to be addressed by future investigations . as mentioned earlier , eimd is associated with prolonged deterioration of muscle strength , doms , edema , increased temperature , and leakage of muscle proteins into the circulation.56,57 prolonged ( 114 days ) muscle strength reduction ( 10%70% ) is considered as one of the most valid markers of eimd since among all markers they exhibit the highest correlation with histological evidence of muscle disruption.8,56,58,59 recovery kinetics of muscle strength depend on genetics , the type of exercise , muscle group(s ) involved , intensity , volume , and novelty of exercise.28 strength loss is attributed to the so - called half - sarcomere nonuniformity , which states that most of finer length adjustment is accommodated by the weakest half - sarcomeres , which become weaker as muscle lengthening progresses and advances beyond the point of myofilament overlap and ultimately microtears develop.2,60 repeated stretching further increases traumatized sarcomeres , and injury of muscle fibers is exacerbated causing membrane disruption and perforation of channels stimulated by mechanical stretch.2,60 damage of junctophilins , that is , proteins connecting t - tubules with the membrane of sarcoplasmic reticulum mediating the communication between the calcium release channel and the dihydropyridine receptor , may also contribute to strength losses due to uncoupling of the excitation contraction coupling mechanism and calcium kinetics from sarcoplasmic reticulum resulting in strength reduction.63,64 this mechanism accounts for ~75% of early ( up to 72 h ) muscle strength deterioration with injury of other force - generating and transmitting structures accounting for the remaining 25%.65 damage of junctophilin proteins results in the rise of intracellular calcium which then stimulates calcium - activated proteolytic enzymes ( i.e. , calpains ) that further degrade contractile and other muscle proteins,66 a mechanism also shown by experimentation with dystrophic mdx mice with an inherent stimulation of calcium channels.67 in fact , removal of calcium from muscle s environment or inhibition of calcium channels with pharmacological agents restores or attenuates strength decline following eccentric work in some studies with mdx mice68,69 but not in healthy muscle.70 according to warren et al,65 degradation of contractile proteins may further contribute to strength loss during prolonged ( > 72 h ) muscle injury . although evidence implicates various other structural and contractile proteins , such as -actinin-3 , in strength loss following damaging exercise,71,72 more research is needed in order to understand the mechanical aspect of their contribution . doms usually peaks 12 days postexercise and recovers to baseline levels after 47 days.73,74 in contrast to muscle strength loss , doms demonstrates a moderate to low correlation with histological evidence of eimd probably due to a significant variability of subjects.73 doms is attributed to muscle fiber disruption , disturbance of calcium homeostasis , and firing of iv nerves in response to various inflammatory by - products.75,76 in fact , synthesis and release of inflammatory agents ( e.g. , bradykinin , prostaglandins , histamine , and nerve growth factor ) by proinflammatory cells ( e.g. , immune cells ) postexercise may actually trigger selective pain receptors and produce soreness.77,78 available evidence suggests that nitric oxide ( no ) produced by muscle no synthase may also stimulate nociceptors of c - fibers , thereby inducing a pain sensation.76 generation of free radicals by the nicotinamide adenine dinucleotide phosphate ( nadph ) oxidase mechanism of the immune cells may also contribute to the damage of injured and noninjured muscle fibers and the onset of doms , a phenomenon also known as respiratory burst - induced secondary damage.1,8 allen et al28 suggested that skeletal muscle responses to eimd follow a continuum with the type of eccentric muscle activation , genetic variability , muscle group engaged , and overload characteristics ( intensity , volume , and degree of novelty ) being the main determinants of the type of adaptations induced . eimd is more severe when electrical stimulation is utilized to induce eccentric contraction ( as compared to voluntary eccentric activation ) , genetic background is optimal ( high vs. low respondents ) , and intensity , volume , and novelty are of sufficient magnitude.28 when eimd is less severe , there is an increased likelihood for positive adaptations , whereas in more severe eimd , an intense disruption of muscle fibers occurs and an augmented inflammatory response develops.28 in more extreme cases , a maladaptation occurs resulting in musculoskeletal injuries ( e.g. , strains ) , fiber necrosis , insufficient regeneration , and probably development of scar tissue.4,28,70 other factors may also affect the predisposition and susceptibility to eimd . sex may be related to variations often seen in eimd manifestations with women demonstrating strength decrements , doms , and membrane disruption of smaller magnitude79,80 probably due to a prophylactic effect of estrogens on muscle fiber membranes , calcium homeostasis , and inflammatory reaction.8183 whether doms responses are sex - dependent is less clear . other factors that may attenuate eimd manifestations are regular exercise training and utilization of nutritional supplements ( i.e. , anti - inflammatory agents , antioxidants , and creatine supplementation).7,84,85 since athletes have a heavy schedule , with training or sometimes competing more than once a day , finding ways to maximize or accelerate the recovery process from exercise training becomes crucial for their performance . nutritional strategies , either by using dietary supplements or foods , have been proposed in the literature to encounter this challenge . it has been shown , for example , that protein supplementation , mainly through the ingestion of amino acids , has a positive effect in reducing muscle soreness following an exercise bout and accelerating functional recovery.86 furthermore , subjects receiving food supplements such as cherry or pomegranate juice had a faster recovery in performance following an eimd protocol.8789 in addition , creatine supplementation has been shown to attenuate eimd following an eccentric exercise protocol.90 potential mechanisms that explain the effect of creatine supplementation on eimd include a reduction in the inflammatory response and oxidative stress and regulation in the calcium homeostasis as well as promotion of proliferation and differentiation of satellite cells.91 however , not all supplements have been proven to be effective,92,93 and further research is needed to identify foods and supplements that are effective in either preventing eimd or speeding up the recovery process . the first leukocyte subpopulation that infiltrates traumatized muscle is the neutrophils within 46 h and remains elevated during the 24 h postinjury.17,9496 macrophage infiltration into the injured muscle follows 114 days postinjury depending on the magnitude of the insult.17,95,97 the mission of the leukocyte subsets is to remove cellular debris such as damaged protein molecules by releasing reactive oxygen and nitrogen species during a process called respiratory burst,7,96,98100 proteolytic enzymes,52 and by synthesizing proinflammatory cytokines such as il-1 , il-6 , il-8 , tnf- , and monocyte chemotactic protein-1 which are also expressed by muscle cells probably within the first 24 h after exercise.97,101 at this time , eimd results in changes of the expression of leukocyte receptors as well as their activity thereby regulating their ability to infiltrate into the damaged tissue.102 these changes are paralleled by an upregulation of major adhesion molecules.22,103 proinflammatory cytokines seem to start the disintegration of cellular fragments.104 a second wave of proinflammatory cytokines ( e.g. , il-6 and transforming growth factor ) are produced by damaged skeletal muscle several days within recovery.97,105,106 other proinflammatory cytokines may be expressed and released by infiltrating immune cells such as il-4 and il-1 receptor antagonist ( il-1ra).95,106 removal of cellular debris may be a prerequisite for the subsequent regeneration of injured myofibers.107 consequently , injured muscle develops an intense chemotactic action to attract immune cells that are usually detected first in the extracellular compartment ( 13 days ) and then intracellularly ( 48 days).7,35,39,108 the greater the damage the greater the infiltration of immune cells in the muscle environment.39,108,109 recent evidence suggests that this inflammatory phase may be under redox - dependent regulation through the mediation of key intracellular signaling pathways such as that of nf-b and mapk which control cytokine synthesis and immune activation.7 the acute inflammatory response following damaging exercise elevates leukocyte numbers not only within the muscle milieu but also in the circulation , suggesting that eimd elicits a systemic inflammatory response as well . this immune response is greater following eccentric exercise protocols ( i.e. , eccentric dynamometry , eccentric cycling , and downhill running ) than other types or modes of exercise incorporating both concentric and eccentric actions such as level running , stepping , jumping , resistance exercise training , and sport activity.95,103,104,110120 these differences are largely attributed to the characteristics of exercise ( intensity , duration , and volume ) , muscle groups involved , and types of contraction that affect stress hormones at different degrees.1,22,114,121123 these systemic immune changes are accompanied by alterations in the circulatory levels of proinflammatory cytokines such il-1 and tnf-,7,121,123,124 indicating that eimd may cause cytokine secretion by cells other than muscle ( e.g. , immune cells ) . interestingly , after extensive eimd , il-6 , also produced by skeletal muscle , may remain elevated even after the completion of the inflammatory phase as it has been implicated in the activation of satellite cell during the regeneration phase7,125,126 and release of anti - inflammatory molecules such as cortisol , il-1ra , and il-10.127 collectively , these results suggest that eimd induced both local and systemic inflammatory responses as depicted by changes in immune cells and cytokines . on the other hand , several investigations suggest that during this initial proinflammatory phase , a number of anti - inflammatory molecules such as il-10 , il-12 , il-1ra , soluble tnf- receptors , and granulocyte - colony stimulating factor are secreted.7,110,124,128 systemic levels or muscle expression of leukemia inhibitory factor ( lif ) , interferon ( ifn)- , and cytokines 2 , 5 , 12 , 13 , 15 , and 17 seem to remain unaffected by eimd . whether all types of exercises trigger such a response is questionable since the greatest elevations of these anti - inflammatory cytokines have been seen following extreme types of exercise such as marathon running with il-1 , tnf- , and il-6 rise preceding that of tnf- receptors and il-1ra.129 although some investigators suggested that these anti - inflammatory cytokines may be released by mobilized immune cells,130 the tissues secreting these molecules are unknown . anti - inflammatory cytokines seem to suppress inflammation by hindering expression and activation of pro - inflammatory cytokines and generation of immune cells.131133 several studies have shown a correlation between the systemic levels of proinflammatory cytokines and ck activity and myoglobin levels ( eimd markers ) in the circulation postexercise.134136 although this observation is important , it does not explain the exact role of cytokines in eimd - induced inflammatory response . furthermore , the origin of these cytokines remains obscure despite findings suggesting muscle and immune cells as their potential source of release . paulsen et al8 believe that in vitro studies do not consider the complex interaction between various tissues and humoral agents during and after exercise . consequently , under in vivo conditions , cytokine production and release may occur from specific cell types exposed to specific local inhibitory or stimulating molecules.8 although human studies are valuable , tissue sampling may be problematic since muscle homogenates or immune cell extracts may include various cell types that are difficult to distinguish , and they all may contribute to cytokine production.8 cytokine levels in the circulation are merely product of their appearance and disappearance kinetics in that compartment and explain very little with regard to the tissue of their origin . most of these molecules are produced by damaged myofibers , and they exist only at the local environment and as such their measurement in the blood is not possible.137 serial sampling of muscle biopsies or inhibition of specific cytokines may be more useful in elucidating their role in eimd - induced inflammation.8 animal studies showed that cytokine depletion or deficiency may attenuate recovery , suggesting that these molecules are important for muscle healing.138,139 this is mainly attributed to the inadequate mobilization of neutrophils and macrophages to the site of injury and/or an attenuated myogenic response.140142 interestingly , antioxidant supplementation attenuated the elevation of proinflammatory cytokines in days 13 postexercise and resulted in reduced recovery of muscle strength during the regeneration phase , suggesting that cytokines may be implicated in the recovery process.7 eimd and its associated inflammatory response is of paramount importance for athletes performance for two basic reasons : 1 ) the rate of deterioration and recovery of performance and discomfort ( doms ) after an athletic activity is largely dependent on the magnitude of eimd - induced inflammatory response and 2 ) it affects the frequency of training stimuli , that is , the time needed for optimal recovery in between practices , official events , and/or an event and a practice . a > 20% decline of the force - generating capacity of muscles exhibits a close association with the magnitude of muscle damage and its associated inflammatory response.56,143145 smaller reduction of muscle strength is usually not accompanied by histological evidence of eimd.8 paulsen et al8 suggested that strenuous isolated eccentric activity produces a greater degree of eimd than the so - called eccentrically biased activities such as downhill running or intense - level running . evidence indicates that the magnitude of force , the degree of lengthening , velocity of movement , and overall volume of eccentric load are probably the most important factors dictating the rate of muscle injury in response to exercise.146156 in fact , it has been shown that eimd induced by a football match is associated with the number of explosive types of movement that incorporate a strong eccentric component.57,157 in cases of mild performance decline , defined by paulsen et al8 as a drop of strength by < 20% , creatine kinase activity ( ck ) in blood remains < 1,000 u / l , inflammation is limited , and recovery is usually fast ( within 1248 h ) independent of type of exercise.39,59,74,95,96,112 , in the case of moderate performance decline , defined by paulsen et al8 as a drop of strength by < 20%50% , myofiber necrosis may be observed ( mostly in high - respondents),39 ck exceeds 1,000 u / l , the inflammatory response is more intense with leukocyte infiltration into the injured muscle , and degradation of structural and contractile proteins may be evident and recovery is usually completed within a week.22,35,39,158,159 in the case of severe performance decline , defined by paulsen et al8 as a drop of strength by > 50% , necrosis is usually observed in parts of the myofibers , ck may exceed 10,000 u / l , soreness is quite high , muscle swelling is evident , the inflammatory response is intense and characterized by a marked accumulation of immune cells into the traumatized tissue , and recovery usually takes 13 weeks or even longer if strength loss is > 70%.24,30,39,108,109,160166 in severe eimd , increased proteolysis and muscle disruption is observed , even during early recovery , due to disturbances in calcium homeostasis.145,167 in these three scenarios , athletes may need to use different recovery strategies in order to be able to train or compete as soon as possible . recovery treatments may be classified in four major categories : 1 ) pharmacological ( e.g. , inflammatory agents , nf-b inhibitors , estrogen therapy , phosphodiesterase inhibitors , ace inhibitors ) , 2 ) nutritional approaches and supplements ( e.g. , antiantioxidants , herbal remedies , alcohol ingestion , -3-fatty acids , -hydroxy--methylbutyrate , protease supplementation , nucleotide supplementation , tea consumption , beetroot , caffeine , creatine , enzyme supplementation , and carbohydrate and/or protein supplementation ) , 3 ) rehabilitation and physical therapy methods ( e.g. , cryotherapy , heat - related treatments , compressive loading techniques , trekking poles , ultrasound and electrical current modalities , massage , hyperoxia or hypoxia , laser therapy , spa therapy , mechanomyographical feedback , vibration therapy , acupuncture , and homeopathy ) , and 4 ) exercise - related treatments ( e.g. , stretching and low - intensity exercise ) , and numerous studies and reviews have examined their effectiveness in reducing eimd and accelerating recovery.168171 of those , nonsteroidal anti - inflammatory agents ( depending on dosage and time of ingestion ) , protein supplementation , -hydroxy--methylbutyrate supplementation , massage ( depending on the time of administration and technique used ) , antioxidant supplementation , and exercise - based treatments have been shown to exert positive action.168171 these recovery approaches aim to reduce swelling , improve blood flow , pain sensation , immune cell recruitment , and/or improve healing by activating satellite cells and anabolic factors as well as improving tendon healing . one has to remember that athletes are interested not only in the attenuation of the eimd - related inflammatory response but also and more importantly in effective and timely performance recovery . certain concerns have been raised regarding the use of anti - inflammatory agents based on reports suggesting that these products not only disrupt the inflammatory response but also hinder the adaptive response to training.7,172 however , the periods during which anti - inflammatory agents are used need to be discerned . there are training periods during which athletes need to compete and train with very high frequency ( e.g. , in - season training ) , and cessation would be beneficial . in contrast , there are periods during which athletes train aiming for long - term adaptations , and anti - inflammatory agents may disrupt both eimd and training adaptations . in the latter case , athletes should rethink about using such recovery approaches . however , more research is needed in order to determine whether anti - inflammatory agents interfere with training adaptations . now it is also well - understood that athletes may be low- , moderate- , and high - respondents to eimd , a fact that also explains the substantial interindividual variability in the responses seen following various types of exercise protocols used to induce muscle micro - injury.73,108,155,163,168,173175 this phenomenon may be attributed to a number of factors such as age,154,176,177 preconditioning,178180 gender,80,166 genetics,181,182 physical conditioning level,57,183185 and joint range of motion.186 therefore , sport practitioners should determine whether their athletes are low- , moderate- , or high - respondents , be advised to establish individual normative values for each one , and avoid absolute comparisons among athletes or generalizations . it must also be mentioned that preconditioning , a phenomenon also known as repeated bout effect , may protect athletes from eimd , that is , after a first session of damaging exercise , skeletal muscle tissue adapts and is less vulnerable to injury in subsequent sessions of the same type of exercise.153,180,187 in fact , it has been shown that repetition of a damaging exercise results in less eimd , inflammation , oxidative stress , leukocyte infiltration , and strength loss.5,111,159,187,188 exercise - induced inflammation is caused by eimd which is mainly associated with eccentric type of contractions that lengthen the muscles . under conditions of repetitive lengthening contractions , weaker sarcomeres are compromised initiating micro - tearing of myofibers that spreads out to the cellular membrane and other subsarcolemal structures as well as to extracellular matrix and connective tissue . calcium efflux from disrupted sarcoplasmic reticulum and stretch - dependent channels activate proteolytic enzymes that contribute to muscle damage by degrading structural and contractile proteins . although the exact mechanisms are largely unknown , eimd triggers an inflammatory response characterized by the accumulation and infiltration of neutrophils and macrophages into the damaged tissue . the activation and mobilization of these immune cells are mediated by cytokines released by injured muscle and other cells and may be under redox regulation . immune cells remove cellular debris and protein fragments to allow for the subsequent muscle regeneration and healing . during the inflammatory phase , muscle strength declines and doms develops at a rate dictated by the magnitude of the stretching stimulus . during this phase , athletic performance is compromised for hours to days depending on the magnitude of muscle damage as well as a number of other factors . there is a large interindividual variability of the responses to eimd , and this has to be acknowledged by sport practitioners . although a good number of studies have investigated eimd and inflammation , the exact mechanisms responsible for the onset of doms remain obscure . research needs to unravel the neural pathways triggering the sensation of muscle soreness , the molecular mechanisms associated with immune cell activation , and the mechanisms regulating the transition from the inflammatory phase to the healing phase . so far , there are no clear guidelines regarding recovery strategies following eimd mainly due to inconsistencies in protocols , time of administrations , dosages , exercise protocols , and type of techniques used among studies . future research should provide clear evidence regarding optimal treatments for effective and timely recovery of both eimd and performance following various types of exercise training modes and methods as well as competition . new genetic and molecular methodologies will allow scientists to answer critical questions regarding the pathways regulating muscle damage and healing .

the study of exercise - induced muscle damage ( eimd ) is of paramount importance not only because it affects athletic performance but also because it is an excellent model to study the mechanisms governing muscle cachexia under various clinical conditions . although , a large number of studies have investigated eimd and its associated inflammatory response , several aspects of skeletal muscles responses remain unclear . in the first section of this article , the mechanisms of eimd are reviewed in an attempt to follow the events that result in functional and structural alterations of skeletal muscle . in the second section , the inflammatory response associated with eimd is presented with emphasis in leukocyte accumulation through mechanisms that are largely coordinated by pro- and anti - inflammatory cytokines released either by injured muscle itself or other cells . the practical applications of eimd and the subsequent inflammatory response are discussed with respect to athletic performance . specifically , the mechanisms leading to performance deterioration and development of muscle soreness are discussed . emphasis is given to the factors affecting individual responses to eimd and the resulting interindividual variability to this phenomenon .

Introduction Mechanisms and consequences The inflammatory response associated with EIMD Exercise-induced inflammation and sports performance Conclusion

PMC3715852

all models with heterogeneities in the mechanical properties show large - scale patterns of stress maxima and minima when they are compressed . 1 . the nature of the patterns in stress and how the patterns are impacted by elastic heterogeneity 2 , which shows the equivalent von mises stress for four of the models . in all models illustrated here although this degree of variation in elastic properties is unrealistic , i use this model as an extreme end - member . in fig . 2d the young s moduli of the grains varies from 100 to 120 gpa , which is similar to the degree of variation seen in high - symmetry minerals like beta quartz or halite . the other models have variations in elastic properties that are intermediate ( table 1 ) . although the patterns occur on a scale much larger than the grain - scale , they do not reflect the pattern of grain boundaries because individual grains contain significant stress gradients . in fig . 2d the pattern can be described as isolated islands of low equivalent stress , but in fig . the change in patterning from the most heterogeneous model to the least is in the spacing of the streams and the intensity of the stress concentration in the streams. the vertically oriented patterns originate from variations in the magnitude and direction of the maximum compressive stress ( 3 ) . a subordinate horizontal pattern ( best seen in fig . figure 3 shows a portion of a model with the full stress tensor plotted . with the exception of the softest grains , the orientation of the maximum compressive stress can deviate substantially away from the macroscopic compression direction and the stress state can even become tensile . these variations are produced by the difference in elastic properties of neighboring grains even before plastic flow in the weakest grains initiates . the patterns in stress are established in the first displacement increment and change only in numerical value until the first yielding occurs . as yielding initiates , the patterning in 3 deteriorates , while the patterning in 1 intensifies ( fig . 4 ) . all of the models produce strain localization in the form of shear bands ( fig . 5 ) , and the spacing of the shear bands is coupled to the stress patterning ; denser patterning produces more closely spaced shear bands concentrating smaller degrees of stain . therefore , the location of shear bands is dictated by the spatial distribution of locations where yielding first occurs ( fig . the degree to which shear bands grow and intensify or not as a function of global strain depends on the level of work hardening included in the plasticity model . all models with heterogeneities in the mechanical properties show large - scale patterns of stress maxima and minima when they are compressed . 1 . the nature of the patterns in stress and how the patterns are impacted by elastic heterogeneity 2 , which shows the equivalent von mises stress for four of the models . in all models illustrated here although this degree of variation in elastic properties is unrealistic , i use this model as an extreme end - member . in fig . 2d the young s moduli of the grains varies from 100 to 120 gpa , which is similar to the degree of variation seen in high - symmetry minerals like beta quartz or halite . the other models have variations in elastic properties that are intermediate ( table 1 ) . although the patterns occur on a scale much larger than the grain - scale , they do not reflect the pattern of grain boundaries because individual grains contain significant stress gradients . in fig . 2d the pattern can be described as isolated islands of low equivalent stress , but in fig . the change in patterning from the most heterogeneous model to the least is in the spacing of the streams and the intensity of the stress concentration in the streams. the vertically oriented patterns originate from variations in the magnitude and direction of the maximum compressive stress ( 3 ) . a subordinate horizontal pattern ( best seen in fig . figure 3 shows a portion of a model with the full stress tensor plotted . with the exception of the softest grains , the orientation of the maximum compressive stress can deviate substantially away from the macroscopic compression direction and the stress state can even become tensile . these variations are produced by the difference in elastic properties of neighboring grains even before plastic flow in the weakest grains initiates . the patterns in stress are established in the first displacement increment and change only in numerical value until the first yielding occurs . as yielding initiates , the patterning in 3 deteriorates , while the patterning in 1 intensifies ( fig . 4 ) . all of the models produce strain localization in the form of shear bands ( fig . 5 ) , and the spacing of the shear bands is coupled to the stress patterning ; denser patterning produces more closely spaced shear bands concentrating smaller degrees of stain . therefore , the location of shear bands is dictated by the spatial distribution of locations where yielding first occurs ( fig . the degree to which shear bands grow and intensify or not as a function of global strain depends on the level of work hardening included in the plasticity model . the fem models presented here make predictions about the heterogeneity and spatial distribution of elastic and plastic strain in polycrystalline materials that are consistent with experimental observations as well as results from other numerical studies . many microstructural studies of plastically deformed polycrystals observe intragranular heterogeneity of plastic strain ranging from those conducted on highly anisotropic materials like olivine and ice67 to metals , which generally have more isotropic single - crystal rheological properties2891011 . the degree of strain heterogeneity is thought to be a function of the anisotropy of the material17 . the development of shear - strain localization at very low degrees of strain has been observed in a number of materials781112 . the development of tensile elastic strain during compression has been observed in ice7 and zirconium11 . large variations in stress states within single grains and plastic features that span many grains have been observed in three - dimensional ( 3d ) finite element models of polycrystalline fcc metals1 . however , consistent with what is observed in synchrotron and neutron diffraction studies of deforming materials , elastically stiffer grains on average strain less than more compliant grains ( see , for example , the study by turner and tome13 , or diffraction data within the study by burnley and zhang14 ) ( fig . . referred to as force chains , the patterns are produced by stress percolation20 and consist of semi - linear subsets of particles arranged parallel to compression that bear the applied load through the so - called granular materials are most obviously distinct from fully dense polycrystalline materials in that the constituent grains have limited or no cohesion . both shear tractions and tensile forces there are extreme variations in the mechanical properties of the constituent structural components ( grain boundaries and grain interiors ) , whereas in fully dense polycrystalline materials variations between structural components , which may or may not include rheologically distinct grain boundaries21 , are comparatively muted . thus , granular materials and fully dense polycrystalline materials fall on a continuum of variation in component properties . the value in making this connection is that it suggests new ways of thinking about deformation of polycrystalline materials . for example , force chains are understood to be the underlying mechanism of shear localization in granular materials ( see discussion in the study by tordesilla and muthuswamy22 and references therein ) . therefore , examining the role of stress patterning in shear localization in polycrystalline materials may prove fruitful . the magnitude of stress concentrations in the stress patterns and the regularity with which they occur may well yield to statistical descriptions , as they do in granular materials23 and should lead to a better understanding of phenomena that are tied to stress concentrations , such as the development of stylolites , slatey cleavage and compositional banding in rocks2425 , as well as help explain how these rhythmic features initially arise from unpatterened rocks such as shale and granite . the recognition of stress patterning will have a number of important implications for experimental characterization and modeling of rheological properties . in experimental studies , experimental samples whose size allows only a portion of the stress pattern to be captured , may present difficulties for obtaining reproducible results ; a phenomena that would be most noticeable in high - pressure studies where sample sizes are restricted . changing the grain size or overall size of experimental samples can be cumbersome , but numerically it should be comparatively painless to predict the appropriate rve by increasing the size of the grain ensemble being modeled until the tails of the stress distribution are fully described . another important implication of stress patterning is that it provides a framework for modeling aggregate behavior over multiple - length scales based on single - crystal properties . robust predictive models of the mechanical behavior of rocks are needed for understanding the mechanical behavior of the interior of the earth and other planets because extrapolations in both length scale and strain rate will always be required26 . the need for reliable predictive rheological models is not confined to the geosciences but extends to engineering and materials science problems that present a variety of challenges for direct observation ; for example , shock loading of nuclear weapon components or detonation of plastic - bonded explosives . these extrapolations can be made with more confidence if they are generated by models that are based on the physics of deformation : capturing the behavior of the crystal lattice as well as the short- and long - range interaction between grains within the polycrystal . plastic and viscoplastic self - consistent models describe single - crystal plasticity but assume that a single set of average boundary conditions apply to each grain . these models could be modified such that the boundary conditions applied to each grain are allowed to vary over a range suitable to the elastic and plastic heterogeneity of the structural components of the material . perhaps the most important impact that the recognition of stress patterning can have is if it , as a simple and intuitive paradigm , can replace the default assumption of a reuss state ( isostate ) of stress in the minds of those who work on stress - related phenomena . although it has been long recognized that the reuss state of stress is an oversimplification , the assumption still pervades our thinking on everything from the attenuation of seismic waves to the development of paleopiezometers . by providing a means of leaving the reuss stress state assumption behind , the recognition of patterning in stress should lead to new insights in a number of disciplines . each grain was modeled as a hexagon containing 48 six - noded triangular elements ( fig . 1 ) ; this type of element uses a parabolic interpolation function along each edge . the nodes on the boundaries of each grain are shared with the elements along the edge of the neighboring grain , thus grain boundaries are demarcated only by a change in elastic and plastic properties . as there can be no offset along a grain boundary in the model , the model consists of 2,574 hexagonal grains that have been assigned randomly into 25 subsets . the constitutive equation used for linear elastic solids is : where ij is the stress tensor , ij is the kronecker delta , ij is the strain tensor , is the lame constant , and g is the shear modulus . can be expressed as : and g can be expressed as : where v is the poisson s ratio and e is the young s modulus . plastic behavior initiates when the equivalent von mises stress ( vm ) , which is calculated as follows : equals the specified yield strength . post - yielding behavior was either perfectly plastic or included a work - hardening slope . the young s modulus and poisson s ratio assigned to each grain set for the models presented in the illustrations are given in table 1 . for all materials shown in the figures , the yield strength was set to 1/100th of the young s modulus and no work hardening was included . nodes along the bottom edge were not allowed to move in y ( the vertical axis ) . nodes along the left edge were not allowed to move in x ( the horizontal axis ) . depending on the goal of the experiment , either a constant edge load was placed on the top edge of the model or a constant displacement was applied to each node on the top of the model . each grain was modeled as a hexagon containing 48 six - noded triangular elements ( fig . 1 ) ; this type of element uses a parabolic interpolation function along each edge . the nodes on the boundaries of each grain are shared with the elements along the edge of the neighboring grain , thus grain boundaries are demarcated only by a change in elastic and plastic properties . as there can be no offset along a grain boundary in the model , the model consists of 2,574 hexagonal grains that have been assigned randomly into 25 subsets . the constitutive equation used for linear elastic solids is : where ij is the stress tensor , ij is the kronecker delta , ij is the strain tensor , is the lame constant , and g is the shear modulus . can be expressed as : and g can be expressed as : where v is the poisson s ratio and e is the young s modulus . plastic behavior initiates when the equivalent von mises stress ( vm ) , which is calculated as follows : equals the specified yield strength . post - yielding behavior was either perfectly plastic or included a work - hardening slope . the young s modulus and poisson s ratio assigned to each grain set for the models presented in the illustrations are given in table 1 . for all materials shown in the figures , the yield strength was set to 1/100th of the young s modulus and no work hardening was included . nodes along the bottom edge were not allowed to move in y ( the vertical axis ) . nodes along the left edge were not allowed to move in x ( the horizontal axis ) . depending on the goal of the experiment , either a constant edge load was placed on the top edge of the model or a constant displacement was applied to each node on the top of the model . how to cite this article : burnley , p. c. the importance of stress percolation patterns in rocks and other polycrystalline materials .

a new framework for thinking about the deformation behavior of rocks and other heterogeneous polycrystalline materials is proposed , based on understanding the patterns of stress transmission through these materials . here , using finite element models , i show that stress percolates through polycrystalline materials that have heterogeneous elastic and plastic properties of the same order as those found in rocks . the pattern of stress percolation is related to the degree of heterogeneity in and statistical distribution of the elastic and plastic properties of the constituent grains in the aggregate . the development of these stress patterns leads directly to shear localization , and their existence provides insight into the formation of rhythmic features such as compositional banding and foliation in rocks that are reacting or dissolving while being deformed . in addition , this framework provides a foundation for understanding and predicting the macroscopic rheology of polycrystalline materials based on single - crystal elastic and plastic mechanical properties .

Results Results of finite element models Discussion Methods Finite element models Author contributions Additional information

PMC2686512

future releases of tcsnp will integrate new t. cruzi sequences as they become available . many of these are expected to come from a resequencing effort that is underway in our lab . planned updates to the website functionality include the development of a standardized api and web services through which other databases can consume the information provided by tcsnp and the development of functionality to design primers by interfacing with primer3 ( 19 ) , while using the snp information in this process . the tcsnp database is composed of a web application written in perl , running against a postgresql database . the database schema is based on the genomics unified schema ( gus , http://gusdb.org ) with local customizations . the web application has been developed using a model - view - controller architectural pattern , where the work of each layer is performed by a specialized perl component [ all components are available from cpan ( 20 ) ] . access to the database from perl ( i.e. the model ) is done through a hybrid combination of ( i ) an abstraction of the database schema using perl 's dbix::class package and ( ii ) custom sql queries executed using perl 's database access interface ( dbi ) . the controller component managing user 's requests and dispatching calls to other components is perl 's catalyst ( 21 ) . a number of custom controller modules were developed , which contain the business logic of the tcsnp application . finally , the viewer component is perl 's template toolkit , which uses custom templates to render web pages using information provided by the controller . the database runs on dedicated freebsd servers , with the catalyst web application running under the apache web server . the reference genome sequence of the cl brener strain of t. cruzi was obtained from genbank using the umbrella accession number aahk00000000 ( july , 2005 ) . other t. cruzi sequences ( mrnas and ests ) were also obtained from genbank using custom entrez queries ( may , 2007 ) . before loading into the database this was necessary because of the variations in how different authors write the names of strains in genbank submissions and publications . cl brener strain appears in different genbank sequences as cl brener , cl - brener , clbrener , cl - brenner , cl brenner , cl brenner , or cl brener ( note the different capitalization , use of middle dash , spaces and the writing of the strain name using a single or a double n ) . because the database allows users to perform searches using strain names , it was important to reduce redundancy where possible . in some cases , however , it was important to keep the distinction between similar strain names , for example to discriminate between cloned stocks and their parental uncloned strains . as an example , we have kept caniii and caniii cl1 ( cl1 stands for clone 1 ) , and sc43 and sc43 cl1 as different redundancy is still present , however , and further curation of strain names can be done . we also encourage users of the database to send feedback about this issue . before clustering all sequences were masked against a library of vector sequences and t. cruzi repetitive elements , as described previously ( 22 ) . annotated coding sequences from the reference genome , and other publicly available sequences were mapped against the genome scaffolds using blat . sequences mapping to the same genomic regions were clustered together and multiple sequence alignments were obtained using phrap . this initial clustering allowed us to group mrnas , ests and reference coding sequences to the reference genome assembly sequences . but because allelic variants in the cl - brener genome were separated during assembly ( 12 ) , those initial clusters showed many instances of allelic variants separated into different alignments ( i.e. each mapping to its own contig ) . to obtain alignments between allelic variants , we merged alignments with highly similar consensus sequences ( by blast analysis ) . afterwards , and based on user feedback , we have also merged , splitted and re - analyzed many alignments . users of tcsnp should also be aware of the fact that many sequences from the cl - brener genome assembly are located in contigs with assembly problems or may represent assembly artifacts . for sequences containing assembly warnings in the original genbank records , we have attached similar notes to the corresponding alignments in tcsnp to help users in the interpretation of the snp data . multiple sequence alignments were scanned to identify polymorphic columns . to calculate the probability of these sites being true polymorphisms as opposed to sequencing errors , we have used the software package polybayes , version 5 ( 13 ) . polybayes uses a bayesian statistical framework that relies on allele frequency , alignment depth and base quality values amongst other attributes to calculate a probability score . because chromatogram trace data are not available for many of the sequences in this release , we have devised a scoring strategy that uses arbitrary base quality values . sequence bases obtained from the t. cruzi cl - brener genome ( 19x shotgun coverage ) were arbitrarily assigned a base quality value of 40 ; those from genbank records , a value of 30 ( individual submissions ) ; and those from dbest , a value of 20 ( single - pass , unedited ) . as an example , using this scoring scheme , a single base from an est differing from two allelic variants of cl - brener reference sequence ( depth = 3 ) would give a probability of 0.22 of being a true snp ( see for example : http://snps.tcruzi.org/snps/view/4028216 ) . to analyze the effect of each snp on the corresponding protein product , we noted the codon position of the snp in each reference coding sequence and evaluated the change introduced by the polymorphic base . also , for a subset of the alignments ( those containing coding sequences of similar length , with indels being a multiple of 3 ) , we calculated dn and ds values ( 14 ) using bioperl 's population genetics modules ( 23 ) . the tcsnp database is composed of a web application written in perl , running against a postgresql database . the database schema is based on the genomics unified schema ( gus , http://gusdb.org ) with local customizations . the web application has been developed using a model - view - controller architectural pattern , where the work of each layer is performed by a specialized perl component [ all components are available from cpan ( 20 ) ] . access to the database from perl ( i.e. the model ) is done through a hybrid combination of ( i ) an abstraction of the database schema using perl 's dbix::class package and ( ii ) custom sql queries executed using perl 's database access interface ( dbi ) . the controller component managing user 's requests and dispatching calls to other components is perl 's catalyst ( 21 ) . a number of custom controller modules were developed , which contain the business logic of the tcsnp application . finally , the viewer component is perl 's template toolkit , which uses custom templates to render web pages using information provided by the controller . the database runs on dedicated freebsd servers , with the catalyst web application running under the apache web server . the reference genome sequence of the cl brener strain of t. cruzi was obtained from genbank using the umbrella accession number aahk00000000 ( july , 2005 ) . other t. cruzi sequences ( mrnas and ests ) were also obtained from genbank using custom entrez queries ( may , 2007 ) . before loading into the database , this was necessary because of the variations in how different authors write the names of strains in genbank submissions and publications . cl brener strain appears in different genbank sequences as cl brener , cl - brener , clbrener , cl - brenner , cl brenner , cl brenner , or cl brener ( note the different capitalization , use of middle dash , spaces and the writing of the strain name using a single or a double n ) . because the database allows users to perform searches using strain names , it was important to reduce redundancy where possible . in some cases , however , it was important to keep the distinction between similar strain names , for example to discriminate between cloned stocks and their parental uncloned strains . as an example , we have kept caniii and caniii cl1 ( cl1 stands for clone 1 ) , and sc43 and sc43 cl1 as different redundancy is still present , however , and further curation of strain names can be done . before clustering all sequences were masked against a library of vector sequences and t. cruzi repetitive elements , as described previously ( 22 ) . annotated coding sequences from the reference genome , and other publicly available sequences were mapped against the genome scaffolds using blat . sequences mapping to the same genomic regions were clustered together and multiple sequence alignments were obtained using phrap . this initial clustering allowed us to group mrnas , ests and reference coding sequences to the reference genome assembly sequences . but because allelic variants in the cl - brener genome were separated during assembly ( 12 ) , those initial clusters showed many instances of allelic variants separated into different alignments ( i.e. each mapping to its own contig ) . to obtain alignments between allelic variants , we merged alignments with highly similar consensus sequences ( by blast analysis ) . afterwards , and based on user feedback , we have also merged , splitted and re - analyzed many alignments . users of tcsnp should also be aware of the fact that many sequences from the cl - brener genome assembly are located in contigs with assembly problems or may represent assembly artifacts . for sequences containing assembly warnings in the original genbank records , we have attached similar notes to the corresponding alignments in tcsnp to help users in the interpretation of the snp data . multiple sequence alignments were scanned to identify polymorphic columns . to calculate the probability of these sites being true polymorphisms as opposed to sequencing errors , we have used the software package polybayes , version 5 ( 13 ) . polybayes uses a bayesian statistical framework that relies on allele frequency , alignment depth and base quality values amongst other attributes to calculate a probability score . because chromatogram trace data are not available for many of the sequences in this release , we have devised a scoring strategy that uses arbitrary base quality values . sequence bases obtained from the t. cruzi cl - brener genome ( 19x shotgun coverage ) were arbitrarily assigned a base quality value of 40 ; those from genbank records , a value of 30 ( individual submissions ) ; and those from dbest , a value of 20 ( single - pass , unedited ) . as an example , using this scoring scheme , a single base from an est differing from two allelic variants of cl - brener reference sequence ( depth = 3 ) would give a probability of 0.22 of being a true snp ( see for example : http://snps.tcruzi.org/snps/view/4028216 ) . to analyze the effect of each snp on the corresponding protein product , we noted the codon position of the snp in each reference coding sequence and evaluated the change introduced by the polymorphic base . also , for a subset of the alignments ( those containing coding sequences of similar length , with indels being a multiple of 3 ) , we calculated dn and ds values ( 14 ) using bioperl 's population genetics modules ( 23 ) . national agency for the promotion of science and technology ( anpcyt , argentina ) ( grant pict 38209 ) ; and the university of san martn ( grant s05/22 ) . funding for open access charge : anpcyt .

the tcsnp database ( http://snps.tcruzi.org ) integrates information on genetic variation ( polymorphisms and mutations ) for different stocks , strains and isolates of trypanosoma cruzi , the causative agent of chagas disease . the database incorporates sequences ( genes from the t. cruzi reference genome , mrnas , ests and genomic sequences ) ; multiple sequence alignments obtained from these sequences ; and single - nucleotide polymorphisms and small indels identified by scanning these multiple sequence alignments . information in tcsnp can be readily interrogated to arrive at gene sets , or snp sets of interest based on a number of attributes . sequence similarity searches using blast are also supported . this first release of tcsnp contains nearly 170 000 high - confidence candidate snps , derived from the analysis of annotated coding sequences . as new sequence data become available , tcsnp will incorporate these data , mapping new candidate snps onto the reference genome sequences .

FUTURE WORK METHODS Database and web application Data sources Sequence clustering and alignment Candidate SNP identification and analysis FUNDING

PMC4399525

posterior teeth , particularly maxillary premolars , have an anatomic shape that makes them more likely to fracture the cusps under occlusal load14 . additionally , these teeth when treated endodontically can be easily fractured because of pulp chamber roof removal19,20 , mainly when the marginal ridge is thin or totally removed28 . several studies have emphasized the importance of maintaining dental structure to preserve the strength of remaining tooth . generally , the wider the involvement by caries or cavity preparation , the weaker the tooth19,20 . other authors have suggested an alternative cusp coverage with amalgam to restore the weakened teeth , with satisfactory long - term results20 . this alternative therapy is economically more accessible , easy to perform and has no cement line as conventional cast restorations , which are more costly and time consuming . it has been stated that remaining tooth structure restored with adhesive technology presents higher fracture resistance29 . however , the hypothesis that direct cusp coverage is still necessary even when adhesive procedures are used in large cavities must be confirmed . this study compared the fracture resistance of weakened human premolars restored with direct condensable resin composite with and without cusp coverage . the freshly extracted teeth were cleaned , stored in tymol solution at 0.1% and used within 1 month after extraction . every tooth was examined under a 10x magnification and those presenting visible enamel cracks or fractures were rejected . the selected teeth were embedded in cold - cure plastic resin in metal rings1 , with the resin limit at 1.0 mm below the cementoenamel junction . the specimens were divided into three groups with 10 teeth each : group a ( control ) included sound teeth , group b included restored teeth without cusp reduction and group c had teeth restored with cusp coverage . cavity preparation was initiated by occlusal approach with a spherical diamond bur towards the pulp chamber . removal of the pulp chamber roof and reduction of the mesial and distal walls were done with a cylindrical diamond bur , creating a 4-mm - deep slit cavity design . the buccolingual isthmus was approximately half the intercuspal distance , as well as the mesial and distal boxes . teeth of group c received a further 2.0-mm - high reduction of both buccal and palatal cusps ( figure 1 ) . in those teeth , before cavity preparation and cusp reduction , a silicone matrix was prepared by taking an impression of the original cusp height and inclined planes . the matrix was sectioned into mesial and distal parts and used as a guide to facilitate posterior restoration with resin to the original shape ( figure 2 ) . the floor of the exposed pulp chambers received a layer of glass ionomer cement ( vitrebond , 3 m espe ) . in group b , the cavity was etched with 37% phosphoric acid for 30 seconds enamel and 15 seconds dentin , rinsed , and dried ( moist technique ) with an absorbing paper . the adhesive ( prime & bond nt , dentsply ) was applied and light - cured by 20s . circumferential metal matrix was adapted to the cervical margins with low fusion impression material ( aquasil , dentsply ) . surefil resin composite ( surefil , dentsply ) was inserted in 2.0 mm thick , oblique increments and light - cured for 40 s each at 600 mw / cm ( gnatus , ribeiro preto , so paulo , brazil ) . in group c , tooth restoration was performed in the same way as in group b , with the aid of the matrix to reconstruct the cusp height and slopes ( figure 3 ) . after 24 hours , all surfaces of the restorations were polished with rubber points and were stored again in distilled water at 37c until testing . after 48 hours of storage , the specimens were mounted in an universal testing machine ( emic dl500 ; so jos dos pinhais , pr , brazil ) and subjected to an axial compression load applied parallel to the long axis of the tooth and to the slopes of the cusps by means of a round - end steel device ( 8.0 mm in diameter ) running at a crosshead speed of 0.5mm / minute ( figure 4 ) . a flame - shaped bur was used to create small contact points on the buccal and lingual cusps for preventing lateral deflection of the sphere . the load required to cause fracture of the specimens the results were analyzed by oneway anova and tukey 's test at 5% significance level . the freshly extracted teeth were cleaned , stored in tymol solution at 0.1% and used within 1 month after extraction . every tooth was examined under a 10x magnification and those presenting visible enamel cracks or fractures were rejected . the selected teeth were embedded in cold - cure plastic resin in metal rings1 , with the resin limit at 1.0 mm below the cementoenamel junction . the specimens were divided into three groups with 10 teeth each : group a ( control ) included sound teeth , group b included restored teeth without cusp reduction and group c had teeth restored with cusp coverage . cavity preparation was initiated by occlusal approach with a spherical diamond bur towards the pulp chamber . removal of the pulp chamber roof and reduction of the mesial and distal walls were done with a cylindrical diamond bur , creating a 4-mm - deep slit cavity design . the buccolingual isthmus was approximately half the intercuspal distance , as well as the mesial and distal boxes . teeth of group c received a further 2.0-mm - high reduction of both buccal and palatal cusps ( figure 1 ) . in those teeth , before cavity preparation and cusp reduction , a silicone matrix was prepared by taking an impression of the original cusp height and inclined planes . the matrix was sectioned into mesial and distal parts and used as a guide to facilitate posterior restoration with resin to the original shape ( figure 2 ) . the floor of the exposed pulp chambers received a layer of glass ionomer cement ( vitrebond , 3 m espe ) . in group b , the cavity was etched with 37% phosphoric acid for 30 seconds enamel and 15 seconds dentin , rinsed , and dried ( moist technique ) with an absorbing paper . the adhesive ( prime & bond nt , dentsply ) was applied and light - cured by 20s . circumferential metal matrix was adapted to the cervical margins with low fusion impression material ( aquasil , dentsply ) . surefil resin composite ( surefil , dentsply ) was inserted in 2.0 mm thick , oblique increments and light - cured for 40 s each at 600 mw / cm ( gnatus , ribeiro preto , so paulo , brazil ) . in group c , tooth restoration was performed in the same way as in group b , with the aid of the matrix to reconstruct the cusp height and slopes ( figure 3 ) . after 24 hours , all surfaces of the restorations were polished with rubber points and were stored again in distilled water at 37c until testing . after 48 hours of storage , the specimens were mounted in an universal testing machine ( emic dl500 ; so jos dos pinhais , pr , brazil ) and subjected to an axial compression load applied parallel to the long axis of the tooth and to the slopes of the cusps by means of a round - end steel device ( 8.0 mm in diameter ) running at a crosshead speed of 0.5mm / minute ( figure 4 ) . a flame - shaped bur was used to create small contact points on the buccal and lingual cusps for preventing lateral deflection of the sphere . the load required to cause fracture of the specimens the results were analyzed by oneway anova and tukey 's test at 5% significance level . teeth restored with condensable resin composite without cusp coverage presented a significant decrease in strength as compared to sound teeth . restorations with cusp coverage recovered the strength of the teeth to values similar to the sound teeth ( p<0.05 ) . different letters indicate statistically significant difference all teeth of group c showed fractures that occurred only within condensable resin , without fracture of the remaining structure . teeth of group b presented cusp fracture mostly at cusps base level , starting in the adhesive interface towards the apical third . the natural and drastic consequence of dental weakness is cusp fracture , and the study of this pathology is relevant because it is considered a common occurrence in clinics3 - 5,7,15 . some authors have investigated the incidence of these dental fractures in oral cavity and found that is more concentrated in upper premolars5,7,15 . sedgley and messer26 studied the biomechanical properties of non - vital teeth in tests of tenacity , microhardness and shear and fracture resistance . they concluded that these properties do not change , suggesting that cumulative loss of dental structure by caries , trauma , restorative and endodontic procedures lead susceptibility to fracture . it has been suggested that cusp elongation due to cavity preparation may be the major factor in fracture susceptibility , mainly in endodontically treated upper premolars whose anatomy tends to separate the buccal and palatal cusps under occlusal load7,19 . some authors have emphasized that endodontically treated premolars with class ii mod cavity designs have a drastic decrease in fracture resistance . after receiving an indirect metallic restoration with cusp protection , these teeth placement of amalgam restorations in weakened premolars with cusp coverage significantly increased the fracture resistance of the teeth ( 63% ) as compared to teeth restored without cusp coverage20 . although metallic restorations with cusp coverage are a reference in the rehabilitation of weakened teeth and cracked tooth syndrome10 . some authors12,13,16,23,24,32 have confirmed that resin composite could be an viable alternative to amalgam , with better results in posterior endodontically treated teeth with mod preparations2 . however , in larger cavities , cusp reduction and posterior restoration with direct or indirect procedures seems to be a more secure option8 , 25 . although cusp reduction promotes more dental tissue reduction27 , this procedure leads the restoration margins to buccal and palatal surfaces , protecting the adhesive interface from early marginal discrepancies30 . in a previous finite element analysis , it was stated that stress value in the restorative material and remaining tooth structure was mainly influenced by the restorative material itself ( 95.49% ) and cavity design ( > 80% ) . when cuspal - coverage treatment is considered , the cuspal height should be reduced in at least 1,5 mm to significant reduce the stress values17 . the present study confirm these results showing that 2.0-mm cusp reduction and posterior restoration with direct resin composite can restore the fracture resistance of weakened teeth . these results seems to be also confirmed clinically6 . as seen in table 1 , the results of the present study do not agree with those of previous investigations2,9,13,18,21,23 that found fracture resistance of weakened teeth restored with resin composite without cusp coverage been similar of sound teeth . this probably occurred because the cavity size used in the present study was larger than those of other studies . the anchorage promoted by resin composite , protecting the buccal and lingual cusps of group c specimens , recovered the fracture resistance in similar values of sound teeth . the adhesive procedure was clearly not the only responsible for this resistance , but also cusp protection that avoided the separation in consequence of the wedge effect caused by cusp elongation14,19,20 . no cusp fracture was seen in group c. however , group b presented some fractures indicating that only the adhesive interface may be insufficient to avoid these fractures . despite failures mentioned by different authors , resin composites improved and are now indicated for posterior teeth as an alternative to amalgam , especially condensable resins , which have higher wear resistance31 . the results of the present study showed that cusp coverage with condensable resin might be an option for restoring weakened endodontically treated teeth because cusp coverage resulted in similar fracture resistance to that sound teeth . cusp coverage with condensable resin might be a safe option for restoring weakened endodontically treated teeth .

objectives : this in vitro study evaluated the fracture resistance of weakened human premolars ( mod cavity preparation and pulp chamber roof removal ) restored with condensable resin composite with and without cusp coverage.material and methods : thirty human maxillary premolars were divided into three groups : group a ( control ) , sound teeth ; group b , wide mod cavities prepared and the pulp chamber roof removed and restored with resin composite without cusp coverage ; group c , same as group b with 2.0 mm of buccal and palatal cusps reduced and restored with the same resin . the teeth were included in metal rings with self - curing acrylic resin , stored in water for 24 h and thereafter subjected to a compressive axial load in a universal testing machine at 0.5 mm / min.results : the mean fracture resistance values standart deviation ( kgf ) were : group a : 151.40 55.32 , group b : 60.54 12.61 , group c : 141.90 30.82 . statistically significant differences were found only between group b and the other groups ( p<0.05 ) . the condensable resin restoration of weakened human premolars with cusp coverage significantly increased the fracture resistance of the teeth as compared to teeth restored without cusp coverage.conclusion:the results showed that cusp coverage with condensable resin might be a safe option for restoring weakened endodontically treated teeth .

INTRODUCTION MATERIAL AND METHODS Tooth Selection Tooth Preparation and Restoration Testing Procedures RESULTS DISCUSSION CONCLUSIONS

PMC4882474

in recent decades participation in long - distance endurance exercise events ( e.g. , ironman triathlons , ultra - marathons , and ultra - distance cycling events ) , has increased substantially among recreational exercisers and competitive athletes ( 7 , 17 , 34 ) . many of these events occur in extreme environmental conditions including high altitude or low or high ambient temperature . such extreme environmental conditions provide substantial additional physiological stress beyond that induced by the long - distance exercise alone and thus can provide additional perturbations to homeostasis . high intensity endurance exercise and environmental stress independently can affect immune system function , but the combination of multiple stressors likely has a more drastic effect on the immune system ( 23 ) . long - distance exercise alone affects populations of circulating immune cells that function in non - specific immunity . marathon ( 42.195 km ) runners exhibit increases in total leukocytes ( ~4 fold ) , monocytes ( ~2.5 fold ) , neutrophils ( ~5.5 fold ) ( 8 , 14 ) , with only a minor increase ( ~1.25 fold ) ( 8) in lymphocytes from before to immediately after the event . ultramarathon ( 160 km ) runners display a similar increase in total leukocytes and monocyte / neutrophil subsets , but exhibit a less dramatic increase in circulating lymphocytes compared to traditional marathon runners ( 27 , 37 ) . similar results of a 12 fold increase in leukocytes , monocytes , neutrophils , has also been observed for 75-km cycling events ( 19 , 27 , 46 ) . such long - distance exercise events impose a substantial mechanical and systemic physiological demand on the body , even in thermoneutral environments ( 46 ) . it is likely that micro - trauma to musculature and vascular system , leakage of endotoxins from the gut to the circulation , and glycogen depletion ( 13 , 20 , 39 , 40 , 43 ) occur during ultra - endurance exercise and can instigate activation , differentiation , and demargination of leukocytes and greater systemic inflammation ( 13 ) . hot and humid environmental conditions magnify the effect of exercise that occurs in thermoneutral environments . studies of shorter duration running and cycling during hot and cold water immersion ( 40 min ) ( 6 , 33 ) and in temperature controlled laboratory settings ( 40 min to 4 h ) ( 3 , 16 , 21 , 23 , 38 ) demonstrate that exercise in a hot environment ( 3539c ) elicits greater increases in total leukocytes , neutrophils , and lymphocytes than exercising in a cold to moderate temperature environment ( 1823c ) ( 6 , 21 , 33 ) . one study emphasized the inefficacy of a thermal clamp ( 16 ) in reducing the exercise - induced changes in circulating leukocyte populations . however , it is interesting to note that the exercise protocol in this study did not elicit core body temperature increases above and beyond the passive heating control in which participants did not exercise . it is likely that the core temperature increase associated with high intensity exercise alone has a role in instigating immune responses , in addition to the damage associated with long duration , eccentric exercise . despite the growing popularity of recreational long - distance exercise events no field or controlled laboratory study appears to have investigated the nonspecific immune response to such prolonged endurance events ( > 4 h ) in a thermally stressful hot environment . long - distance exercise in moderate temperature conditions induces an increase in leukocytes and in laboratory settings a hot environment augments the leukocyte response to shorter - distance exercise . therefore , the purpose of this observational study was to determine the leukocyte subset response to completing a recreational 164-km road cycle event in an extremely hot and humid environment . exercise duration and relative exercise intensity ( 42 ) likely have significant effects on the immune response , independent of the extreme environmental conditions . thus , a secondary purpose of this study was to examine the associations among the time to complete the event , relative intensity ( % vo2max ) and changes in leukocyte subsets . in order to determine the leukocyte subsets response to completing a recreational 164-km road cycle event in a hot ambient temperature we recruited adults registered for participating in the 2013 hottern hell hundred cycling event ( hhh ) . the procedures of the study were reviewed and approved by the university institutional review board and undertaken in compliance with the helsinki declaration . on the day of the hhh , participants provided a blood sample in the morning 12 hours prior to the start of the event ( pre ) and immediately upon completion of the event ( post ) . blood samples were analyzed for total leukocyte as well as leukocyte subsets : neutrophils , monocytes , and lymphocytes . in order to describe the immune response that recreational riders experience when completing the hhh , we aimed to minimize any effect of study involvement on participant behavior before and during the event . therefore , no instructions or restrictions were provided with regard to ride strategies ( e.g. , hydration / diet intake , number rest stops , pace of the ride , or length of the rest stops ) . twenty - eight men and four women volunteered to participate and were enrolled in this study . participants demographic information and ride characteristics ( e.g. , pace ) are presented in table 1 and 2 , respectively . core temperature was not measured in the present study but based on findings from a prior year s hhh event ( same course and similar populations , environmental conditions ) it was expected to rise approximately 1.6 c from before to after the ride ( 1 ) . participants were recruited through the hhh official event registration website , through invitation emails sent to all hhh entrants , and at the official hhh onsite event registration center ( wichita falls , tx ) . upon review of the medical history , and with follow - up questions as needed , all participants were approved for participation by the on - site medical doctor . to be included in the study , volunteers were required to 1 ) be apparently healthy , 2 ) be 1862 years of age , 3 ) have previously completed a 160-km cycle ride , and 4 ) intent to complete the hhh ride within 9 hours . participants were excluded if they met any of the following criteria : 1 ) tobacco product user ; 2 ) taking cholesterol - lowering , blood pressure , or anticoagulant medications ; 3 ) healing from a musculoskeletal injury ; 4 ) liver , kidney , cardiovacular , gastrointestinal or blood disease or severe metabolic or endocrine disorders ( e.g. , diabetes ) ; 5 ) past history of exertional heatstroke or exercise - heat inotlerance ; 6 ) use of anabolic hormonal substances . a detailed description of the anthropometrics measurements used in this study has previously been published ( 15 ) . briefly , anthropometric measurements were obtained on one of the two days preceding the event . body weight and height was measured using a floor scale and a tape measure attached to the wall , respectively . percent body fat was estimated using three - site ( men : chest , abdomen , thigh ; women : triceps , suprailiac , thigh ) skinfold thickness equations ( 18 ) using calibrated skinfold calibers ( bodycare harpenden caliper , england ) . the hhh is among the largest recreational road cycle events ( > 10,000 participant annually ) in the usa and held every late - august in wichita falls , tx . the 164-km course covers largely flat terrain ( 15 ) and is usually held under hot and humid conditions ( average wichita falls environmental conditions for late - august is 35c and humidity is 59% , with little to no cloud cover ) . on event day , dry bulb temperature , relative humidity , and wet bulb globe temperature were recorded at the start / finish line ( 7:31am to 3:24pm ) and at the 97-km aid station ( 8:17am to 12:32pm ) using the kestrel meter 4400 heat meter ( nielson - kellerman co. , boothwyn , pa , usa ) . the ride began at 0700h and depending on starting position a particular rider might not pass the start line until as late as 0800h . participants without a computer - downloadable heart rate monitoring device were provided one to wear during the ride ( polar electro inc . , lake success , ny ) ; participants having their own computer - downloadable heart rate monitoring device were requested to provide heart rate data files via electronic mail within 7d after the event . participant total ride time was determined using either the participant s own timer or a timer provided by the research team and was confirmed using official hhh ride electronic chip monitoring system . participants were instructed to record the number of stops and the duration of each stop . the rolling time was calculated as the difference between the total event time and the resting time . heart rate was collected by standard telemetry using either participants own device or one provided by the investigators . whole blood samples and urine sample were obtained 12 hours prior to the start of the ride ( pre ) and immediately after ( post ) completion of the event without fasting . upon arrival , participants were weighted and then sat for a few minutes after which 30ml of blood were collected from an antecubital vein using venipuncture by a 21 gauge needle with an evacuated tube containing edta , while participants remained in a seated position . after blood collection , participants provided a small urine sample for determination of urine specific gravity . participants were instructed to be arrive for the pre time point before performing their event warm - up . the edta - treated whole blood samples were refrigerated and stored at 4c until biochemical analyses on the following morning . participants were instructed to remember the food consumption during the ride and if possible , kept the food labels . upon arrival at the medical tent after the ride , participants recorded the quantity and the type of food that were consumed or participants provided the food labels to investigators for them to record . prior to analysis , blood samples were diluted with an equal volume of phosphate buffered saline and results were adjusted to account for this dilution . then complete blood count was measured in duplicate using a 20-l sample of diluted edta - whole blood loaded into an automatic hematology analyzer ( bc-3200 ; mindray , mahwah , nj ) . in this automated procedure , total ( total leukocytes ) wbc and three - part differentiation ( lymphocytes , monocytes , and neutrophils ) of wbc are measured using electrical impedance . the analyzer has a precision of 2.5% and a linear range from 0.3~99.9 10cellsl for wbc . the complete blood count analysis also included determination of hemoglobin ( hb ) concentration ( using a cyanide free method ) and hematocrit ( hct ) . plasma volume change ( pv ) was determined by the change in hct and hb from pre to post using established methods ( 9 ) ( pv : 7.1 12.6% ) . the post immune cell ( total leukocytes , lymphocytes , monocytes , and neutrophils ) concentration was adjusted according to individual pv . the percent of body mass loss ( % bml ) was the fluid loss as percent of pre - body mass , and it was corrected for the weight of food consumed during the ride . estimated relative exercise intensity during the ride was calculated through the estimation of % vo2max from exercise heart rate using an equation which was validated by franklin et al . ( 11 ) ( i.e. % vo2max = 1.33 x ( % hrmax ) - 37.3 ) . the percent of maximum heart rate ( % hrmax ) was calculated using the maximum heart rate ( hrmax ) and the average heart rate ( hravg ) recorded during the cycling event . although it is not possible to determine if the recorded hrmax represents the true hrmax , the recorded hrmax ( 167 10 bpm ) was similar to but slightly less than the age predicted hrmax ( i.e. 208 ( 0.7 x age ) ; 174 6 bpm ) ( 44 ) . data were analyzed using spss version 20.0 ( ibm , chicago , il ) . tests for normality of distribution and homogeneity of variance were performed for all data sets . data sets that did not meet assumptions of linear statistics were log10 transformed and retested ; after transformation all data sets met these assumptions . paired t - tests were used to evaluate the mean pre and post differences in total leukocytes , and each leucocyte subset ( neutrophils , monocytes , and lymphocytes ) concentrations . to examine the effect intensity and duration on total leukocytes and each leucocyte subset , first , pearson s product - moment correlation coefficients between pre to post changes and ride characteristics ( estimated relative exercise intensity and total rolling time ) were calculated . then data for the fastest and slowest quartiles were examined using 2 ( time point : pre vs post ) 2 ( rolling time : fastest vs slowest quartile ) anovas with repeated measures on time point . in order to determine the leukocyte subsets response to completing a recreational 164-km road cycle event in a hot ambient temperature we recruited adults registered for participating in the 2013 hottern hell hundred cycling event ( hhh ) . the procedures of the study were reviewed and approved by the university institutional review board and undertaken in compliance with the helsinki declaration . on the day of the hhh , participants provided a blood sample in the morning 12 hours prior to the start of the event ( pre ) and immediately upon completion of the event ( post ) . blood samples were analyzed for total leukocyte as well as leukocyte subsets : neutrophils , monocytes , and lymphocytes . in order to describe the immune response that recreational riders experience when completing the hhh , we aimed to minimize any effect of study involvement on participant behavior before and during the event . therefore , no instructions or restrictions were provided with regard to ride strategies ( e.g. , hydration / diet intake , number rest stops , pace of the ride , or length of the rest stops ) . twenty - eight men and four women volunteered to participate and were enrolled in this study . participants demographic information and ride characteristics ( e.g. , pace ) are presented in table 1 and 2 , respectively . core temperature was not measured in the present study but based on findings from a prior year s hhh event ( same course and similar populations , environmental conditions ) it was expected to rise approximately 1.6 c from before to after the ride ( 1 ) . participants were recruited through the hhh official event registration website , through invitation emails sent to all hhh entrants , and at the official hhh onsite event registration center ( wichita falls , tx ) . upon review of the medical history , and with follow - up questions as needed , all participants were approved for participation by the on - site medical doctor . to be included in the study , volunteers were required to 1 ) be apparently healthy , 2 ) be 1862 years of age , 3 ) have previously completed a 160-km cycle ride , and 4 ) intent to complete the hhh ride within 9 hours . participants were excluded if they met any of the following criteria : 1 ) tobacco product user ; 2 ) taking cholesterol - lowering , blood pressure , or anticoagulant medications ; 3 ) healing from a musculoskeletal injury ; 4 ) liver , kidney , cardiovacular , gastrointestinal or blood disease or severe metabolic or endocrine disorders ( e.g. , diabetes ) ; 5 ) past history of exertional heatstroke or exercise - heat inotlerance ; 6 ) use of anabolic hormonal substances . a detailed description of the anthropometrics measurements used in this study has previously been published ( 15 ) . briefly , anthropometric measurements were obtained on one of the two days preceding the event . body weight and height was measured using a floor scale and a tape measure attached to the wall , respectively . percent body fat was estimated using three - site ( men : chest , abdomen , thigh ; women : triceps , suprailiac , thigh ) skinfold thickness equations ( 18 ) using calibrated skinfold calibers ( bodycare harpenden caliper , england ) . the hhh is among the largest recreational road cycle events ( > 10,000 participant annually ) in the usa and held every late - august in wichita falls , tx . the 164-km course covers largely flat terrain ( 15 ) and is usually held under hot and humid conditions ( average wichita falls environmental conditions for late - august is 35c and humidity is 59% , with little to no cloud cover ) . on event day , dry bulb temperature , relative humidity , and wet bulb globe temperature were recorded at the start / finish line ( 7:31am to 3:24pm ) and at the 97-km aid station ( 8:17am to 12:32pm ) using the kestrel meter 4400 heat meter ( nielson - kellerman co. , boothwyn , pa , usa ) . the ride began at 0700h and depending on starting position a particular rider might not pass the start line until as late as 0800h . participants without a computer - downloadable heart rate monitoring device were provided one to wear during the ride ( polar electro inc . , lake success , ny ) ; participants having their own computer - downloadable heart rate monitoring device were requested to provide heart rate data files via electronic mail within 7d after the event . participant total ride time was determined using either the participant s own timer or a timer provided by the research team and was confirmed using official hhh ride electronic chip monitoring system . participants were instructed to record the number of stops and the duration of each stop . the rolling time was calculated as the difference between the total event time and the resting time . heart rate was collected by standard telemetry using either participants own device or one provided by the investigators . whole blood samples and urine sample were obtained 12 hours prior to the start of the ride ( pre ) and immediately after ( post ) completion of the event without fasting . upon arrival , participants were weighted and then sat for a few minutes after which 30ml of blood were collected from an antecubital vein using venipuncture by a 21 gauge needle with an evacuated tube containing edta , while participants remained in a seated position . after blood collection , participants provided a small urine sample for determination of urine specific gravity . participants were instructed to be arrive for the pre time point before performing their event warm - up . the edta - treated whole blood samples were refrigerated and stored at 4c until biochemical analyses on the following morning . participants were instructed to remember the food consumption during the ride and if possible , kept the food labels . upon arrival at the medical tent after the ride , participants recorded the quantity and the type of food that were consumed or participants provided the food labels to investigators for them to record . prior to analysis , blood samples were diluted with an equal volume of phosphate buffered saline and results were adjusted to account for this dilution . then complete blood count was measured in duplicate using a 20-l sample of diluted edta - whole blood loaded into an automatic hematology analyzer ( bc-3200 ; mindray , mahwah , nj ) . in this automated procedure , total ( total leukocytes ) wbc and three - part differentiation ( lymphocytes , monocytes , and neutrophils ) of wbc are measured using electrical impedance . the analyzer has a precision of 2.5% and a linear range from 0.3~99.9 10cellsl for wbc . the complete blood count analysis also included determination of hemoglobin ( hb ) concentration ( using a cyanide free method ) and hematocrit ( hct ) . plasma volume change ( pv ) was determined by the change in hct and hb from pre to post using established methods ( 9 ) ( pv : 7.1 12.6% ) . the post immune cell ( total leukocytes , lymphocytes , monocytes , and neutrophils ) concentration was adjusted according to individual pv . the percent of body mass loss ( % bml ) was the fluid loss as percent of pre - body mass , and it was corrected for the weight of food consumed during the ride . estimated relative exercise intensity during the ride was calculated through the estimation of % vo2max from exercise heart rate using an equation which was validated by franklin et al . ( 11 ) ( i.e. % vo2max = 1.33 x ( % hrmax ) - 37.3 ) . the percent of maximum heart rate ( % hrmax ) was calculated using the maximum heart rate ( hrmax ) and the average heart rate ( hravg ) recorded during the cycling event . although it is not possible to determine if the recorded hrmax represents the true hrmax , the recorded hrmax ( 167 10 bpm ) was similar to but slightly less than the age predicted hrmax ( i.e. 208 ( 0.7 x age ) ; 174 6 bpm ) ( 44 ) . tests for normality of distribution and homogeneity of variance were performed for all data sets . data sets that did not meet assumptions of linear statistics were log10 transformed and retested ; after transformation all data sets met these assumptions . paired t - tests were used to evaluate the mean pre and post differences in total leukocytes , and each leucocyte subset ( neutrophils , monocytes , and lymphocytes ) concentrations . to examine the effect intensity and duration on total leukocytes and each leucocyte subset , two different analyses were utilized . first , pearson s product - moment correlation coefficients between pre to post changes and ride characteristics ( estimated relative exercise intensity and total rolling time ) were calculated . then data for the fastest and slowest quartiles were examined using 2 ( time point : pre vs post ) 2 ( rolling time : fastest vs slowest quartile ) anovas with repeated measures on time point . the concentrations of total leukocytes and of each leukocyte subset increased significantly from pre to post . the data for leukocyte subset is presented in table 4 and individual responses are presented in figures 14 . both leukocytes and neutrophils increased in concentration uniformly , while monocytes and lymphocytes did not show this pattern . thirteen of 32 participants had a reduction from pre to post in monocyte concentration ( range : 0.05 to 0.88 10l ) and eight of 32 showed a reduction from pre to post in lymphocyte concentration ( range : 0.11 to 0.83 10l ) . the cause of these findings is unclear but could represent naturally occurring individual differences or be due to differences in training state , heat acclimation , and/or immune function . no significant relationship was observed between changes in total leukocytes or leukocyte subset concentrations from pre to post and ride characteristics ( estimated relative exercise intensity and rolling time ) . circulating total leukocytes and leukocyte subset concentrations were not different between the fastest and slowest quartile of riders . the primary purpose of this study was to determine the circulating leukocyte response to completing a recreational 164-km road cycle event in a hot ambient temperature . our data indicated that leukocytes ( 134% ) and the each leukocyte subset ( neutrophils : 319% ; monocytes : 24% ; lymphocytes : 53% ) increased from pre to post , but these increases were not affected by estimated relative exercise intensity . considering the growing popularity of long - distance cycling , this is important new knowledge since this substantial immune response might affect the acute and long - term health of recreational cyclist who regularly perform long - distance cycling in the heat . better understanding of the acute physiological effects of such exercise is critical to defining proper recovery and monitoring health following rides and over the course of training . to our knowledge these results are the first to describe the non - specific immune response to a completion of a self - paced recreational long - distance road cycling in a hot ( > 30 c ) environment . overall , this study provides unique physiological insight by showing that completing a 164-km cycling event ( 58h ) without instructions or restrictions with regard to pace or food and fluid intake during the exercise induces substantial leukocytosis . these findings adds to the previous findings of increases in leukocyte subsets concentrations with prolonged endurance exercise in more moderate temperature conditions : including standard marathon running ( 8 , 14 , 46 ) ; 160-km running ( 27 ) ; 24h cycling ( 28 , 35 ) ; and 3h treadmill running ( 26 ) . long - distance endurance exercise induces leukocytosis ( 8 , 14 , 19 , 2628 , 35 , 37 ) . although all circulating leukocyte subsets increase with exercise , the largest increase is observed in neutrophils . neutrophils are involved in phagocytosis of damaged tissue and thus are important to recovery from exercise . despite of its importance to the host defense , neutrophils release reactive oxygen species , which further the process of muscle damage following exercise and contribute to inflammation ( 29 ) . the exercise - induced increase in neutrophils has been suggested to be the result of demargination of cells from endothelial tissues and bone marrow to the circulation , a response which is mediated by catecholamine and cortisol , respectively ( 30 ) . the absolute resting ( pre ) circulating of total leukocytes and each leukocyte subset observed in the present study were all within the normal reference range for the general population ( 2 ) and for elite , albeit younger , cycle athletes ( 12 ) ; however , the absolute concentration of neutrophil was near the low end of the normal range . this low - end pre neutrophil concentration likely explain the low relative proportion of neutrophils ( 33% ) and the high relative proportion of lymphocytes ( 60% ) among total circulating leukocytes . it is uncertain why the neutrophil proportion was low in the study population ; however , it has been suggested that that plasma volume expansion , which is commonly observed among trained endurance athletes ( 5 ) , might cause a selective reduction in neutrophil concentration but not in lymphocytes ( 12 ) . following 164-km of cycling in the heat , post neutrophil concentrations were greater than 4-fold those observed for pre . the magnitude of the increase in neutrophils is similar to the increase found for 100-km ( 31 ) and 160-km ( 27 ) running in a moderate ( ~23c ) ambient temperature although these events were likely performed at a lower relative intensity ( 10 ) since running induces greater muscle damage than cycling at the same relative intensity ( 41 ) , the similar neutrophil response suggests that a hot environment provides added stress to the immune system during exercise . this is supported by the previous finding that cycle exercise induced a greater increase in neutrophils during shorter - term heat exposure ( 40 min submersion in 39c water ) than cold exposure ( 1823c water ) ( 6 , 33 ) . a potential mechanism underlying this augmented neutrophil response when exercising in the heat exercise in the heat induces a greater increase in the concentration of cortisol ( 2 fold ) , epinephrine ( 4 fold ) , and norepinephrine ( 23 fold ) compared to exercise in the cold ( 6 , 33 , 38 ) . cortisol and catecholamine interact with glucocorticoid receptors ( 22 ) and 2-andrenergic receptors ( 24 ) of neutrophils , respectively , and influence the development , trafficking , and function of immunocytes ( 25 , 36 , 47 ) . monocytes with inflammatory profiles migrate to damaged tissue and mature into macrophages after entering that tissue . similar to neutrophils , macrophages are involved in phagocytosis of damaged tissue and thus facilitate muscle remodeling ( 45 ) , but macrophages also serve an important role as a link between the non - specific and specific response by releasing cytokines into the circulation to attract other immune cells . the increase in monocytes ( 24% ) in the present study is similar to that observed for 4 hours of ergometer cycling at room temperature ( 29%)(35 ) and for 40 min water immersed cycling at 39c ( 15% ) and 18c ( 20%)(33 ) . the similarity in these monocyte responses to exercise suggests that , unlike neutrophils and lymphocytes , the monocytes response is ambient temperature independent during cycle exercise . in contrast to the moderate increase in monocytes found with cycling , long distance running across a range of relative intensities , including intensities similar to that of the current study , induces a substantial increase in monocytes : marathon ( 150% ) ( 8) , 3 hour treadmill ( 67% ) ( 26 ) , 100 km ( 148% ) ( 31 ) , and 160 km ( 213% ) ( 27 ) running in moderate temperature ; no study appears to have investigated the monocyte response to long distance running in the heat . running induces greater muscle damage than cycling at the same intensity ( 41 ) thus , it appears likely that the smaller monocyte increase observed in this and other cycling studies compared to that observed for running is due to the difference in muscle damage from the two exercise modes . the increase in lymphocytes in response to endurance exercise observed in this and previous studies is less pronounced than that of neutrophils ( 6 , 8 , 26 , 32 , 33 , 38 ) . the increase in lymphocytes ( 53% ) in the present study following the completion of a 164-km cycling event in the heat ( average temperature 35c ) , is similar to the increase found in studies involving shorter - term exercise under heat exposure : 40 min cycling immersed in 39c water ( 5354%)(6 , 33 ) and 90 min cycling at 70% vo2peak at 35c ( 38 ) . in contrast , exercise in a cold environment ( 1823c water ) induced only a modest increase in lymphocyte concentration ( 2125% ) ( 6 , 33 ) . using multiple regression , cross et al . ( 6 ) found that cortisol concentration , growth hormone concentration , and rectal temperature accounted for 93% of the variance in lymphocyte concentrations following exercise in the heat and 87% of the variance following exercise in the cold(6 ) . thus as with neutrophils , the greater lymphocyte response to exercise in the heat than in the cold is likely , at least in part , the result of core temperature mediated stimulation of stress hormone release(6 , 33 ) . the results of present study showed no significant relationship between the change in leukocyte subsets concentrations and time to complete the event ( rolling time ) or intensity ( % vo2max ) . it was hypothesized that the slower riders would have a greater leukocyte response since they were exposed to the high temperature for longer . although there was a large spread in the time to complete the event ( 4.78.2 hours ) even the fastest rider spent more than 4 hours under heat exposure and thus the additional heat exposure for the slower riders appears to have not affected the leukocyte subsets response . it is possible that any potential effects of prolonged heat exposure on leukocyte subsets concentrations were counteracted by the lower absolute intensity of exercise performed by the slower riders . in contrast to the large range in time to complete the event , the range of the estimated relative intensity was rather small ( 6185% ) and thus not well suited for investigations of bivariate correlations . it appears that when completing a 164-km cycling event in the heat , the rolling time and self - determined relative intensity do not affect the leukocyte response . it should be noted that the age range of participants in the current study was large ( 2561 years ) although most of the participants were between 40 and 58 . previous studies have found that age can significantly affect neutrophils ( 4 ) , thus the findings might not apply to young adults . gender can also affect immune cell volume , but since only 4 women volunteered for the current study , it is not possible to examine a potential gender effect on changes in immune cell volume during the 164 km ride in a hot ambient temperature . although in this observational study , we do not have clear information about the activational state and characterization within each immune cell subpopulation , the increases observed , most notably , in neutrophil and monocyte sub - populations vs. lymphocyte sub - populations , suggest that the exercise in combination with the environmental stress posed a significant stimulus for a circulating immune response . furthermore , the lack of correlation to relative exercise intensity or duration suggests that in this event , the participants might have experienced a dramatic enough environmental stressor or overall exercise stress such that differences on the order of hours for duration time had no apparent effect on circulating concentrations of immune cells . this secondary finding suggests an intriguing hypothesis that there is a threshold of heat stress exposure beyond which any additional effects of exercise are either non - significant or negligible with regard to the leukocyte subset response . long - distance endurance events , and the training preceding these events , which often occur in extreme environmental conditions , might affect the acute and long - term health of even in well - trained participants . considering the growing popularity of these types of events , better understanding the acute effects of such events is critical to defining proper recovery and monitoring health over the course of training and post - ride .

the purpose of this observational study was to determine the circulating leukocyte subset response to completing the 2013 hottern hell hundred recreational 164-km road cycle event in a hot and humid environmental condition . twenty - eight men and four women were included in this study . whole blood samples were obtained 12 hours before ( pre ) and immediately after ( post ) the event . electronic sizing / sorting and cytometry were used to determine complete blood counts ( cbc ) including neutrophil , monocyte , and lymphocyte subsets . the concentration of circulating total leukocytes ( 103l1 ) increased 134% from pre to post with the greatest increase in neutrophils ( 319% , p<0.0001 ) . circulating monocytes ( including macrophages ) increased 24% ( p=0.004 ) and circulating lymphocytes including b and t cells increased 53% ( p<0.0001 ) . no association was observed between rolling time or relative intensity and leukocyte subset . completing the hotter n hell hundred ( hhh ) , a 100 mile recreational cycling race in extreme ( hot and humid ) environmental conditions , induces a substantial increase in total leukocytes in circulation . the contribution of increases in specific immune cell subsets is not equal , with neutrophils increasing to greater than 4-fold starting values from pre to post race . it is likely that exercise in stressful environmental conditions affects the complement of circulating immune cells , although activational state and characterization of specific leukocyte subsets remains unclear . the observed increase in circulating cell sub - populations suggests that the circulating immune surveillance system may be acutely affected by exercise in hot and humid conditions .

INTRODUCTION METHODS Participants Protocol Statistical Analysis RESULTS DISCUSSION

PMC3010682

maintaining fidelity of dna replication is essential to the survival of even the simplest organisms . based on mammalian models , lack of faithful dna replication by dna polymerases is associated with a large number of human diseases including , but not limited to , cancer and a variety of precancerous conditions [ 17 ] . a number of repair mechanisms exist to correct errors that occur during dna synthesis , including mismatch repair , base and nucleotide excision repair , and recombination repair , and these mechanisms have been reviewed recently elsewhere [ 811 ] . despite the existence of these repair mechanisms , a major determinant of replication fidelity this ability is promoted not only by the high fidelity with which the polymerase selects correct versus incorrect nucleotide , but also by the reduced capacity for the polymerase to extend or replicate through primer / templates ( p / ts ) containing distortions such as mismatches , abasic ( ap ) sites , or oxidative or bulky lesions [ 1217 ] . this kinetic barrier to extension causes the replicative polymerase to stall . in many organisms , including prokaryotes , archea , and eukaryotes , stalling of the replicative polymerase is required to permit a short - term exchange with a low - fidelity polymerase ( including one or more y - family polymerases , or the b - family dna polymerase ) . this occurs via exclusive interactions of the respective polymerases with processivity factor in order to permit lesion bypass [ 1827 ] . although these lesion bypass polymerases are generally more error - prone and do not repair the lesion , they rescue the organism from lethal replication failure when dna is damaged [ 28 , 29 ] . the stalling of a replicative polymerase at dna lesions or following misincorporation also appears to be important for self - correction by allowing engagement of the intrinsic 3 to 5 exonuclease ( exo ) activity to proof - read replication errors as they occur or to sense the presence of dna lesions [ 14 , 3037 ] . pre - steady - state and steady - state kinetic analysis of polymerases with or without a functional exo activity have been instrumental in understanding the mechanisms that permit dna polymerases to copy dna with high fidelity , as well as how they avoid the permanent mutagenic effects of misincorporated nucleotides or dna damage [ 14 , 15 , 33 , 34 , 3840 ] . this review analyzes the different mechanisms that control replication fidelity that are intrinsic to replicative dna polymerases , with a specific focus on those that are utilized by the polymerase encoded by herpes simplex virus type 1 ( hsv-1)the prototypic member of a family of viruses which cause high morbidity in the human population . because of the ease with which they can be genetically manipulated , viruses have also proven to be excellent models to better understand the functions required for faithful genome duplication [ 4145 ] . moreover , viral polymerases , including the hsv-1 dna polymerase and the human immunodeficiency virus ( hiv ) reverse transcriptase , are important therapeutic targets of antiviral drugs [ 4648 ] . a summary of studies that have reported the pre - steady - state and steady - state kinetics of hsv-1 dna polymerase are provided and compared to those of other replicative polymerases to gain insight into the similarities and differences by which these polymerases control fidelity . by understanding how activities required for high - fidelity dna replication are modulated and coordinated by the hsv-1 dna polymerase , it may be possible to design novel antiviral approaches to thwart replication of this important human pathogen . the single most important contributor to fidelity of most dna polymerases is nucleotide selectivity that is , the propensity with which the polymerase incorporates correctly versus incorrectly base - paired nucleotide [ 33 , 34 , 38 , 4953 ] . for replicative polymerases , nucleotide selectivity has been shown to account for an error rate in the range of > 10 to 10 [ 33 , 34 , 49 , 52 ] . the structures of a variety of replicative dna polymerases have been solved , including the structures for the a - type polymerases that replicate t7 bacteriophage and mammalian mitochondria ( pol ; ) , and the b - type replicative polymerases of the t4-related bacteriophage rb69 , hsv-1 , and yeast ( saccharomyces cerevisiae ) dna polymerase . it is clear that the nucleotide - binding pocket is able to accommodate or snuggly fit the correctly paired nucleotide via space and geometry [ 59 , 60 ] . this promotes a higher ground - state binding affinity of the correct , compared to incorrect , nucleotide . in addition , evidence points to an induced fit model , whereby the binding of correct nucleotide better promotes the conformational change necessary to form the closed structure required for catalysis [ 34 , 49 , 50 , 54 , 56 , 61 ] . for the bulk of these polymerases , it is this conformational change , rather than the chemical catalytic step , which is rate - limiting for correct nucleotide incorporation under pre - steady - state conditions [ 34 , 39 , 6264 ] . for incorrect nucleotide incorporation , there may be contributions from conformational and chemical steps as well as from the propensity for rapid dissociation of mispaired dntp from the polymerase . by contrast , the structure of the active site of the y - family polymerases reveals that the relatively high frequency for mismatch incorporation ( > 10 ) and for bypass of dna lesions can be attributed to a more open active site than that formed by a or b type polymerases [ 6669 ] . most replicative polymerases possess or are closely associated with a 3 to 5 exo activity , and loss of this inherent or associated activity results in an increased mutation frequency in vitro and in vivo [ 7074 ] . the contribution of the exo activity to increasing overall fidelity of dna replication varies for different sequence contexts , but is on the order of 10- to 100-fold for most of the replicative polymerases [ 31 , 33 , 51 , 63 , 75 , 76 ] . the importance of polymerase - associated exo activity in maintaining genomic stability in higher eukaryotes is also confirmed by the increased cancer incidence in transgenic mice containing an exo - deficient polymerase [ 5 , 6 ] and by the premature aging that occurs in transgenic mice containing an exo - deficient pol . because the exo activity competes with the polymerizing activity for the 3 primer terminus , high - fidelity replication in its presence might be expected to have a real cost in terms of polymerizing speed and/or efficiency . however , pre - steady - state kinetic experiments have demonstrated that the means by which both rapid polymerization and correct nucleotide incorporation can be maintained reflects the ability of a polymerase to partition the primer terminus to the polymerizing or exo sites as needed [ 15 , 31 , 33 , 34 , 62 , 63 , 77 ] . the relative efficiencies for polymerization and proofreading during active dna synthesis are influenced by a variety of parameters , including the relative affinities of a matched or mismatched primer terminus for the polymerizing or exo site , the rates of polymerization at matched versus mismatched primer termini , the rates of excision of matched versus mismatched primer termini , and the ability of a primer end to switch between the polymerizing and exo site with or without dissociation of the template from the polymerase . donlin and coworkers demonstrated that the t7 dna polymerase holoenzyme ( with e. coli thioredoxin processivity factor ) possesses the same binding affinity for matched or mismatched p / t although the t7 pol binds p / t tighter to the polymerization domain than to the exo site . in addition , the polymerization rate of incorporation of correct nucleotides is fast ( 300/sec ) and the excision rate on ssdna is even faster ( > 700/sec ) [ 33 , 62 ] . however , transfer of the primer to the physically distinct exo site is slower by 10-fold when the dna is matched compared to when it is mismatched . when a misincorporation event occurs , the polymerization rate for t7 polymerase is slowed , permitting a transfer of the primer strand from the pol to the exo site . the rapid transfer of the mismatched nucleotide to the exo site permits excision of that nucleotide . for the t7 holoenzyme , the transfer of the now - matched p / t back to the polymerization site occurs without dissociation . thus , the kinetic partitioning of the primer between the polymerizing and exo sites , without a rate - limiting dissociation of the holoenzyme from the p / t , permits rapid repair with little cost to overall rate of extension . the structurally similar a - type mitochondrial polymerase ( pol ) possesses an inherently poorer fidelity than the t7 polymerase [ 31 , 63 ] . compared to t7 polymerase , excision of mismatched dna occurs at a much slower maximum rate ( 9/sec ) . nevertheless , error correction without enzyme dissociation occurs with an efficiency of 80% . by contrast , another a - type enzyme , klenow fragment of dna polymerase i , has an even slower rate of excision ( 0.003/sec ) , which would favor dissociation when that enzyme stalls at a mismatch [ 15 , 77 ] . the latter results suggest that proofreading by klenow is a less efficient means for error correction than by either bacteriophage t7 dna polymerase or mammalian pol . the t4 dna polymerase catalytic subunit ( a b - type polymerase ) also incorporates correct nucleotides at a rapid rate ( > 400/sec ) whereas the exo activity excises ssdna at a rate of approximately 100/sec . as for the t7 polymerase , transfer of primer from the polymerization to the exo site is slower ( 5-fold for t4 polymerase ) for matched compared to mismatched p / t . however , because the rate constant for dissociation of the t4 polymerase catalytic subunit from the p / t ( 68/sec ) competes with that for transfer of a mismatched primer to the exo site ( 5/sec ) , direct editing can occur only 40% of the time . therefore , editing by the t4 polymerase catalytic subunit is less efficient than by the t7 dna polymerase holoenzyme [ 33 , 34 ] . as shown for the t4-like rb69 bacteriophage dna polymerase , the ability of a mispaired end to access the exo site requires a ~40 rotation relative to its position at the polymerase active site [ 56 , 81 ] . because the template strand is not firmly anchored during this movement to the exo site for klenow enzyme , the dna must diffuse to the exo site , although one - way diffusion could be facilitated by retention of the primer in the thumb domain during the conformational change [ 56 , 82 ] . a -hairpin within the exo domain of the b - type polymerase encoded by bacteriophage rb69 has been proposed to hold the template in place as the primer terminus is transferred from the polymerizing to the exo active sites . the analogous hairpin of s. cerevisiae pol interacts extensively with the template strand which could stabilize the enzyme - dna interaction during the conformational transition . by contrast , the -hairpin of rb69 polymerase does not associate with normal dna and interacts with only a portion of the ssdna template containing an abasic site [ 56 , 58 ] . this could account for the relative inefficiency by which the rb69-like t4 dna polymerase can edit mismatched dna in a single association event [ 34 , 58 , 84 ] . it is becoming increasingly clear that the exo activity associated with polymerases increases fidelity by mechanisms in addition to nucleotide editing . for example , the presence of exo activity also appears to be important for preventing translesion dna synthesis or strand displacement synthesis by a number of polymerases , including those of pol , bacteriophage t4 , and hsv-1 [ 14 , 8589 ] . this is because the ability of a polymerase to partition the dna primer between the polymerizing and exo sites allows the enzyme to engage in idling turnover that is , successive rounds of excision and incorporation , with or without dissociation whenever it encounters a kinetic barrier to extension . this idling - behavior increases overall dna replication fidelity because it prevents extension through abasic sites or oxidized nucleotides , such as 8-oxyguanine , which would have a high probability for introducing mutations . idling turnover by a polymerase also limits strand displacement synthesis . failure to limit strand displacement synthesis by yeast pol during okazaki fragment maturation has been shown to lead to the production of aberrant or improperly processed lagging strand intermediates in vitro and higher mutation frequency in vivo [ 86 , 87 ] . there has been much disagreement concerning the role of polymerase accessory proteins in maintaining fidelity . parameters that could be altered by processivity factors include kinetic barriers to extending a mismatch or bypassing a lesion , the ability of the polymerase to partition its activity between the physically distinct exo and pol sites , the relative accessibility of the exo and pol sites , and dissociation of polymerase upon encountering a barrier to extension [ 33 , 72 , 76 , 90 , 91 ] . proposed that the t4 polymerase processivity factor would enhance fidelity by limiting the dissociation of enzyme from mismatched p / t , thereby increasing the efficiency for direct editing by the enzyme . however , for several polymerases , an inverse relationship was shown between processivity or dna binding and fidelity , at least under some circumstances [ 9093 ] . for t7 dna polymerase , the processivity factor was found to enhance fidelity by retaining the polymerase long enough to edit misincorporated nucleotides . for t4 dna polymerase , the addition of processivity factors to the wild - type enzyme had no impact on fidelity of dna replication and did not permit lesion bypass . however the addition of processivity factors to an exo - deficient t4 polymerase did permit lesion bypass with low concentrations of enzyme , which did not permit efficient translesion synthesis in the absence of processivity factors . proliferating cell nuclear antigen ( pcna ) was also demonstrated to increase misinsertion frequency and to promote the ability of mammalian pol to bypass dna lesions [ 90 , 93 , 94 ] , thereby reducing fidelity . likewise , the addition of processivity factor to mitochondrial polymerase ( pol ) reduced the incorporation fidelity of both the wild - type and exo - deficient enzyme in gap - filling assays , predominantly by enhancing the efficiency for extending mismatches . hsv-1 encodes a b - type dna - dependent dna polymerase that is the product of the ul30 gene and is essential for origin - dependent dna synthesis in cell culture and for the production of infectious progeny virus in vivo [ 95100 ] . in vitro , the hsv-1 dna polymerase is also required together with 5 other viral encoded proteins for leading strand synthesis on model templates . in addition to a 5 to 3 polymerizing activity , the polymerase catalytic subunit ( pol ) possesses an intrinsic 3 to 5 exo domain [ 57 , 99 , 102 , 103 ] . in infected cells , hsv-1 pol forms a stable heterodimer with the product of the ul42 gene ( ul42 ) [ 104106 ] . the ul42 protein is a double - stranded dna - binding protein that serves as an accessory protein to increase the processivity of the hsv-1 pol catalytic subunit [ 104 , 107 , 108 ] . the ability of the ul42 processivity subunit to form a stable heterodimer with the hsv-1 pol catalytic subunit resembles the type of arrangement observed for the a - type polymerases , t7 bacteriophage dna polymerase and mitochondrial pol , with their respective processivity factors . however , the structure of hsv-1 ul30 pol catalytic subunit most closely resembles that for the eukaryotic replicative polymerase , pol [ 57 , 58 ] . interestingly , the structure of the ul42 processivity factor resembles that for a subunit of the pol processivity factor , pcna [ 109 , 110 ] . however , the two globular domains of hsv-1 ul42 are oriented differently from those of a pcna monomer such that they could not easily form the toroidal structure of the homotrimeric pcna processivity factor . the co - crystal structure of ul42 with a pol c - terminal peptide suggests a flexible attachment to the unstructured c - terminal tail of hsv-1 pol , similar to what has been observed for other processivity factors with their cognate b - type polymerase [ 5658 , 81 , 82 , 109 ] . since the ul42 processivity factor does not require energy or other proteins to load it onto dna or to form the heterodimeric holoenzyme [ 104 , 105 , 107 , 111 ] , it is easier to investigate the role of processivity factor in controlling the fidelity of this viral replicative polymerase compared to that of its close homolog , pol . epidemiological studies suggest that hsv-1 dna replication is not as faithful as that of its human host . for example , mutations occur frequently during hsv-1 replication in vivo , such that many nucleotide polymorphisms can be observed even in epidemiologically related strains of virus [ 112114 ] . the genetic diversity of viral progeny that results from this poor fidelity is likely to contribute to selection of virus mutants capable of evading natural host immune mechanisms as well as antiviral drugs . indeed , clinical isolates from humans not exposed to antiviral drugs have been shown to contain a preexisting subpopulation of virus resistant to high concentrations of acyclovir . for random hsv-1 isolates from japan , the average number of nucleotide differences observed in the 1,131 bp thymidine kinase ( tk ) gene was 3.3 . in another study , the hsv-1 tk gene mutation frequency ( estimated at 5 10 ) was determined by measuring the average frequency of acyclovir - resistant plaques that arose under nonselective growth conditions during a single replication cycle for independent cultures of an acyclovir - sensitive ( wild - type ) laboratory strain of hsv-1 . examining the role of the 3-5 exo activity in maintaining the fidelity of hsv-1 dna replication in culture has been challenging since it has not been possible to isolate viral mutants by altering conserved residues within either the exo i or exo ii sites [ 117 , 118 ] . most of the hsv-1 exo ii site pol mutations created by gibbs and coworkers failed to complement the replication of a pol null virus or to synthesize any viral dna in these complementation assays . biochemical analysis of similar exo ii mutants by another group revealed that these mutations compromised not only the exo activity , but also the extension activity of the polymerase , perhaps accounting for their lethal phenotype . mutations that map in the conserved exo i residues that are involved in the coordination of the metal ions required for catalysis ( e.g. , d368a and e370a ) also possess little if any exo activity , though their polymerase activities are similar to or greater than that of the wild - type hsv-1 pol [ 75 , 119 ] . nevertheless , the d368a exo i mutation was lethal for virus replication . it has been suggested that the exo and polymerizing domains of the hsv-1 pol may be fundamentally different and less distinct from each other than those domains for other polymerases . however , the resolved crystal structure of the hsv-1 pol shows that the organization of the exo domain with respect to the palm , fingers , and thumb subdomains is similar to that shown for s. cereviseae pol [ 57 , 58 ] . the only exo - deficient hsv-1 polymerases capable of supporting virus replication are those that map to the conserved exo iii site . these exo iii site mutant proteins ( e.g. , y577h , a double mutant y577h / d581a , and y577f ) were somewhat compromised for polymerizing activity , but the 3 to 5 exo activity was reduced from 6- to 50-fold , depending on the mutation [ 116 , 119 ] . transient expression of the y577h and y577h / d581a mutant pol genes complemented the replication of an hsv-1 pol null mutant virus , and viable virus progeny could be isolated when the mutations were introduced into the viral genome . these viruses displayed a mutator phenotype with mutation frequencies that were 300- and 800-fold higher , respectively , than that for the parental wild - type virus . the mutator phenotype of exo iii mutant viruses clearly demonstrates an important role for the intrinsic exo activity of the hsv-1 dna polymerase in maintaining fidelity of viral dna replication in culture . however , the inability of other exo - deficient polymerases to support virus replication , particularly the exo i site mutants with wild - type polymerizing activity , suggests a more complex role for this domain during viral dna replication . pre - steady - state kinetic analysis can provide a better understanding of the complex interactions between the polymerizing and exo functional domains by determining the individual parameters that control correct and incorrect nucleotide incorporation with undamaged template or a template with a noninformative lesion , such as an ap site . table 1 summarizes the results of several different pre - steady - state kinetic studies of single nucleotide incorporation by various hsv-1 polymerases that is , the wild - type and the exo i site mutant ( d368a ) pols with or without the ul42 processivity factor . for better comparisons , only the apparent kds for dntp are shown since true kd for dntp was determined in only one study . the rate constants for correct nucleotide incorporation by the wild - type hsv-1 pol in the absence or presence of ul42 processivity factor were not significantly different in two different sequence contexts ( table 1 and ) . this contrasts with the ability of pcna to increase the rates of polymerization by mammalian pol approximately 3-fold . for wild - type hsv-1 pol , the rate constant for misincorporation of datp opposite da template ( 106/sec ) was only moderately less than for correct incorporation of dttp ( 157/sec ) into the same p / t ( table 1 ) . discrimination between the correct and incorrect nucleotide for hsv-1 pol occurs predominantly at the level of ground state binding affinity of dntp , as indicated by a > 20-fold decrease in affinity of datp ( incorrect ) compared to dttp ( correct ) for da - containing template . although the processivity factor slowed the rate of incorporation of mismatched dntp by the wild - type pol approximately 4-fold , the pol / ul42 complex had an even greater difference in affinity between incorrect and correct dntp compared to the wild - type pol catalytic subunit . thus , hsv-1 pol , with or without processivity factor , discriminates between correct versus incorrect dntp with similar relative efficiencies ( table 1 ) . the relative discrimination values shown in table 1 ( 34 and 130 ) differ somewhat from published values , since the latter used the lower true kd ( dntp ) values to estimate a misincorporation frequency of 1 in 300 for both pol and pol / ul42 . this misincorporation frequency is approximately 10 times higher than that reported for the exo - deficient klenow fragment of e. coli dna polymerase i , suggesting that hsv-1 pol is one of the least faithful dna polymerases with intrinsic exo activity . it is interesting to note that a misincorporation frequency of 1 in 300 is similar to the nucleotide variation within the hsv-1 tk locus ( an average of 3.3 changes for a gene of 1,000 bp ) noted among japanese isolates . most other studies that have estimated lower hsv-1 mutation frequencies ( between 5 10 and 5 10 ) have relied on the frequencies for isolating mutations in genes that are scored by function , such as acyclovir - resistance or plaque color due to mutation in a lac z locus introduced into the viral genome [ 120 , 121 ] . such studies naturally underestimate misincorporation frequency because they fail to score silent mutations that do not alter phenotype . the d368a mutation renders the polymerase completely deficient in exo function but with a pre - steady - state polymerization rate constant for incorporation of correct nucleotide that is approximately 20% higher for the mutant catalytic subunit ( 199/sec ) compared to the wild - type pol on the same p / t ( table 1 ) . although the relative differences in ground state binding affinity for mismatched versus matched nucleotide were similar for the exo and wild - type pols , the pre - steady - state rate constant for incorporating an incorrect nucleotide by this mutant pol was 23 times lower than that for incorporating a correct one ( table 1 ) . this results in an overall 600-fold reduced efficiency for incorporation of incorrect compared to correct dntp by the exo pol . overall , the hsv-1 exo pol misincorporates nucleotides > 5-times less efficiently than does the wild - type pol using the same p / t ( table 1 ) . thus , the hsv-1 exo pol misincorporation efficiency differs from that of exo - deficient klenow enzyme by a factor of only 2 . as discussed in detail in a subsequent section , the lower misincorporation efficiencies observed by the hsv-1 exo pol , compared to the wild - type pol , may reflect a defect in the ability of the enzyme , or enzyme : incorrect dntp ternary complex , to transfer the primer terminus between the polymerizing and exo sites . the specific impact of the ul42 processivity factor on the efficiency with which the exo - deficient pol can discriminate between incorrect and correct nucleotides for incorporation can not be determined from table 1 . however , the ground - state binding affinity of the antiviral compound , acyclovir ( acv ) triphosphate , for the exo pol / ul42 complex , is roughly the same as the affinity of dgtp for incorporation opposite a dc templating residue [ 78 , 122 ] ( table 1 ) . by contrast , the rate constant for incorporation of acv - tp by the exo pol / ul42 complex is considerably slower than the rate constant for incorporation of matched dntp by the exo pol , but similar to that for incorporation of mismatched nucleotide by the exo catalytic subunit ( table 1 ) . thus , it is likely that the slow rate - limiting step for incorporation of acv - tp by the exo pol / ul42 reflects a slow conformational change and/or chemistry step , as suggested for mismatched dntp incorporation . the kinetic parameters that govern nucleotide incorporation by the hsv-1 pol opposite an ap site on the template differ from those for either correct or incorrect incorporation with undamaged templates . for example , for wild - type pol , there is no significant difference in the rate constant for incorporation ( kpol ) of datp opposite the matched dt compared to that for incorporation opposite an ap site ( table 1 ) . pre - steady - state kinetic experiments also revealed that the productive binding of the hsv-1 pol to a p / t containing an ap site located 1 nucleotide downstream ( + 1 ) from the p / t interface ( true kd ( dna ) = 42.1 7.4 nm ; ) was indistinguishable from that for undamaged p / t ( true kd ( dna ) = 44 3.0 nm ; ) . these results suggest that the enzyme can not sense the ap site on the template strand ahead of attempts at incorporation . nevertheless , the ground state binding affinity for datp opposite the noncoding ap site ( 1/kd ) was reduced approximately 5-fold for wild - type pol compared to that for incorporation opposite a matched nucleotide ( table 1 ) . under single turnover conditions in which the wild - type pol is bound to p / t at equilibrium and excision is initiated by the addition of mgcl2 with high - excess cold dna trap , the kinetics for excision of a mismatched p / t , or a p / t containing da opposite an ap site , approach that for excision of frayed ssdna ends by the wild - type pol / ul42 holoenzyme ( table 2 , [ 64 , 78 ] ) . the fast burst rate constant for excision of matched p / t by wild - type pol is considerably slower than that for mismatched or a : ap p / ts ( table 2 ) . in addition , a smaller proportion of matched primer was cleaved , compared to primer opposite a mismatched or ap - containing template . wild - type pol / ul42 also cleaved a smaller proportion of matched ends compared to frayed ones , and with a smaller burst rate constant ( table 2 ) . these results indicate that most of the matched p / t is held at equilibrium in the polymerization site with little difference in the relative distribution in the presence ( 12.5% ) versus absence ( 6% ) of processivity factor . because the presence of excess unlabeled dna trap prevents access of labeled p / t to any polymerase that dissociates , the slow burst rate constant for excision , when present , is indicative of the rate by which the primer strand is destabilized and transferred to the exo active site in a single association event ( 2.2/sec for pol and 0.25/sec for pol / ul42 ; table 2 ) . from these data alone , it is difficult to determine whether the differences observed in both fast and slow burst rate constants for cleavage of a da / dt matched p / t by pol , compared to those for cleavage of dg / dc matched p / t by pol / ul42 , reflect a greater thermal stability of the dg / dc base - pair or a significant slowing of excision rate by ul42 . however , in side - by - side comparisons on the same matched p / t , chaudhuri et al . demonstrated no more than a 2-fold reduction in fast burst rates for cleavage of matched p / t in the presence versus the absence of ul42 . although hsv-1 pol excises the 3 nucleotide from mismatched or a : ap p / ts at rates similar to those for ssdna ( unpublished results , but exemplified by the fast rates of excision of frayed ends by pol / ul42 ) , only a portion of the p / ts occupied by the pol are cleaved at the fast burst rate in a single - turnover ( table 2 ) . since no slow burst rate was observed for wild - type pol with either p / t , the results indicate that the remainder of the p / ts were either not bound to the enzyme or dissociated from it at a rate faster than that required to transfer the 3 primer terminus from the polymerizing to the exo site . in the presence of pol / ul42 , not only is a large proportion of frayed primer cleaved with a fast burst rate constant , but the remainder that bound initially at the polymerization site ( 27% ; table 2 ) was transferred to the exo active site for cleavage prior to dissociation . these results suggest that ul42 might increase the overall fidelity of dna synthesis by increasing the probability that a mismatched p / t will be excised prior to its dissociation from wild - type pol during processive synthesis , when most of the primer would be expected to be located at the polymerizing site . as previously indicated by steady - state kinetic analysis , pre - steady - state kinetic analysis reveals that the excision of acv - tp opposite the matched dc template is extremely slow , with a burst rate constant approximately 2000-fold slower than for the fast burst rate constant for excision of dg on matched template ( table 2 ) . the excision rate is slower than the rate of transfer from the pol to the exo site and/or the rate of dissociation from the p / t since no slower burst rate was observed . the association of next correct dntp has been reported to lock the polymerase into a dead - end complex with acv - tp . however , the comparatively small difference in excision burst rate constants in the presence versus absence of dntp following acv incorporation may suggest that the dntp stabilizes a replicating p / t within the polymerizing site , thereby reducing its rate of transfer from the pol to exo site [ 57 , 78 ] . taken together , these results suggest the importance of the hsv-1 pol to possess a dynamic ability to switch the p / t between the polymerizing and exo sites for its ability to function properly , and a role for ul42 in preventing dissociation of pol from a mismatched primer terminus during active dna extension . early steady - state experiments were used to determine the ability of the hsv-1 pol to extend mismatches . despite potent exo activity , the wild - type hsv-1 pol was observed to extend mismatches at high concentrations of next correct single nucleotide . however , the efficiency ( vmax / km ) for wild - type pol extension of mismatches with next correct nucleotide was considerably lower than for the exo pol [ 75 , 123 ] . this was due in large part to cleavage of the mismatched primer terminus by the exo activity of the wild - type pol , resulting in an incorrectly matched p / t for extension with the tested nucleotide . indeed , when the template nucleotide located at position 1 with respect to the mismatched primer terminus was complementary to the incoming nucleotide , efficiency for extending the mismatched p / t was virtually the same as for matched [ 75 , 119 ] . because dissociation would effectively reduce the vmax , these results demonstrate the effective ability of the wild - type hsv-1 pol to switch the primer to the exo site , cleave the mismatch , switch it back to the polymerizing site , and incorporate new nucleotide , all without dissociating . it also suggests that with this kinetic partitioning , there is little cost to extension efficiency when proof - reading by the hsv-1 polymerase is required . nevertheless , steady - state experiments do not provide an accurate measure of the ability of a polymerase to extend uncorrected mismatches . running - start single - turnover experiments have been used to gain a better understanding of the kinetic barrier for hsv-1 pol to extend mismatches and how the polymerase responds to misincorporation during active polymerization . the data in have been adapted and included in figure 1 for comparative purposes . in these experiments , the terminus of a 5 end - labeled primer was located 3 nucleotides upstream ( 3 ) of a target site that forced misincorporation due to the absence of matched nucleotide in the pool ( figure 1(a ) ) . critical to these experiments was ( 1 ) a saturating or close to saturating concentration of all other nucleotides , ( 2 ) equilibrium binding of the polymerase to the p / t prior to initiation ( in the presence of edta ) , and ( 3 ) initiation of reactions with mgcl2 and excess dna to trap any polymerase molecules that dissociate from the p / t following initiation . because active polymerization would occur for several nucleotides , the p / t would be expected to be firmly bound to the polymerase active site for several cycles of processive correct nucleotide incorporations prior to a forced misincorporation event . surprisingly , three - fourths of the p / t productively engaged by the wild - type pol misincorporated nucleotide at the target position , and two - thirds of those were extended by at least one additional nucleotide prior to dissociation ( accounting for 48% of total p / t productively bound by the pol , see figure 1(b ) ) . the presence of a strong pause site suggests that the wild - type pol switches the primer end to the exo site , cleaves the mismatch to form a matched p / t , then rapidly switches back to the pol site for another forced misincorporation event . repeated cycles of excision and incorporation ( idling ) then ensue in a single association of the pol catalytic subunit with p / t , until the mismatch is successfully extended ( figure 1(b ) ) . these results indicate that the rate of transfer of the mismatched primer terminus between the polymerizing and exo sites ( one or more times ) is greater than the rate of dissociation of the wild - type pol from the p / t during forced misincorporation . the exo hsv-1 pol also misincorporated and extended mismatches , but the kinetics for misincorporation were markedly slower compared with wild - type pol , with only half of the productively engaged exo pol misincorporating within 2 sec ( figure 1(c ) ) . the latter results are in excellent agreement with the less efficient ability of the exo pol , compared to the wild - type pol , in incorporating an incorrect nucleotide in standing start experiments ( table 1 ) . however , a similar proportion of available mismatched p / t was extended by the exo pol , compared with the wild - type pol , albeit at a slower rate . the near linear kinetics for mismatch extension by the exo pol are not the result of multiple turnovers of enzyme with p / t because of the presence of dna trap . instead , the absence of a burst likely indicates that the rate - limiting step is the slow rate of primer switching ( 0.24/sec ) from the polymerizing to the exo site and back when the mismatch can not be cleaved . the impact of the ul42 processivity factor in preventing mismatch extension is clearly observed in these running start experiments . not only do fewer mismatched p / t accumulate in the presence ( 26% ) versus absence ( 76% ) of ul42 , but only 5.6% of all p / t ( 21% of those that misincorporated ) are extended one nucleotide beyond the mismatch and none were extended further ( figure 1(d ) ; ) . because the rates of incorporation of incorrect nucleotide and excision of mismatches or frayed ends ( tables 1 and 2 , resp . ) do not differ substantially for wild - type hsv-1 pol in the presence or absence of processivity factor , the results suggest that ul42 increases the efficiency and/or rates by which the mismatched / frayed primer terminus is transferred between the polymerizing and exo sites in a single association event during active replication . similar running - start single - turnover experiments have been performed on p / ts in which the target site on the template was an ap site , located 4 nt downstream of the primer terminus ( figure 1(f ) ) . the data have been adapted from a previously published report to permit side - by - side comparisons with mismatch extension data . the wild - type hsv-1 pol extended the primer to the ap target site with more rapid burst kinetics than it did to a misincorporation target ( compare figures 1(g ) and 1(b ) ) . however , compared to mismatches , ap sites posed a greater kinetic barrier to extension by the wild - type hsv-1 pol , since no lesion bypass was detected ( figure 1(g ) ; ) . with such a high kinetic barrier , the rate of dissociation of the pol from the damaged p / t exceeded that required for extension , resulting in a smaller percentage ( 30% ) of primer extended to the ap target site ( figure 1(g ) ) compared to that which resulted from misincorporation ( 76% ; figure 1(b ) ) . the absence of exo activity prevents excision of the residue that is incorporated opposite an ap site and , as observed following misincorporation , this slows the transfer of primer between the exo and polymerizing sites . although the exo pol was able to bypass ap lesions , bypass synthesis was extremely inefficient ( figure 1(h ) ) . these results are in agreement with the requirement for exo - deficiency in order for the hsv-1 pol to incorporate nucleotide opposite a cisplatin cross - link . it was also shown in those studies that neither the wild - type nor exo pol , with or without ul42 processivity subunit , could completely bypass the cisplatin lesion . however , ul42 did increase the ability of hsv-1 exo pol to incorporate nucleotide opposite the cisplatin lesion . no studies to date have examined the ability of ul42 to permit ap lesion bypass synthesis . ap sites have been shown to be present in hsv-1 replicating and virion dna at a frequency of 2.85.9/genome equivalent . given that wild - type pol is unable to bypass these lesions , there must be other mechanisms to permit synthesis through or around ap lesions , such as recombination or repair . the newly discovered ap and 5-deoxyribose phosphate lyase activities of the hsv-1 pol , both of which are involved in base excision repair , might perform such a task to repair ap lesions . it is also possible that other viral dna replication components would permit lesion bypass . the kinetic barrier to extending a mismatched p / t prevents most misincorporation events from becoming fixed mutations . the exo activity of the hsv-1 pol reinforces the kinetic barrier by permitting excision of the mismatched primer terminus when the polymerase stalls . in reality , the polymerase does not truly stall when it encounters a kinetic barrier , but rather engages in idling at that site through successive rounds of incorporation and excision , even in the absence of the ul42 processivity factor . the idling activity is made possible by the ability of the polymerase to dynamically switch the p / t from the polymerizing site to the exo site and back . pre - steady - state kinetic analysis has demonstrated that ul42 increases the processivity of the hsv-1 pol from 1700 to > 5,000 per association event . ul42 also increases the efficiency of excision of mismatches formed during processive dna synthesis by increasing the rate and/or efficiency of transfer of the mismatched primer between the polymerizing and exo active sites . this makes ul42 an unusual fidelity factor because it affects neither the rates of polymerization nor the rates of excision . moreover , ul42 does not promote mismatch extension or lesion bypass , distinguishing it from the functions of the processivity factors of polymerases in the same family , such as t4 dna polymerase and mammalian pol [ 90 , 91 , 94 ] . mounting evidence suggests that altering the ability of the hsv-1 pol to switch between the exo and pol active sites will have an impact on fidelity . it is interesting to note that mutations in ul42 that either increase or decrease affinity for dna result in decreased replication fidelity and reduced numbers of viable virus progeny [ 120 , 121 ] . although classical measures of this idling activity depend on the presence of a functional exo activity , the running - start single - turnover experiments described in figure 1 demonstrate the ability of the hsv-1 polymerase to switch the primer terminus between the exo and pol sites even in the absence of exo catalytic activity . however , the increased affinity of the exo site for mismatched primer ( ssdna ) , and the corresponding decreased affinity of mismatched dna for the polymerizing site , renders the catalytically inactivated exo i site mutant pol ( d368a ) less efficient in switching behavior and in extending mismatched primers [ 75 , 123 ] . the fact that the exo i mutant pol is also slower in its ability to incorporate incorrect dntp than the wild - type pol suggests that binding of an incorrectly matched dntp to the hsv-1 pol may destablize the association of the primer terminus with the polymerizing active site . ul42 slows the rate of switching of the exo pol on mismatched termini during processive synthesis even more ( figure 1(e ) ; and ) . if the rate of switching is slower than that for dissociation of the polymerase from the p / t , then extension would be disfavored , which could explain why no viable progeny have been isolated with this particular mutation . mutations outside the exo domain may also impact p / t switching by the hsv-1 pol . kinetic analysis of hsv-1 pol containing a finger domain mutation ( l774f ) showed significant stalling during normal elongation and a decreased efficiency for extending mismatches [ 125 , 126 ] . when introduced into the pol gene of the virus , this mutation increased fidelity of dna synthesis compared to that of the parental virus , regardless of the presence or absence of an exo iii site mutation [ 125 , 126 ] . these properties are consistent with enhanced idling - turnover ability and increased primer switching compared to the wild - type pol . for t4 dna polymerase , mutations that increase the switching rate have also been shown to possess antimutator activity , consistent with the importance of the ability of the pol to efficiently transfer the primer end between the polymerizing and exo active sites to maintain fidelity . since the t4 switch mutations do not map to the same domain as the hsv-1 l774f mutation , additional studies will be required to ascertain the mechanism by which the hsv-1 finger domain mutant enhances replication fidelity . the ability of the hsv-1 dna polymerase to engage in dynamic switching of p / t between the pol and exo sites when kinetic barriers to extension are encountered is likely to be essential to the viability of virus . it is interesting to note that part of the effectiveness of the chain - terminator , acyclovir , now appears to be its influence on the ability of the hsv-1 pol to switch the primer terminus between the polymerizing and exo sites . as suggested by hanes et al . , the addition of dntp to an hsv-1 pol that has incorporated acv may favor retention of the acv - containing p / t at the pol site , making acv less likely to be removed by the exo activity . we propose that compounds that specifically target the hsv-1 pol exo activity for inactivation , or that significantly inhibit the ability of replicating viral dna to be transferred between the polymerizing and exo sites , would be effective antiviral agents . by holding the primer terminus in the exo site , extension by the polymerase would be slowed and could lead to chain termination and replication failure . holding the primer in the polymerase active site would be expected to decrease the ability of the polymerase to excise mismatches that are frequently formed by the polymerase and to increase the probability for mismatch extension . this would lead to the accumulation of a lethal number of mutations during replication . due to the conserved nature of the polymerase among the herpesvirus family members , it should be possible to target other herpesvirus polymerases in the same way , particularly those for which no effective nontoxic inhibitor has been identified to date .

we discuss how the results of presteady - state and steady - state kinetic analysis of the polymerizing and excision activities of herpes simplex virus type 1 ( hsv-1 ) dna polymerase have led to a better understanding of the mechanisms controlling fidelity of this important model replication polymerase . despite a poorer misincorporation frequency compared to other replicative polymerases with intrinsic 3 to 5 exonuclease ( exo ) activity , hsv-1 dna replication fidelity is enhanced by a high kinetic barrier to extending a primer / template containing a mismatch or abasic lesion and by the dynamic ability of the polymerase to switch the primer terminus between the exo and polymerizing active sites . the hsv-1 polymerase with a catalytically inactivated exo activity possesses reduced rates of primer switching and fails to support productive replication , suggesting a novel means to target polymerase for replication inhibition .

1. Introduction and Overview 2. General Parameters that Control Polymerase Fidelity 3. Herpes Simplex Virus Type 1 as a Model Eukaryotic Replicative DNA Polymerase

PMC4758412

radiative transfer is unique among parameterization problems for global atmospheric models because the governing equations are deeply grounded in fundamental physics , the approximations ( e.g. , of onedimensional radiative transfer ) applicable across many relevant scales , and the result entirely deterministic . in aerosolfree clear skies , where scattering is small relative to absorption and emission , the problem is defined by the profile of extinction of the gaseous atmosphere , which is itself determined by the profiles of temperature , pressure , and the concentrations of radiatively active gases . fluxes of longwave or terrestrial radiation also depend on how the extinction profile is related to the profile of local emission ( which also depends on temperature ) , while for fluxes of shortwave or solar radiation local emission can be neglected , but rayleigh scattering , which increases extinction and singlescattering albedo , changes the problem slightly . for monochromatic problems these calculations are straightforward so that the main challenge for atmospheric models is treating the spectral dependence of radiative fluxes . the best available information for the spectral variation of radiation in the atmosphere comes from socalled linebyline models with full spectral detail . when the state of the atmosphere is well characterized , such models are now able to match carefully calibrated observations to within fractions of percent at full spectral resolution [ see , for example , turner et al . , 2004 ; this is sufficiently accurate , for example , that the very small spectrally dependent signal from a decade 's increase in co2 concentrations can be teased from surface measurements of spectral intensity that are dominated by secular changes in temperature and water vapor [ feldman et al . , the absolute accuracy of these models in individual cases is difficult to judge because it is quite difficult to separate errors in the characterization of the atmosphere from errors in the spectral variation of optical thickness ( vertically integrated extinction ) . estimates from remaining spectrally resolved differences against observations suggest that absolute errors in broadband fluxes ( i.e. , fluxes integrated over all energy emitted by the sun or earth ) are no larger than about 1 w / m [ oreopoulos and mlawer , 2010 ] in the longwave . comparisons in the shortwave between spectrally resolved measurements and model calculations have been less extensive but suggest that broadband flux errors are somewhat larger . broadband fluxes are required by dynamical models of the atmosphere to compute heating and cooling rates within the atmosphere and surface fluxes of energy . the opacity of the cloud and aerosolfree atmosphere ( e.g. , the clear , clean conditions of ghan ) depends strongly enough on temperature and radiatively active gas concentrations that the extinction of the gaseous atmosphere is computed anew at each location and time . linebyline models are impractically slow for this task because absorption coefficients due to gases vary by many orders of magnitude over very narrow spectral intervals . computationally efficient parameterizations therefore use techniques such as exponential sum fitting of transmissivities [ hunt and grant , 1969 ; wiscombe and evans , 1977 ] , correlated kdistributions [ lacis and oinas , 1991 ; fu and liou , 1992 ] , or the simplified exchange approximation [ fels and schwarzkopf , 1981 ; schwarzkopf and ramaswamy , 1999 ] to approximate the spectral integral using a relatively small number of quadrature points minimizing some measure of error across a distribution of atmospheric states . ( other simplifications frequently used in parameterizations , such as low angular resolution or the neglect of scattering in the longwave , do not introduce significant errors in aerosolfree clear skies . ) systematic assessments of parameterizations against the reference models on which they are based date back more than two decades [ ellingson and fouquart , 1991 ; cess et al . , parameterizations have improved over time such that the best now reproduce linebyline flux calculations in presentday conditions to within about 1 w / m [ oreopoulos et al . , 2012 ] , although a surprisingly large range of error still exists for such a wellunderstood problem . parameterizations are substantially less accurate with respect to forcing calculations [ collins et al . , 2006 ; forster et al . , 2011 ] ( i.e. , the change in flux due to a change in atmospheric composition ) in part because the sets of atmospheric states used to develop parameterizations often do not include large variations in composition [ see , e.g. , mlawer et al . , 1997 , section 4 ] . but changes in radiative flux that arise from increasing levels of greenhouse gases are precisely the radiative forcing that drives climate change . parameterization errors in estimates of clearsky instantaneous radiative forcing , a purely radiative quantity that might be determined to high precision , are one reason that different models participating in coordinated experiments ( e.g. , the coupled model intercomparison project or cmip ; see taylor et al . , which describes the recently concluded cmip5 ) may be subject to different instantaneous forcing for the same change in atmospheric composition [ zhang and huang , 2014 ; chung and soden , 2015 ] . this is especially true for large excursions from presentday conditions such as 4co2 ( carbon dioxide concentrations quadrupled from their preindustrial value ) . despite this concern , abrupt 4co2 experiments are widely used to obtain estimates of sensitivity and effective radiative forcing from timeevolving flux imbalance and temperature [ gregory et al . , here we examine the accuracy of the greenhouse gas component of a set of parameterizations used in atmospheric models . we consider both fluxes under a range of presentday conditions and the forcing from quadrupled concentrations of carbon dioxide under the same range of conditions . we focus on parameterizations used in climate models ( roughly , those that participate in cmip experiments ) because these models are often used to understand the response to forcing ; here we seek to identify errors in the forcing itself . , 2013 ] communities have focused on more processspecific metrics using a different set of models though with broadly similar results . ) we demonstrate below that recent revisions to parameterizations at several modeling centers have reduced error ( at least in our test cases ) but that errors in absorption relative to surface and topofatmosphere fluxes imply biases in presentday model hydrologic cycles and in the hydrologic sensitivity to increasing greenhouse gas concentrations . we also show that parameterization accuracy often depends on atmospheric state and composition , suggesting that estimates of accuracy at the global scales needed to estimate radiative forcing error require largescale assessments across a wideranging set of conditions . we report on calculations following a variant of the protocol used by the first phase of the continual intercomparison of radiation codes ( circ ) [ see oreopoulos et al . , 2012 ] in which broadband fluxes are computed for atmospheric and surface conditions ( temperature , pressure , and greenhouse gas concentrations ) specified in between 55 and 66 layers , roughly comparable to the vertical resolutions used in climate models . in most cases participants performed the calculations with radiative transfer codes extracted from the host model ( see the recommendation by forster et al . ) , although at least one implemented diagnostic calculations in a singlecolumn version of the full model . we focus on the treatment of gases by considering four clearsky cases , with precipitable water vapor ranging from 0.32 to 4.85 cm , originally chosen for good agreement between spectral measurements at the surface and reference model calculations using estimates of the overlying atmospheric state . we further simplify the problem by specifying no aerosols and spectrally uniform surface albedo and emissivity . we perform two sets of experiments , one under the initially observed atmospheric conditions ( essentially repeating the shortwave case b calculations of oreopoulos et al . ) and a second in which the observed concentrations of co2 have been quadrupled . fluxes are reported at the top of atmosphere and the surface . atmospheric absorption or emission ( radiative divergence ) many provided results from the current version of their radiation parameterization ; some also provided results from the version used in cmip5 [ taylor et al . , 2012 ] . calculations made with version 12.2 of the linebyline radiative transfer model ( lblrtm ) model [ clough et al . , 2005 ] provide the benchmark , as they did for circ ( though circ used a slightly older version ) . although our assessment does not include every parameterization used by models participating in cmip , several parameterizations are used in more than one model , so that the radiation component is even less diverse than the already interdependent set of models itself [ knutti et al . , as with past assessments , we find that reference models agree with one another to within 1 w / m , or to fractions of a percent , for almost all calculations , though this level of agreement is not a measure of uncertainty . the absolute accuracy of linebyline models , as measured by comparisons of highresolution spectra against observations [ e.g. , delamere et al . , 2010 ; mlawer et al . , 2012 ; alvarado et al . , 2013 ] , is improving over time . in most cases improvements are due to refined information about the presence , position , and strength of individual absorption lines or continua . better spectral information introduces changes in broadband fluxes , illustrated in figure 1 by results from an older version ( 9.4 + ) of lblrtm ( orange squares in this and all subsequent figures ) which has less complete spectroscopy and different models for the water vapor continuum , and agrees less well with spectrally resolved observations , than the more recent version ( 12.2 ) we use as our benchmark . ( broadband fluxes and forcings are far more similar than spectrally resolved differences ; see alvarado et al . . ) more sophisticated treatment of line interactions is also useful : the primary difference between the arts 2.2 ( small black squares ) and arts 2.3 ( large black squares ) is the treatment of line mixing by co2 ( revisions to continua , also present , have a small impact ) . full names of the radiative transfer codes and the institutions using them is provided in the supporting information . ( left ) errors in topofatmosphere net downward broadband longwave flux under presentday conditions and ( right ) error in forcing from co2 concentrations quadrupled from presentday values for a variety of reference models ( squares , including one highresolution kdistribution model ) and parameterizations used in climate models ( circles ) . calculations for four atmospheric profiles are ordered by columnintegrated water vapor ranging from 0.32 to 4.85 cm . histograms along the right edge of each panel show the error by model type ( black for reference models , dark grey for current parameterizations , and light grey for parameterizations used in cmip5 ; normalized across model categories ) in intervals of 1 ( figure 1 , left ) or 0.5 ( figure 1 , right ) w / m ) centered on 0 . parameterization error is larger but still modest , with error in some parameterizations depending strongly on the water vapor path . parameterizations that have been updated since cmip5 are more accurate than their predecessors ( compare filled circles and dark grey histogram bars to open circles and light grey bars . ) nonetheless , linebyline model results are almost entirely determined by the underlying spectroscopic data including models for absorption continua . all the linebyline models in this study use the same continua in the longwave , so spread among the reference models does not reflect true uncertainty . indeed , linebyline models have agreed well with one another for several decades [ e.g. , ridgway and arking , 1991 ] , often to a greater degree than they have agreed with observations . whatever the underlying uncertainty in linebyline calculations , parameterizations are constructed to reproduce these reference model results across a range of atmospheric conditions . by this measure parameterization accuracies vary more widely across quantities ( e.g. , topofatmosphere versus surface flux ) , spectral intervals , and atmospheric conditions than do reference models . for topofatmosphere longwave flux and forcing , as one example , parameterization error is relatively low ( figure 1 ) [ see also oreopoulos et al . , 2012 ] , but even here errors of 2 w / m ( roughly 1% , depending on the case ) in presentday net flux and 0.5 w / m ( 10% ) in forcing from quadrupled co2 concentrations are common . figure 1 is meant to be illustrative ; readers interested in details will find an exhaustive set of figures in the supporting information . revisions to parameterizations since cmip5 have reduced errors , in some cases dramatically , with respect to both flux and forcing ( compare the filled circles and dark grey histogram bars in figure 1 to the open circles and light grey histogram bars ; see also figures s1 and s2 in the supporting information ) . in most cases the reduced error is due to improved parameterizations of spectroscopy , i.e. , more accurate treatment of how the optical thickness of the gaseous component of the atmosphere depends on temperature , pressure , and composition . ( other changes may be relevant for more complicated atmospheric conditions including aerosols and clouds . ) perhaps more importantly , errors depend to some extent on atmospheric state including the profile of temperature and humidity . this is evident in how the error in presentday flux in the giss parameterizations ( shown in brown circles ) , and the error in forcing in all models but especially mstrnx(green circles ) , depend on columnintegrated water vapor , which increases from left to right in figure 1 . as we discuss below , the state dependence of parameterization error makes it hard to extrapolate from relatively few cases , even those constructed to sample , say , latitudinal dependence [ myhre and stordal , 1997 ; forster et al . , relationships among errors at the top of atmosphere , surface , and within the atmosphere can have important impacts on the host model : because globally averaged surface radiative fluxes affect the planet 's surface temperature while net radiative cooling controls the global amount of precipitation , radiation parameterization errors can impact presentday energy and water cycles nearly independently . in general , errors in longwave fluxes at the topofatmosphere and surface often partially compensate for one another ( figure 2 , left ) . for some parameterizations the reasons for this compensation are understood : the gfdl longwave parameterization , for example , includes energy in a narrower spectral interval than do the reference calculations , resulting in smaller upwelling fluxes ( positive error ) at both the surface and top of the atmosphere and hence very small impacts on atmospheric cooling . errors in surface net downward ( left ) longwave and ( right ) shortwave flux in the present day as a function of errors in net topofatmosphere flux , along with ( diagonal ) isolines of constant error in atmospheric absorption inferred from fluxes at the boundary . axis tick marks are every 1 w / m and absorption isolines every 5 w / m . errors in longwave boundary fluxes and radiative divergence are generally within a few w / m , especially for current parameterizations , while errors in shortwave fluxes and absorption are twice as large . calculations in the shortwave are generally less accurate in absolute terms than in the longwave [ see also oreopoulos et al . , 2012 ] , though for boundary fluxes the relative errors are similar ( within a few percent ) . more importantly , shortwave errors at the top of atmosphere tend to be of opposite sign to those at the surface , leading to errors in radiative divergence as large as 10 w / m or 5% even for current parameterizations . errors for the giss and lmdz cmip5era parameterizations are even more dramatic . in all but one parameterization shortwave absorption tends to be underestimated , implying a bias toward increased global precipitation in many host models . the exception is the gfdl parameterization , for which slightly greater solar absorption is consistent with the reference model on which the parameterization is based . this linebyline model absorbs more than our benchmark due to more recent underlying spectroscopic data and to a different treatment of water vapor continua due to paynter and ramaswamy . thus , the overestimate of absorption by gfdl relative to our reference is more properly viewed as a legitimate difference than an error . similarly , the partitioning of forcing errors within and at the boundaries of the atmosphere means that parameterization errors may project differently onto climate ( temperature ) and hydrologic sensitivities to increasing concentrations of greenhouse gases . in the shortwave most 4 co2 forcing errors , like errors in presentday fluxes , are of opposite sign at the boundaries ( see figure 3 , left ) so that errors in radiative divergence are relatively larger than at the surface or the top of the atmosphere . this implies that radiation parameterization errors will have larger impacts on hydrologic sensitivity than on climate sensitivity . the situation is much better in the longwave as shown in figure s3 . in practice relationships between forcing at the boundaries of and within the atmosphere will be modified by tropospheric and stratospheric adjustments , normally considered as part of the total forcing , that may dampen or amplify one error without affecting others . ( left ) errors in surface net downward shortwave forcing from quadrupled presentday co2 concentrations as a function of error in net topofatmosphere forcing , along with ( diagonal ) isolines of constant error in atmospheric absorption forcing . axis tick marks are every 1 w / m and absorption isolines every 5 w / m . for many parameterizations errors in topofatmosphere forcing are smaller than errors in surface forcing , implying errors in absorption forcing with impacts on the host model 's hydrologic cycle . ( right ) error in solar absorption forcing as a function of error in the presentday estimates of solar absorption . parameterizations have characteristic relationships among presentday and forcing errors that outweigh the impact of atmospheric state . it is tempting to think of parameterizations as being better or worse depending on some measure of the error in , for example , presentday fluxes , but the size of the error under presentday conditions says nearly nothing about the magnitude of errors in forcing ( figures 3 , right , and s4 ) . instead , relationships between errors in presentday and 4 co2 conditions are determined more by the parameterization being used than by atmospheric state . to the extent that these results hold on a global scale , it suggests both that presentday biases do not imply errors in forcing ( e.g. , the met office or gfdl parameterizations ) and , conversely , that parameterizations with small errors in the present day may still have significant forcing errors ( e.g. , the ccc and rrtmg parameterizations ) a focus on clear skies may seem narrow given that roughly two thirds of the planet 's surface at any given time is covered by some kind of cloud [ e.g. , stubenrauch et al . but clearsky errors exist even in cloudy skies ( though they need not add linearly to errors introduced in the treatment of clouds ) , and clouds do not affect forcing directly , so that characterizing the error in aerosolfree clear skies remains a useful starting point for assessing errors in radiative forcing . in particular , the models to be used in cmip6 [ meehl et al . , 2014 ] are still under construction and the radiation parameterizations are subject to revision before simulations are made and shared . one goal of intercomparison exercises is to identify errors so they may be fixed ( see discussions in cahalan et al . , for example ) , and this study has already motivated two changes reflected in our results : spectroscopy was updated in the giss parameterizations , and an important simplification ( the assumption that surface skin and surface air temperature are identical ) was relaxed in the lmdz and cnrm longwave parameterizations after initial calculations demonstrated relatively large errors in two cases . some , we hope , will expand the range of atmospheres used to train the parameterization to include conditions such as large concentrations of greenhouse gases . the reference models on which radiation parameterizations are based are themselves moving targets whose results change modestly as knowledge of the underlying spectroscopy improves , especially in the shortwave . all reference models in this study , for example , rely on the hitran ( highresolution transmission ) spectroscopic data ; differences between the 2012 and 2008 versions [ rothman et al . , 2009 , 2013 ] explain half or more ( 1 to 1.5 w / m , depending on the case ) of the difference in absorption calculations with lblrtm ( which uses the older version ) and the gfdl rfm linebyline model . periodic and/or continual assessments of radiation parameterizations help modeling centers from straying too far from current spectroscopic knowledge . radiation parameterization error is a function of the state of the atmosphere , as is clear from figure 1 . the set of conditions used in our calculations was originally determined by the availability of highquality observational data with which linebyline models could be validated [ turner et al . , 2004 ] . we can not extrapolate from these limited results to obtain global estimates of parameterization error in even in aerosol and cloudfree skies . one goal of the radiative forcing model intercomparison project ( rfmip , http://www.wcrp-climate.org/modelling-wgcm-mip-catalogue/modelling-wgcm-mips/418-wgcm-rfmip ) associated with cmip6 is to provide such globally relevant estimates , under presentday and future conditions and in strongly forced scenarios , by performing offline radiative transfer calculations under a much larger range of atmospheric conditions than is discussed here or in past assessments a set of conditions designed to allow for estimates of global parameterization error . these calculations will enable the surprisingly large diversity in instantaneous clearsky radiative forcing from 4 co2 [ zhang and huang , 2014 ; chung and soden , 2015 ] , for example , to be decomposed into correctable parameterization errors and legitimate differences in model climatology . highly accurate calculation of radiative fluxes is of primary interest to the climate modeling community where long freerunning simulations and highly variable greenhouse gas concentrations are the norm . the range of parameterization errors , especially with respect to forcing , implies that specifying changes in greenhouse gas concentrations ( as in cmip ) does not completely determine the instantaneous clearsky radiative forcing to which each model is subject . in fairness it is the effective radiative forcing , including modelspecific rapid adjustments [ see , e.g. , sherwood et al . , 2015 ] and statedependent sensitivities , that is relevant to determining the longterm climate response . 2014 ] will partly mask and may well outweigh the range of parameterization error . but rapid adjustments themselves can be influenced by radiation parameterization error [ ogura et al . , 2014 ] , and , given the maturity of understanding about radiative transfer , global estimates of parameterization error are a necessary first step in efforts to characterize and assess radiative forcing .

abstractthis article reports on the accuracy in aerosol and cloudfree conditions of the radiation parameterizations used in climate models . accuracy is assessed relative to observationally validated reference models for fluxes under presentday conditions and forcing ( flux changes ) from quadrupled concentrations of carbon dioxide . agreement among reference models is typically within 1 w / m2 , while parameterized calculations are roughly half as accurate in the longwave and even less accurate , and more variable , in the shortwave . absorption of shortwave radiation is underestimated by most parameterizations in the present day and has relatively large errors in forcing . error in presentday conditions is essentially unrelated to error in forcing calculations . recent revisions to parameterizations have reduced error in most cases . a dependence on atmospheric conditions , including integrated water vapor , means that global estimates of parameterization error relevant for the radiative forcing of climate change will require much more ambitious calculations .

Assessing the Accuracy of Radiation Parameterizations in Climate Models Building on Previous Assessments Results From CMIP5 and Beyond Tracing Error From a Few AerosolFree Clear Skies to Projections of Climate Change Supporting information

PMC2729898

muscular dystrophies with an autosomal recessive mode of inheritance comprise of a genetically heterogeneous group of disorders . among these , miyoshi myopathy ( mm ) is an early - adult onset , autosomal recessive form of distal muscular dystrophy , characterized by predominant involvement in the calf muscles and highly elevated serum creatine kinase ( ck ) levels ( 1 ) . mm has been known to be caused by mutations in the dysferlin gene ( dysf ) on chromosome 2p13 ( 2 ) . mutations in the same gene were also identified in patients with limb - girdle muscular dystrophy type 2b ( lgmd2b ) ( 3 ) , and distal anterior compartment myopathy ( 4 ) . these diseases caused by mutations in dysf are known by the term ' dysferlinopathy ' . although mm patients and their mutations in the dysf gene have been found from all over the world , only a few cases have been reported in korea ( 5 - 8 ) . considering that there have been many reports of mm among japanese population ( 9 ) , the incidence of mm might be underestimated in korea since both countries are geographically and ethnically related with each other ( 10,11 ) . in the present study , we performed clinical and genetic analysis of three korean patients with mm and found that all patients had compound heterozygous mutations in the dysf gene . three unrelated and non - consanguineous patients with clinical features of mm were evaluated ( table 1 ) . all the patients experienced early adulthood onset of slowly progressive muscle weakness , which preferentially involved muscles in the lower extremities . patients 1 and 2 had a history of steroid medication under the presumptive diagnosis of polymyositis according to the muscle biopsies and both showed transient responses . the genomic dna was extracted from the peripheral blood leukocytes using a wizard genomic dna purification kit according to the manufacturer 's instructions ( promega , madison , wi , u.s.a . ) . all the coding exons as well as the flanking introns of the dysf gene were amplified using the primer sets designed by the authors ( primer sets available on request ) . a polymerase chain reaction was performed with a thermal cycler ( model 9700 , applied biosystems , foster city , ca , u.s.a . ) using the following conditions : 32 cycles of denaturation at 94 for 30 sec , annealing at 60 for 30 sec , and extension at 72 for 30 sec . after the amplicon ( 5 l ) treatment with 10 u shrimp alkaline phosphatase and 2 u exonuclease i ( usb corp . , cleveland , oh , u.s.a . ) , direct sequencing was performed with the bigdye terminator cycle sequencing ready reaction kit ( applied biosystems ) using a abi prism 3,100 genetic analyzer ( applied biosystems ) . three unrelated and non - consanguineous patients with clinical features of mm were evaluated ( table 1 ) . all the patients experienced early adulthood onset of slowly progressive muscle weakness , which preferentially involved muscles in the lower extremities . patients 1 and 2 had a history of steroid medication under the presumptive diagnosis of polymyositis according to the muscle biopsies and both showed transient responses . the genomic dna was extracted from the peripheral blood leukocytes using a wizard genomic dna purification kit according to the manufacturer 's instructions ( promega , madison , wi , u.s.a . ) . all the coding exons as well as the flanking introns of the dysf gene were amplified using the primer sets designed by the authors ( primer sets available on request ) . a polymerase chain reaction was performed with a thermal cycler ( model 9700 , applied biosystems , foster city , ca , u.s.a . ) using the following conditions : 32 cycles of denaturation at 94 for 30 sec , annealing at 60 for 30 sec , and extension at 72 for 30 sec . after the amplicon ( 5 l ) treatment with 10 u shrimp alkaline phosphatase and 2 u exonuclease i ( usb corp . , cleveland , oh , u.s.a . ) , direct sequencing was performed with the bigdye terminator cycle sequencing ready reaction kit ( applied biosystems ) using a abi prism 3,100 genetic analyzer ( applied biosystems ) . patient 1 was a 45 yr - old male patient who had presented with progressive weakness in both lower extremities for the previous 17 yr . he noticed difficulty in running and atrophy of the lower leg muscles at 27 yr of age . the muscle weakness and atrophy progressed . a neurological examination showed bilateral motor weakness and symmetric atrophy of the entire lower extremity muscles , which were more severe on the distal part than on the proximal part . marked atrophy of the lower leg muscles with relatively spared pelvic girdle and upper thigh muscles resulted in a stalk - leg appearance . he had been diagnosed of having a polymyositis at the age of 28 yr as a result of a muscle biopsy of the medial gastrocnemius showing myopathic changes with inflammatory cell infiltration . his 43 yr - old younger sister was affected in the same way in her early thirties and found it difficult to walk up and downhills . since then , the muscle weakness worsened so that walking was still possible but only for short distances using a cane . she also had difficulty in holding her baby due to extended weakness of her upper extremity muscles . patient 2 was a 30 yr - old female who presented with difficulty in stair walking and pain in her low back and knee . difficulty in climbing stairs developed at 26 yr of age and this difficulty in walking gradually worsened so that she could not climb stairs without the support of her upper extremities . a neurological examination showed atrophy of both calf muscles , decreased strength of the lower extremity muscles , and an absent tendon reflex . she could not stand on her tip - toe , but standing on her heels was possible . the emg revealed the myopathic changes in gastrocnemius , rectus femoris , and biceps brachii muscles . a muscle biopsy without immunohistochemial stain from the biceps brachii suggested a diagnosis of inflammatory myopathy . magnetic resonance imaging of her lower leg disclosed symmetric fatty degeneration and decreased muscle volume of the posterior compartment muscles in both lower legs . the anterior and lateral compartment muscles had either a normal or a relatively normal signal intensity and morphology . patient 3 was a 37-yr - old man with quadriparesis who first became aware of periodic weakness in his lower extremities at the age of 18 yr . he noticed difficulties in standing and climbing stairs at the age of 21 and could not walk by himself by the age of 33 . an examination at the age of 37 showed incomplete quadriparesis and he could walk with a four - point crutch . his older sister developed lower extremity weakness and atrophy at the age of 18 yr and could not walk by the age of 36 . direct sequencing analysis identified five heterozygous mutations in the dysf gene including three novel mutations ( fig . patient 1 and his sister had compound heterozygous mutations of a novel c to t transition ( c.2494c > t ) in exon 24 resulting in a nonsense mutation ( gln832x ) along with a previously reported missense mutation ( c.2997g > t ; trp999cys ) . the gln832x mutation was also found in the patient 2 with a novel missense mutation ( c.2974c > t ; trp992arg ) . patient 3 had two mutations including a novel a to t transversion ( c.3307a > t ) in exon 30 , which resulted in a nonsense mutation ( lys1103x ) , and a previously reported insertion mutation ( c.4200dupc ) in exon 39 resulting in a frame shift mutation ( ile1401hisfsx8 ) . three novel mutations ( gln832x , trp992arg , and lys1103x ) were not detected in 100 control chromosomes . patient 1 was a 45 yr - old male patient who had presented with progressive weakness in both lower extremities for the previous 17 yr . he noticed difficulty in running and atrophy of the lower leg muscles at 27 yr of age . the muscle weakness and atrophy progressed . a neurological examination showed bilateral motor weakness and symmetric atrophy of the entire lower extremity muscles , which were more severe on the distal part than on the proximal part . marked atrophy of the lower leg muscles with relatively spared pelvic girdle and upper thigh muscles resulted in a stalk - leg appearance . he had been diagnosed of having a polymyositis at the age of 28 yr as a result of a muscle biopsy of the medial gastrocnemius showing myopathic changes with inflammatory cell infiltration . his 43 yr - old younger sister was affected in the same way in her early thirties and found it difficult to walk up and downhills . since then , the muscle weakness worsened so that walking was still possible but only for short distances using a cane . she also had difficulty in holding her baby due to extended weakness of her upper extremity muscles . patient 2 was a 30 yr - old female who presented with difficulty in stair walking and pain in her low back and knee . difficulty in climbing stairs developed at 26 yr of age and this difficulty in walking gradually worsened so that she could not climb stairs without the support of her upper extremities . a neurological examination showed atrophy of both calf muscles , decreased strength of the lower extremity muscles , and an absent tendon reflex . she could not stand on her tip - toe , but standing on her heels was possible . the emg revealed the myopathic changes in gastrocnemius , rectus femoris , and biceps brachii muscles . a muscle biopsy without immunohistochemial stain from the biceps brachii suggested a diagnosis of inflammatory myopathy . magnetic resonance imaging of her lower leg disclosed symmetric fatty degeneration and decreased muscle volume of the posterior compartment muscles in both lower legs . the anterior and lateral compartment muscles had either a normal or a relatively normal signal intensity and morphology . patient 3 was a 37-yr - old man with quadriparesis who first became aware of periodic weakness in his lower extremities at the age of 18 yr . he noticed difficulties in standing and climbing stairs at the age of 21 and could not walk by himself by the age of 33 . an examination at the age of 37 showed incomplete quadriparesis and he could walk with a four - point crutch . his older sister developed lower extremity weakness and atrophy at the age of 18 yr and could not walk by the age of 36 . direct sequencing analysis identified five heterozygous mutations in the dysf gene including three novel mutations ( fig . patient 1 and his sister had compound heterozygous mutations of a novel c to t transition ( c.2494c > t ) in exon 24 resulting in a nonsense mutation ( gln832x ) along with a previously reported missense mutation ( c.2997g > t ; trp999cys ) . the gln832x mutation was also found in the patient 2 with a novel missense mutation ( c.2974c > t ; trp992arg ) . patient 3 had two mutations including a novel a to t transversion ( c.3307a > t ) in exon 30 , which resulted in a nonsense mutation ( lys1103x ) , and a previously reported insertion mutation ( c.4200dupc ) in exon 39 resulting in a frame shift mutation ( ile1401hisfsx8 ) . three novel mutations ( gln832x , trp992arg , and lys1103x ) were not detected in 100 control chromosomes . this study identified five different mutations in three korean mm patients , and all patients had compound heterozygous mutation in the dysf gene . the c.2997g > t ( trp999cys ) mutation is reported to be one of the most frequent mutations found in japanese mm patients , and has been associated with a significantly later onset and a relatively mild form of mm ( 9 ) . the age at onset in patient 2 with the c.2997g > t mutation was delayed compared with the other patients , but was earlier than the mean age at onset reported elsewhere ( 9 ) . the c.4200dupc mutation ( ile1401hisfsx8 ) , which was found in two patients with mm or lgmd2b , appeared in the dysferlin sequence variations in the leiden muscular dystrophy pages . the c.2494c > t ( gln832x ) mutation was novel and was found in 2 unrelated patients , which might be due to a possible founder effect in the korean population . however , it will be necessary to perform haplotype analysis and collect more data on mm patients in korea to address this issue . some patients with mm can be easily misdiagnosed as having an inflammatory myopathy because inflammation is frequently observed in patients with mm and the inflammatory changes found in patients with mm are indistinguishable from those observed in patients with inflammatory myopathy ( 12 ) . indeed , two patients in this study had previously been suspected of having a form of inflammatory myopathy based on the muscle biopsy findings . ( 13 ) reported that the presence of inflammation is not an exclusion criterion in muscular dystrophy related to a dysferlin deficiency , while the muscle biopsies of mm or lgmd2b patients generally demonstrates signs of a dystrophy with a dysferlin deficiency . it is possible that some mm patients have been erroneously diagnosed with polymyositis based on the absence of dystrophic features and the presence of inflammation in the muscle biopsies . therefore , mm should be considered when making a differential diagnosis of patients exhibiting distal myopathy and muscular inflammation on muscle biopsies , and dysf gene analysis should also be considered .

miyoshi myopathy ( mm ) is an autosomal recessive distal muscular dystrophy caused by mutations in the dysferlin gene ( dysf ) on chromosome 2p13 . although mm patients and their mutations in the dysf gene have been found from all over the world , there is only one report of genetically confirmed case of mm in korea . recently , we encountered three unrelated korean patients with mm and two of them have previously been considered as having a type of inflammatory myopathy . the clinical and laboratory evaluation showed typical features of muscle involvement in mm in all patients but one patient initially had moderate proximal muscle involvement and another showed incomplete quadriparesis with rapid progression . direct sequencing analysis of the dysf gene revealed that each patient had compound heterozygous mutations ( gln832x and trp992arg , gln832x and trp999cys , and lys1103x and ile1401hisfsx8 , respectively ) among which three were novel . although mm has been thought to be quite rare in korea , it should be considered in a differential diagnosis of patients exhibiting distal myopathy .

INTRODUCTION MATERIALS AND METHODS Subjects Mutation analysis RESULTS Clinical findings Mutation analysis DISCUSSION

PMC5102570

behavioral and neurophysiological data were collected from two exotropic juvenile rhesus ( macaca mulatta ) monkeys ( 6 years , 910 kg ) . strabismus was previously induced by optical prism - rearing of infant monkeys . during the prism - rearing paradigm , infant monkeys wore a helmet like device that housed a horizontally oriented fresnel prism in front of one eye and a vertically oriented fresnel prism ( 20 prism diopter ) in front of the other eye , starting from day 1 after birth to 4 months of age , after which they were allowed unrestricted vision . disruption of binocular vision due to prism - viewing during this initial period leads to strabismus as it is the critical period for development of eye alignment , stereopsis , and binocular sensitivity . all procedures were carried out in accordance with national institutes of health guidelines and were approved by the university of houston institutional animal care and use committee . prior to commencement of experiments associated with this study , each juvenile monkey ( 4 years of age ) underwent surgical procedures carried out under aseptic conditions under isoflurane anesthesia ( 1.25%2.5% ) . in the first surgery , a head stabilization post was implanted to restrict head movements during the experiments . in subsequent procedures , scleral search coils were implanted in both eyes , using the technique of judge et al . , to record binocular eye movements . after collecting baseline data , strabismus correction surgery was performed by an expert strabismus surgeon ( also one of the study authors ) on the left eye only of m1 and right eye only of m2 and consisted of lateral rectus recession ( weakening the lateral rectus muscle ) and medial rectus resection ( strengthening the medial rectus muscle ) to improve their strabismus angle . we specifically chose to operate on only one eye , because we wanted to use the fellow eye as a control . the goal of data acquisition and analysis was to perform a longitudinal evaluation of eye alignment and eye movements following strabismus correction surgery . therefore , binocular eye movement data were collected as the animals performed central and eccentric fixation , saccades ( 15 horizontal and vertical target locations ) , and smooth pursuit ( 0.3 hz , 15 ) tasks under monocular left eye or right eye viewing conditions . alignment data were collected at five time points : before surgical treatment ( pre ) , the day after surgical treatment ( p1d ) , 1 week after treatment ( p1w ) , 1 month after treatment ( p1 m ) , and 6 months after treatment ( p6 m ) . saccade and smooth - pursuit data were analyzed at pre , p1w , p1 m , and p6 m because there were not enough experimental trials collected at p1d . binocular data were collected using the magnetic search coil technique ( primelec industries , regensdorf , switzerland ) . eye coils were calibrated at the beginning of each experiment by rewarding the animal with small amounts of juice as they looked at a series of targets projected along the horizontal or vertical meridian on a screen at distance of 57 cm . calibration of each eye was performed independently during monocular viewing forced by occluding one or the other eye using liquid crystal shutter goggles ( citizen fine devices , nagano , japan ) that were under computer control . visual stimuli were generated using the bits # visual stimulus generator ( cambridge research systems , cambridge , united kingdom ) and psychtoolbox 3 operated under computer control and presented using a depthq projector running at a 120-hz frame rate ( lightspeed design , inc . , bellevue , wa , usa ) . the target and binocular eye position signals were passed through anti - aliasing filters with a cutoff of 400 hz before digitization at 2.79 khz with 12-bit precision ( alphalab snr system ; alpha - omega engineering , nazareth , israel ) . were additionally calibrated offline and filtered using a software finite impulse response ( fir ) low - pass filter with a cutoff of 80 hz prior to further analysis . analysis of eye movement data was partially automated using custom scripts written in matlab ( mathworks , natick , ma , usa ) . for saccade data , the computer displayed target position , binocular eye position , eye velocity , and eye acceleration traces of a single saccade trial . velocity and acceleration signals were generated by digital differentiation of the position signal using a central difference algorithm . the investigator viewed the traces and decided whether the saccade trial was to be accepted or rejected . trials that were rejected were usually those in which the animal was not fixating prior to the target step , the saccade did not appear to be directed toward the target , or the saccade did not occur within 400 ms of the target step . once a decision to accept the trial was made , mean and sd of control eye acceleration prior to the saccade were calculated over a 70-ms fixation period . saccade onset was automatically determined by the software as the first time point at which eye acceleration was greater than 5 sds away from the control eye acceleration , and saccade offset was determined as the last time point at which eye deceleration was less than 5 sds away from the same mean eye acceleration . although detection of saccade onset and offset was automated , the investigator visually examined the velocity and acceleration traces of every saccade and had the option of either accepting or changing the computer selection , although this was not typical . once the saccade was identified , saccade metric parameters ( amplitude and peak velocity ) for each eye were calculated . main sequence plots of horizontal saccade peak velocity versus horizontal saccade amplitude were fitted with the following exponential rise to maximum equation : where amp = horizontal saccade amplitude , pv = horizontal saccade peak velocity , a = constant determining exponential rise , and pvmax = asymptotic peak velocity . for sinusoidal smooth - pursuit analysis then smooth - pursuit gain ( ratio of peak eye velocity to peak target velocity ) was calculated separately for nasalward and temporalward movements . to assess fixation stability , 10 seconds of eye movement data from both the eyes were analyzed as the monkey maintained monocular fixation at a stationary straight - ahead target . fixation stability was quantified by calculating the bivariate contour ellipse area ( bcea ) , a metric that quantifies the area of the region over which eye positions are dispersed during attempted fixation . the bcea encompassing 68.2% of fixation points was calculated using the following equation : , where x = sd of horizontal eye position , y = sd of vertical eye position , 2.291 is the value ( 2 df ) corresponding to a probability of 0.68 , and p is the pearson product moment correlation coefficient of horizontal and vertical eye positions . for statistical analysis , the kruskal - wallis 1-way anova on ranks was performed , and if significant , post hoc testing was performed using dunn 's method . behavioral and neurophysiological data were collected from two exotropic juvenile rhesus ( macaca mulatta ) monkeys ( 6 years , 910 kg ) . strabismus was previously induced by optical prism - rearing of infant monkeys . during the prism - rearing paradigm , infant monkeys wore a helmet like device that housed a horizontally oriented fresnel prism in front of one eye and a vertically oriented fresnel prism ( 20 prism diopter ) in front of the other eye , starting from day 1 after birth to 4 months of age , after which they were allowed unrestricted vision . disruption of binocular vision due to prism - viewing during this initial period leads to strabismus as it is the critical period for development of eye alignment , stereopsis , and binocular sensitivity . all procedures were carried out in accordance with national institutes of health guidelines and were approved by the university of houston institutional animal care and use committee . prior to commencement of experiments associated with this study , each juvenile monkey ( 4 years of age ) underwent surgical procedures carried out under aseptic conditions under isoflurane anesthesia ( 1.25%2.5% ) . in the first surgery , a head stabilization post was implanted to restrict head movements during the experiments . in subsequent procedures , scleral search coils were implanted in both eyes , using the technique of judge et al . , to record binocular eye movements . after collecting baseline data strabismus correction surgery was performed by an expert strabismus surgeon ( also one of the study authors ) on the left eye only of m1 and right eye only of m2 and consisted of lateral rectus recession ( weakening the lateral rectus muscle ) and medial rectus resection ( strengthening the medial rectus muscle ) to improve their strabismus angle . we specifically chose to operate on only one eye , because we wanted to use the fellow eye as a control . the goal of data acquisition and analysis was to perform a longitudinal evaluation of eye alignment and eye movements following strabismus correction surgery . therefore , binocular eye movement data were collected as the animals performed central and eccentric fixation , saccades ( 15 horizontal and vertical target locations ) , and smooth pursuit ( 0.3 hz , 15 ) tasks under monocular left eye or right eye viewing conditions . alignment data were collected at five time points : before surgical treatment ( pre ) , the day after surgical treatment ( p1d ) , 1 week after treatment ( p1w ) , 1 month after treatment ( p1 m ) , and 6 months after treatment ( p6 m ) . saccade and smooth - pursuit data were analyzed at pre , p1w , p1 m , and p6 m because there were not enough experimental trials collected at p1d . binocular data were collected using the magnetic search coil technique ( primelec industries , regensdorf , switzerland ) . eye coils were calibrated at the beginning of each experiment by rewarding the animal with small amounts of juice as they looked at a series of targets projected along the horizontal or vertical meridian on a screen at distance of 57 cm . calibration of each eye was performed independently during monocular viewing forced by occluding one or the other eye using liquid crystal shutter goggles ( citizen fine devices , nagano , japan ) that were under computer control . visual stimuli were generated using the bits # visual stimulus generator ( cambridge research systems , cambridge , united kingdom ) and psychtoolbox 3 operated under computer control and presented using a depthq projector running at a 120-hz frame rate ( lightspeed design , inc . , the target and binocular eye position signals were passed through anti - aliasing filters with a cutoff of 400 hz before digitization at 2.79 khz with 12-bit precision ( alphalab snr system ; alpha - omega engineering , nazareth , israel ) . all eye movement data were additionally calibrated offline and filtered using a software finite impulse response ( fir ) low - pass filter with a cutoff of 80 hz prior to further analysis . analysis of eye movement data was partially automated using custom scripts written in matlab ( mathworks , natick , ma , usa ) . for saccade data , the computer displayed target position , binocular eye position , eye velocity , and eye acceleration traces of a single saccade trial . velocity and acceleration signals were generated by digital differentiation of the position signal using a central difference algorithm . the investigator viewed the traces and decided whether the saccade trial was to be accepted or rejected . trials that were rejected were usually those in which the animal was not fixating prior to the target step , the saccade did not appear to be directed toward the target , or the saccade did not occur within 400 ms of the target step . once a decision to accept the trial was made , mean and sd of control eye acceleration prior to the saccade were calculated over a 70-ms fixation period . saccade onset was automatically determined by the software as the first time point at which eye acceleration was greater than 5 sds away from the control eye acceleration , and saccade offset was determined as the last time point at which eye deceleration was less than 5 sds away from the same mean eye acceleration . although detection of saccade onset and offset was automated , the investigator visually examined the velocity and acceleration traces of every saccade and had the option of either accepting or changing the computer selection , although this was not typical . once the saccade was identified , saccade metric parameters ( amplitude and peak velocity ) for each eye were calculated . main sequence plots of horizontal saccade peak velocity versus horizontal saccade amplitude were fitted with the following exponential rise to maximum equation : where amp = horizontal saccade amplitude , pv = horizontal saccade peak velocity , a = constant determining exponential rise , and pvmax = asymptotic peak velocity . for sinusoidal smooth - pursuit analysis then smooth - pursuit gain ( ratio of peak eye velocity to peak target velocity ) was calculated separately for nasalward and temporalward movements . to assess fixation stability , 10 seconds of eye movement data from both the eyes were analyzed as the monkey maintained monocular fixation at a stationary straight - ahead target . fixation stability was quantified by calculating the bivariate contour ellipse area ( bcea ) , a metric that quantifies the area of the region over which eye positions are dispersed during attempted fixation . therefore , a smaller value for bcea is indicative of greater fixation stability . the bcea encompassing 68.2% of fixation points was calculated using the following equation : , where x = sd of horizontal eye position , y = sd of vertical eye position , 2.291 is the value ( 2 df ) corresponding to a probability of 0.68 , and p is the pearson product moment correlation coefficient of horizontal and vertical eye positions . for statistical analysis , the kruskal - wallis 1-way anova on ranks was performed , and if significant , post hoc testing was performed using dunn 's method . prior to treatment , both monkeys had an exotropia whose magnitude depended on the eye of fixation , a likely indicator for dissociated horizontal deviation . m1 had an exotropia of 27 under left eye viewing ( lev ) conditions and 30 under right eye viewing ( rev ) conditions . m2 had an exotropia of 37 under lev conditions and 30 during rev . immediately after surgery ( p1d ) , exotropia in m1 reduced to 10 ( lev ) and 11 ( rev ) and exotropia in m2 reduced to 9 ( lev ) and 12 ( rev ) . the initial decrease in strabismus angle after the surgery was followed by a gradual reversal toward presurgical exotropia by the end of 6 months . figure 1 is a hess plot showing alignment at different time points as the animals fixated a series of targets along the horizontal and vertical meridian . by 6 months after the correction surgery , m1 showed an exotropia of 20 during either eye viewing and m2 showed an exotropia of 30 ( lev ) and 20 ( rev ) . thus , it appeared that both animals reverted to 60%80% of their original strabismus angle within 6 months . alignment was also examined at 10 months , and the strabismus angle for m1 was still 20 but had changed slightly in m2 to 24 ( lev ) and 18 ( rev ) . longitudinal change in eye misalignment following strabismus treatment in m1 ( a d ) and m2 ( e h ) . plots show eye positions of the viewing eye ( b , c , e , h ) and the nonviewing eye ( a , d , f , g ) during monocular viewing of a series of targets along the horizontal or vertical meridian . a , b ( m1 ) and e , f ( m2 ) show data collected when the animal viewed with their untreated eye ( treated eye under cover ) and c , d ( m1 ) and g , h ( m2 ) show data collected when the animals viewed with their treated eye . each plot shows data collected at five time points : before surgery ( pre ; blue circle ) , 1 day after surgery ( p1d ; red triangle ) , 1 week after surgery ( p1w ; yellow square ) , 1 month after surgery ( p1 m ; gray diamond ) , and 6 months after surgery ( p6 ; green inverted triangle ) . pairs of plots on each row show that the viewing eye is on target while the covered eye is abducted . rightward and upward eye positions are positive and leftward and downward eye positions are negative . we analyzed 4021 saccades at four time points ( pre [ 1449 saccades ] , p1w [ 648 saccades ] , p1 m [ 1103 saccades ] , and p6 m [ 821 saccades ] ) . because the numbers of saccadic trials acquired on any single day were relatively low , data from multiple days near the indicated time point were pooled for analysis . average saccade gains ( ratio of saccade amplitude to target amplitude ) of the two animals are summarized in figure 2 . in general , saccade gain of the viewing eye , irrespective of whether the animal viewed with the treated or untreated eye , did not show significant longitudinal changes ( blue bars in 2a , 2c ; red bars in 2b , 2d ) , as might be expected if the goal of the viewing eye is to saccade onto the target . longitudinal changes in the nonviewing eye were more striking , although slightly different in the two monkeys . 2a ) , the treated eye ( red bars ) showed a significant increase in saccade gain after surgery that decreased to presurgery values by p6 . on the other hand , when m2 viewed with the untreated eye ( fig . 2c ) , there was an immediate drop in saccade gain in the treated eye ( red bars ) following surgery that reverted to presurgical values by p1m p6 . note that when viewing with the untreated eye , any changes in treated eye saccade gains can be attributed to adaptive muscle or neural changes . 2b ) , the untreated eye saccade gain ( blue bars ) decreased or remained the same at p1w and thereafter increased by p6 ( greater than pre values for leftward saccades ) . in m2 ( fig . 2d ) , the untreated eye saccade gain increased at p1w and thereafter decreased by p6 for leftward saccades only . note that when viewing with the treated eye , any changes in untreated eye saccade gains are likely driven by a combination of hering 's law based neural mechanisms and adaptive untreated eye muscle or neural changes triggered by the surgical intervention of the treated muscle . longitudinal change in horizontal saccade gains in m1 ( a , b ) and m2 ( c , d ) following strabismus treatment . bars in each plot show means and sds of saccade gains of the untreated eye ( blue ) and treated eye ( red ) at four time points ( pre , p1w , p1 m , and p6 ) . the asterisks represent statistically significant difference compared with presurgical values ( blue , untreated eye ; red , treated eye ) . main sequence plots are shown in figure 3 along with the mean and 95% upper and lower confidence limits . longitudinal changes in saccade peak velocity were relatively small and difficult to identify because of the large variability in saccade velocities . there was a slight but significant increase in saccade velocity in both the treated and untreated eyes as animal m1 viewed with the treated eye ( figs . 3c , 3d ) as p6 velocities are higher than they were before surgery . a similar , albeit greater magnitude , increase in peak saccade velocity was observed in both the treated and untreated eyes as animal m2 viewed with the treated eye ( more so for rightward than leftward saccades ; figs . estimated fit parameters for pvmax and a from equation 1 for each of the eyes under the different viewing conditions and at the different time points are provided in the table . h ) showing horizontal saccade amplitude on the x - axis and horizontal saccade peak velocity on the y - axis . negative saccade amplitudes indicate leftward saccades and positive amplitudes indicate rightward saccades . left column panels ( a , c , e , g ) show data from the untreated eye and right column panels ( b , d , f , g ) show data from the treated eye . each data point represents a single saccade , and lines represent exponential rise - to - maximum fits to the main sequence data . for these plots , viewing and nonviewing eyes ' data were collected when the animals viewed monocularly with either treated eye or untreated eye . data points and curve fits are overlaid for saccade data collected at pre ( blue circle ) , p1w ( red triangle ) , p1 m ( gray diamond ) , and p6 ( green inverted triangle ) . fit parameters for main sequence plots derived from equation 1 summary of leftward and rightward smooth - pursuit gains obtained from 15 to 30 cycles of smooth pursuit at each time point for each monkey are shown in figure 4 along with results of statistical comparison to presurgical smooth - pursuit gains . longitudinal trends in smooth - pursuit gains reflected the trends observed for saccadic eye movement gains . gains of viewing eye remained more or less constant following surgery ( blue bars in figs . 4a ) , there was a significant increase in gain in the treated eye ( red bars ) for both rightward and leftward pursuit that was mostly reversed by p6 . on the other hand , when m2 viewed with the untreated eye ( fig . 4c ) , there was a significant decrease in gain in the treated eye that mostly reversed by p6 . smooth - pursuit gains calculated when the animals viewed with their treated eyes ( figs . 4b , 4d ) also followed trends observed during saccade analysis . longitudinal change in horizontal smooth - pursuit gains in m1 ( a , b ) and m2 ( c , d ) following strabismus treatment . bars in each plot show mean smooth - pursuit gains and sds of the untreated eye ( blue ) and treated eye ( red ) at four time points ( pre , p1w , p1 m , and p6 ) . leftward and rightward smooth - pursuit was analyzed separately and are represented by the directional arrows . the asterisks represent statistically significant difference compared with presurgical values ( blue , untreated eye ; red , treated eye ) . strabismic humans and monkeys often exhibit increased monocular viewing smooth - pursuit tracking gains for nasalward target motion compared with temporalward motion ( naso - temporal asymmetry ) . both the monkeys in the current study displayed a naso - temporal ( n / t ) asymmetry prior to surgery ( except m2 during left eye viewing ; fig . 5b , blue bars ) , and we wondered whether surgical manipulation of eom resulted in any modification of this feature of strabismus . a naso - temporal asymmetry index was calculated as the ratio of viewing eye temporalward smooth - pursuit gain to viewing eye nasalward smooth - pursuit gain for both treated and untreated eyes viewing conditions . in m1 , the naso - temporal asymmetry persisted mostly unchanged following surgery during either untreated or treated eye viewing . in m2 also , n / t asymmetry was unchanged during treated ( right ) eye viewing . during untreated ( left ) eye viewing , there was a slight improvement in the index ( reduced asymmetry ) that reversed by p1 m . bars in each plot show a naso - temporal asymmetry index calculated as the ratio of the viewing eye temporalward and nasalward smooth - pursuit gain for m1 ( a ) and m2 ( b ) at four time points ( pre , p1w , p1 m , p6 ) . n / t asymmetry index during untreated eye viewing is shown in blue and during treated eye viewing is shown in red . fixation stability was assessed by calculating the bcea metric for each eye during monocular viewing ( fig . when the untreated eye was viewing , fixation stability of the nonviewing treated eye ( red bars ) improved ( lower bcea ) after surgery but regressed by p6 in m2 only . these data were not amenable for statistical testing because each bar represents a single data point for the fixation stability metric . fixation stability of the viewing untreated eye ( blue bars ) did not show consistent longitudinal effects . when the treated eye was viewing , fixation stability of nonviewing untreated eye ( blue bars ) showed postsurgical improvement that regressed by p6 in m1 but no consistent longitudinal effects in m2 . fixation stability of the nonviewing eye was worse at all time points for both the monkeys , consistent with previous studies of fixation stability in strabismic monkeys . longitudinal change in fixation stability ( m1 , a ; m2 , b ) following strabismus treatment . bars in each plot show bcea values of the treated eye ( red ) and untreated eye ( blue ) during either eye viewing at the four time points ( pre , p1w , p1 m , p6 m ) . prior to treatment , both monkeys had an exotropia whose magnitude depended on the eye of fixation , a likely indicator for dissociated horizontal deviation . m1 had an exotropia of 27 under left eye viewing ( lev ) conditions and 30 under right eye viewing ( rev ) conditions . m2 had an exotropia of 37 under lev conditions and 30 during rev . immediately after surgery ( p1d ) , exotropia in m1 reduced to 10 ( lev ) and 11 ( rev ) and exotropia in m2 reduced to 9 ( lev ) and 12 ( rev ) . the initial decrease in strabismus angle after the surgery was followed by a gradual reversal toward presurgical exotropia by the end of 6 months . figure 1 is a hess plot showing alignment at different time points as the animals fixated a series of targets along the horizontal and vertical meridian . by 6 months after the correction surgery , m1 showed an exotropia of 20 during either eye viewing and m2 showed an exotropia of 30 ( lev ) and 20 ( rev ) . thus , it appeared that both animals reverted to 60%80% of their original strabismus angle within 6 months . alignment was also examined at 10 months , and the strabismus angle for m1 was still 20 but had changed slightly in m2 to 24 ( lev ) and 18 ( rev ) . longitudinal change in eye misalignment following strabismus treatment in m1 ( a d ) and m2 ( e h ) . plots show eye positions of the viewing eye ( b , c , e , h ) and the nonviewing eye ( a , d , f , g ) during monocular viewing of a series of targets along the horizontal or vertical meridian . a , b ( m1 ) and e , f ( m2 ) show data collected when the animal viewed with their untreated eye ( treated eye under cover ) and c , d ( m1 ) and g , h ( m2 ) show data collected when the animals viewed with their treated eye . each plot shows data collected at five time points : before surgery ( pre ; blue circle ) , 1 day after surgery ( p1d ; red triangle ) , 1 week after surgery ( p1w ; yellow square ) , 1 month after surgery ( p1 m ; gray diamond ) , and 6 months after surgery ( p6 ; green inverted triangle ) . pairs of plots on each row show that the viewing eye is on target while the covered eye is abducted . rightward and upward eye positions are positive and leftward and downward eye positions are negative . we analyzed 4021 saccades at four time points ( pre [ 1449 saccades ] , p1w [ 648 saccades ] , p1 m [ 1103 saccades ] , and p6 m [ 821 saccades ] ) . because the numbers of saccadic trials acquired on any single day were relatively low , data from multiple days near the indicated time point were pooled for analysis . average saccade gains ( ratio of saccade amplitude to target amplitude ) of the two animals are summarized in figure 2 . in general , saccade gain of the viewing eye , irrespective of whether the animal viewed with the treated or untreated eye , did not show significant longitudinal changes ( blue bars in 2a , 2c ; red bars in 2b , 2d ) , as might be expected if the goal of the viewing eye is to saccade onto the target . longitudinal changes in the nonviewing eye were more striking , although slightly different in the two monkeys . when m1 viewed with the untreated eye ( fig . 2a ) , the treated eye ( red bars ) showed a significant increase in saccade gain after surgery that decreased to presurgery values by p6 . on the other hand , when m2 viewed with the untreated eye ( fig . 2c ) , there was an immediate drop in saccade gain in the treated eye ( red bars ) following surgery that reverted to presurgical values by p1m p6 . note that when viewing with the untreated eye , any changes in treated eye saccade gains can be attributed to adaptive muscle or neural changes . 2b ) , the untreated eye saccade gain ( blue bars ) decreased or remained the same at p1w and thereafter increased by p6 ( greater than pre values for leftward saccades ) . in m2 2d ) , the untreated eye saccade gain increased at p1w and thereafter decreased by p6 for leftward saccades only . note that when viewing with the treated eye , any changes in untreated eye saccade gains are likely driven by a combination of hering 's law based neural mechanisms and adaptive untreated eye muscle or neural changes triggered by the surgical intervention of the treated muscle . longitudinal change in horizontal saccade gains in m1 ( a , b ) and m2 ( c , d ) following strabismus treatment . bars in each plot show means and sds of saccade gains of the untreated eye ( blue ) and treated eye ( red ) at four time points ( pre , p1w , p1 m , and p6 ) . the asterisks represent statistically significant difference compared with presurgical values ( blue , untreated eye ; red , treated eye ) . main sequence plots are shown in figure 3 along with the mean and 95% upper and lower confidence limits . longitudinal changes in saccade peak velocity were relatively small and difficult to identify because of the large variability in saccade velocities . there was a slight but significant increase in saccade velocity in both the treated and untreated eyes as animal m1 viewed with the treated eye ( figs . 3c , 3d ) as p6 velocities are higher than they were before surgery . a similar , albeit greater magnitude , increase in peak saccade velocity was observed in both the treated and untreated eyes as animal m2 viewed with the treated eye ( more so for rightward than leftward saccades ; figs . a from equation 1 for each of the eyes under the different viewing conditions and at the different time points are provided in the table . h ) showing horizontal saccade amplitude on the x - axis and horizontal saccade peak velocity on the y - axis . negative saccade amplitudes indicate leftward saccades and positive amplitudes indicate rightward saccades . left column panels ( a , c , e , g ) show data from the untreated eye and right column panels ( b , d , f , g ) show data from the treated eye . each data point represents a single saccade , and lines represent exponential rise - to - maximum fits to the main sequence data . for these plots , viewing and nonviewing eyes ' data were collected when the animals viewed monocularly with either treated eye or untreated eye . data points and curve fits are overlaid for saccade data collected at pre ( blue circle ) , p1w ( red triangle ) , p1 m ( gray diamond ) , and p6 ( green inverted triangle ) . summary of leftward and rightward smooth - pursuit gains obtained from 15 to 30 cycles of smooth pursuit at each time point for each monkey are shown in figure 4 along with results of statistical comparison to presurgical smooth - pursuit gains . longitudinal trends in smooth - pursuit gains reflected the trends observed for saccadic eye movement gains . gains of viewing eye remained more or less constant following surgery ( blue bars in figs . 4a ) , there was a significant increase in gain in the treated eye ( red bars ) for both rightward and leftward pursuit that was mostly reversed by p6 . 4c ) , there was a significant decrease in gain in the treated eye that mostly reversed by p6 . smooth - pursuit gains calculated when the animals viewed with their treated eyes ( figs . 4b , 4d ) also followed trends observed during saccade analysis . longitudinal change in horizontal smooth - pursuit gains in m1 ( a , b ) and m2 ( c , d ) following strabismus treatment . bars in each plot show mean smooth - pursuit gains and sds of the untreated eye ( blue ) and treated eye ( red ) at four time points ( pre , p1w , p1 m , and p6 ) . leftward and rightward smooth - pursuit was analyzed separately and are represented by the directional arrows . the asterisks represent statistically significant difference compared with presurgical values ( blue , untreated eye ; red , treated eye ) . strabismic humans and monkeys often exhibit increased monocular viewing smooth - pursuit tracking gains for nasalward target motion compared with temporalward motion ( naso - temporal asymmetry ) . both the monkeys in the current study displayed a naso - temporal ( n / t ) asymmetry prior to surgery ( except m2 during left eye viewing ; fig . 5b , blue bars ) , and we wondered whether surgical manipulation of eom resulted in any modification of this feature of strabismus . a naso - temporal asymmetry index was calculated as the ratio of viewing eye temporalward smooth - pursuit gain to viewing eye nasalward smooth - pursuit gain for both treated and untreated eyes viewing conditions . in m1 , the naso - temporal asymmetry persisted mostly unchanged following surgery during either untreated or treated eye viewing . in m2 also , n / t asymmetry was unchanged during treated ( right ) eye viewing . during untreated ( left ) eye viewing , there was a slight improvement in the index ( reduced asymmetry ) that reversed by p1 m . longitudinal change in naso - temporal asymmetry following strabismus treatment . bars in each plot show a naso - temporal asymmetry index calculated as the ratio of the viewing eye temporalward and nasalward smooth - pursuit gain for m1 ( a ) and m2 ( b ) at four time points ( pre , p1w , p1 m , p6 ) . n / t asymmetry index during untreated eye viewing is shown in blue and during treated eye viewing is shown in red . fixation stability was assessed by calculating the bcea metric for each eye during monocular viewing ( fig . when the untreated eye was viewing , fixation stability of the nonviewing treated eye ( red bars ) improved ( lower bcea ) after surgery but regressed by p6 in m2 only . these data were not amenable for statistical testing because each bar represents a single data point for the fixation stability metric . fixation stability of the viewing untreated eye ( blue bars ) did not show consistent longitudinal effects . when the treated eye was viewing , fixation stability of nonviewing untreated eye ( blue bars ) showed postsurgical improvement that regressed by p6 in m1 but no consistent longitudinal effects in m2 . fixation stability of the nonviewing eye was worse at all time points for both the monkeys , consistent with previous studies of fixation stability in strabismic monkeys . longitudinal change in fixation stability ( m1 , a ; m2 , b ) following strabismus treatment . bars in each plot show bcea values of the treated eye ( red ) and untreated eye ( blue ) during either eye viewing at the four time points ( pre , p1w , p1 m , p6 m ) . to our knowledge , this study is the first attempt at investigating longitudinal changes in eye alignment and eye movements following strabismus treatment surgery in either humans or animal models . in addition to providing a detailed account of time course of change in alignment and movements , these data will provide a basis for examination of neural and muscle plasticity that is likely to occur as a consequence of correction surgery . our main findings in this study were that 1 ) eye misalignment reduced as a consequence of resect recess surgery , but regressed to close to presurgical values by 6 months after surgery ; 2 ) patterns of longitudinal changes in saccadic and smooth - pursuit eye movements also showed immediate postsurgical changes that reversed by 6 months after treatment , although naso - temporal asymmetry of smooth - pursuit tracking was unchanged ; 3 ) there were only small changes in fixation stability ; and 4 ) the overall similarity in response to human strabismus surgical treatment validates our monkey model for strabismus . below we discuss each of our findings in the context of longitudinal adaptive changes occurring in strabismus . compared with dynamic changes in eye movements that were generally small but significant ( discussed later ) , longitudinal changes in eye alignment were clear and consistent in the two monkeys in our study . both the exotropic monkeys in the study responded to treatment in that exotropia was reduced significantly to 30% of presurgical misalignment immediately after surgery as might be expected following a resect ( day after surgery ) , the reduction in misalignment observed when viewing with the untreated eye is possibly only a consequence of the change in contractility of the treated eom as the brain may not have had sufficient time to adapt . in other words , the neural signals to the eom of viewing ( untreated ) and nonviewing ( treated ) eyes are expected to be the same as in the presurgical condition . when the monkey is forced to view with the treated eye , the brain must alter the neural signals to the treated muscles to compensate for their altered contractility to allow fixation of the target , for example , increase in neuronal drive of recessed lateral rectus from ipsilateral abducens nucleus . the anatomical connection between abducens nucleus and contralateral oculomotor nucleus ( underpinnings of hering 's law ) leads to increase in neuronal drive to the medial rectus muscle of the contralateral ( in this case untreated ) eye . note that the change in neural drives to the treated and untreated eye eom when the monkey is forced to view with the treated eye is not a long - term adaptive response per se as the brain is only momentarily adjusting signals to enable target acquisition and fixation . following the initial reduction in strabismus angle , the angle of misalignment reverted to 60%80% of presurgical value by 6 months after treatment , again similar to outcomes of strabismus correction surgery in a proportion of human subjects whose initial procedure resulted in undercorrection . the lack of persistence of the initial improvement in strabismus angle may be because improvement in alignment in the two study animals was beyond the range at which central binocular factors would be expected to be able to maintain the initial improvement in postsurgical ocular alignment . therefore , remodeling at the level of the eom along with central neural adaptation could act to fight the intent of surgery ( pullela m , et al . additional studies directly examining neural drives and muscle contractility are needed to understand the relative plastic changes due to these two factors . another point to note is that the patterns of alignment change following treatment , which are similar to human strabismus response to treatment , also help to validate the monkey model as suitable for investigating strabismus mechanisms . there were significant small magnitude changes in saccadic and smooth - pursuit gains following treatment , although it was different in the two monkeys ( figs . thus , saccade and smooth - pursuit gain for m1 showed an increase after surgery , whereas saccade and smooth - pursuit gains for m2 showed a decrease after surgery , although both animals regained presurgical gain values by p6 . analysis of performance of saccades in human subjects after undergoing strabismus surgery showed an improvement after surgery that was proposed as due to improvement in binocular coordination because eye misalignment is corrected . because full alignment was not achieved in our monkeys even on p1d , we suggest that the observed changes in saccade and smooth - pursuit gain in our monkeys are more likely to be driven by brain and muscle adaptation that is unrelated to binocular coordination . it is unclear why eye movement gains increased after surgery in m1 but decreased after surgery in m2 . we suggest that this likely represents variability that might be observed in humans as well and could represent differences in relative adaptation ( neural or muscle related ) of the recessed versus the resected muscle . confirmation of this hypothesis would come from future studies that measure neuronal drive to the treated and untreated muscles . the most likely explanation is that saccade velocities are inherently too variable to show significant changes following surgical treatment . we also did not find any consistent changes in n / t asymmetry during monocular smooth - pursuit . because n / t asymmetry is fundamentally due to developmental loss of binocular inputs from sensory areas such as middle temporal / middle superior temporal ( mt / mst ) to brainstem areas such as the nucleus of the optic tract ( not ) , any longitudinal changes in n / t asymmetry would have been , in fact , surprising . fixation stability or lack thereof in strabismic animals and humans is usually influenced by presence of visual problems such as amblyopia and oculomotor instabilities such as nystagmus and excessive drifts . in addition , fixation stability may be influenced by several visual factors such as target size , shape , contrast , and luminance . we measured fixation stability in these animals at the different time points and found a temporary improvement in fixation stability of the treated eye when the untreated eye was viewing but no consistent changes when the treated eye was viewing . dell'osso et al . suggested that tenotomy surgery reduces nystagmus intensity in humans with infantile nystagmus syndrome due to manipulation of proprioceptive receptors in the eom . it is possible that similar mechanisms are at play following the strabismus correction surgery that we performed , although another study that attempted tenotomy surgery in monkeys with infantile nystagmus showed inconsistent results . our data suggest that surgical treatment of strabismus is followed by an adaptive response that evolves and reaches a steady state within the first 6 months after treatment . because adaptation can either oppose or facilitate the outcome of treatment strategies , the fundamental challenge to improving treatment methods is to identify and devise methods that will induce adaptation that promotes desired treatment effects while avoiding adaptation that may oppose or reverse desired treatment effects . as suggested earlier , both central neural adaption and peripheral muscle remodeling are likely driving the final state of alignment and ultimately determining the success or failure of the surgical treatment . immediate ( p1d ) changes in alignment and eye movements are likely driven by changes in muscle contractility alone , but later changes ( p1 m and p6 m ) are likely due to a combination of neural plasticity and peripheral remodeling . behavioral studies alone can not readily differentiate between these mechanisms , but they are critical to develop a framework to conduct and interpret neural recording studies or studies that evaluate muscle function . such neuronal studies are underway in our laboratory in the same animals as in this study . major portions of the strabismus treatment literature are accounts of outcomes following different types of surgical procedures . the strength of these studies is the relatively large number and large variety ( e.g. , esotropes versus exotropes and patients with residual binocularity versus patients with no binocularity ) of patients that are evaluated . due to several ( mostly practical ) reasons , monkey studies can not hope to replicate the large n of human studies . however , the strength of our monkey investigation lies in the ability to perform careful and controlled longitudinal evaluation in the same animals that is not possible in human clinical investigation . moreover , these behavioral studies provide a framework to perform a neurophysiologic investigation that is not possible in humans and is critical to extend our understanding of strabismus mechanisms including plasticity . it is possible that outcomes in the current study might have been different ( i.e. , monkeys ' exotropia does not revert ) if we had chosen to treat both eyes and attempted to more closely align the eyes , given the general notion that surgical realignment in the vicinity of 510 pd helps facilitate binocularity . however , on the other hand , both the animals in this study were reared with prisms from the first day of birth , likely preventing the development of even coarse stereopsis . also , we purposely did not attempt a second procedure on the fellow eye because we wanted to be able to use the fellow eye as a control and thereby ascertain the nature and time course of plasticity that follows surgical treatment . using the fellow eye as an untreated control is also quite critical when evaluating neural responses that are driving the extraocular muscles . compared with dynamic changes in eye movements that were generally small but significant ( discussed later ) , longitudinal changes in eye alignment were clear and consistent in the two monkeys in our study . both the exotropic monkeys in the study responded to treatment in that exotropia was reduced significantly to 30% of presurgical misalignment immediately after surgery as might be expected following a resect ( day after surgery ) , the reduction in misalignment observed when viewing with the untreated eye is possibly only a consequence of the change in contractility of the treated eom as the brain may not have had sufficient time to adapt . in other words , the neural signals to the eom of viewing ( untreated ) and nonviewing ( treated ) eyes are expected to be the same as in the presurgical condition . when the monkey is forced to view with the treated eye , the brain must alter the neural signals to the treated muscles to compensate for their altered contractility to allow fixation of the target , for example , increase in neuronal drive of recessed lateral rectus from ipsilateral abducens nucleus . the anatomical connection between abducens nucleus and contralateral oculomotor nucleus ( underpinnings of hering 's law ) leads to increase in neuronal drive to the medial rectus muscle of the contralateral ( in this case untreated ) eye . note that the change in neural drives to the treated and untreated eye eom when the monkey is forced to view with the treated eye is not a long - term adaptive response per se as the brain is only momentarily adjusting signals to enable target acquisition and fixation . following the initial reduction in strabismus angle , the angle of misalignment reverted to 60%80% of presurgical value by 6 months after treatment , again similar to outcomes of strabismus correction surgery in a proportion of human subjects whose initial procedure resulted in undercorrection . the lack of persistence of the initial improvement in strabismus angle may be because improvement in alignment in the two study animals was beyond the range at which central binocular factors would be expected to be able to maintain the initial improvement in postsurgical ocular alignment . therefore , remodeling at the level of the eom along with central neural adaptation could act to fight the intent of surgery ( pullela m , et al . additional studies directly examining neural drives and muscle contractility are needed to understand the relative plastic changes due to these two factors . another point to note is that the patterns of alignment change following treatment , which are similar to human strabismus response to treatment , also help to validate the monkey model as suitable for investigating strabismus mechanisms . there were significant small magnitude changes in saccadic and smooth - pursuit gains following treatment , although it was different in the two monkeys ( figs . 2 , 4 ) . thus , saccade and smooth - pursuit gain for m1 showed an increase after surgery , whereas saccade and smooth - pursuit gains for m2 showed a decrease after surgery , although both animals regained presurgical gain values by p6 . analysis of performance of saccades in human subjects after undergoing strabismus surgery showed an improvement after surgery that was proposed as due to improvement in binocular coordination because eye misalignment is corrected . because full alignment was not achieved in our monkeys even on p1d , we suggest that the observed changes in saccade and smooth - pursuit gain in our monkeys are more likely to be driven by brain and muscle adaptation that is unrelated to binocular coordination . it is unclear why eye movement gains increased after surgery in m1 but decreased after surgery in m2 . we suggest that this likely represents variability that might be observed in humans as well and could represent differences in relative adaptation ( neural or muscle related ) of the recessed versus the resected muscle . confirmation of this hypothesis would come from future studies that measure neuronal drive to the treated and untreated muscles . the most likely explanation is that saccade velocities are inherently too variable to show significant changes following surgical treatment . we also did not find any consistent changes in n / t asymmetry during monocular smooth - pursuit . because n / t asymmetry is fundamentally due to developmental loss of binocular inputs from sensory areas such as middle temporal / middle superior temporal ( mt / mst ) to brainstem areas such as the nucleus of the optic tract ( not ) , any longitudinal changes in n / t asymmetry would have been , in fact , surprising . fixation stability or lack thereof in strabismic animals and humans is usually influenced by presence of visual problems such as amblyopia and oculomotor instabilities such as nystagmus and excessive drifts . in addition , fixation stability may be influenced by several visual factors such as target size , shape , contrast , and luminance . we measured fixation stability in these animals at the different time points and found a temporary improvement in fixation stability of the treated eye when the untreated eye was viewing but no consistent changes when the treated eye was viewing . dell'osso et al . suggested that tenotomy surgery reduces nystagmus intensity in humans with infantile nystagmus syndrome due to manipulation of proprioceptive receptors in the eom . it is possible that similar mechanisms are at play following the strabismus correction surgery that we performed , although another study that attempted tenotomy surgery in monkeys with infantile nystagmus showed inconsistent results . our data suggest that surgical treatment of strabismus is followed by an adaptive response that evolves and reaches a steady state within the first 6 months after treatment . because adaptation can either oppose or facilitate the outcome of treatment strategies , the fundamental challenge to improving treatment methods is to identify and devise methods that will induce adaptation that promotes desired treatment effects while avoiding adaptation that may oppose or reverse desired treatment effects . as suggested earlier , both central neural adaption and peripheral muscle remodeling are likely driving the final state of alignment and ultimately determining the success or failure of the surgical treatment . immediate ( p1d ) changes in alignment and eye movements are likely driven by changes in muscle contractility alone , but later changes ( p1 m and p6 m ) are likely due to a combination of neural plasticity and peripheral remodeling . behavioral studies alone can not readily differentiate between these mechanisms , but they are critical to develop a framework to conduct and interpret neural recording studies or studies that evaluate muscle function . such neuronal studies are underway in our laboratory in the same animals as in this study . major portions of the strabismus treatment literature are accounts of outcomes following different types of surgical procedures . the strength of these studies is the relatively large number and large variety ( e.g. , esotropes versus exotropes and patients with residual binocularity versus patients with no binocularity ) of patients that are evaluated . due to several ( mostly practical ) reasons , monkey studies can not hope to replicate the large n of human studies . however , the strength of our monkey investigation lies in the ability to perform careful and controlled longitudinal evaluation in the same animals that is not possible in human clinical investigation . moreover , these behavioral studies provide a framework to perform a neurophysiologic investigation that is not possible in humans and is critical to extend our understanding of strabismus mechanisms including plasticity . it is possible that outcomes in the current study might have been different ( i.e. , monkeys ' exotropia does not revert ) if we had chosen to treat both eyes and attempted to more closely align the eyes , given the general notion that surgical realignment in the vicinity of 510 pd helps facilitate binocularity . however , on the other hand , both the animals in this study were reared with prisms from the first day of birth , likely preventing the development of even coarse stereopsis . also , we purposely did not attempt a second procedure on the fellow eye because we wanted to be able to use the fellow eye as a control and thereby ascertain the nature and time course of plasticity that follows surgical treatment . using the fellow eye as an untreated control is also quite critical when evaluating neural responses that are driving the extraocular muscles .

purposestrabismus correction surgery is well documented in both the literature and practice with varying levels of success and permanence . our goal was to characterize longitudinal changes in eye alignment and eye movements following strabismus correction surgery in a monkey model for developmental strabismus.methodswe studied two juvenile rhesus monkeys with exotropia previously induced via an optical prism - rearing paradigm in infancy . eye misalignment was corrected via a resection recession surgery of the horizontal rectus muscles of one eye . binocular search coils were used to collect eye movement data during smooth - pursuit , saccades , and fixation tasks before surgical treatment , immediately after surgery , and through 6 months after treatment.resultsboth animals showed an immediate 70% reduction in misalignment as a consequence of surgery that regressed to a 20%40% improvement by 6 months after treatment . significant changes were observed in saccade and smooth - pursuit gain of the nonviewing eye after surgery , which also reverted to presurgical values by 6 months . a temporary improvement in fixation stability of the nonviewing eye was observed after surgery ; naso - temporal ( n / t ) asymmetry of monocular smooth - pursuit remained unchanged.conclusionssurgical realignment is followed by plastic changes that often lead to reversal of surgery effects . immediate improvement in misalignment and changes in eye movement gains are likely a result of contractility changes at the level of the extraocular muscle , whereas longer - term effects are likely a combination of neural and muscle adaptation .

Methods Subjects and Rearing Paradigms Surgical Procedures Data Acquisition and Experimental Procedures Data Analysis Results Longitudinal Changes in Eye Alignment Longitudinal Changes in Saccade Parameters Longitudinal Changes in Smooth-Pursuit Behavior Longitudinal Changes in Fixation Stability Discussion Eye Alignment Saccadic and Smooth-Pursuit Eye Movements Fixation Stability Implications for Plasticity Mechanisms Limitations of Study

PMC3956398

the longevity of a ceramic restoration appears to be linked to its physical and mechanical properties , as well as reliable bonding between the restoration and the tooth . in order to obtain effective bonding between feldspathic ceramics and the tooth substrate , surface treatment is necessary for both the internal aspect of the ceramic and the tooth structure . the use of methods to pretreat the ceramic surface , such as mechanical ( burs and aluminum oxide sandblasting ) , chemical ( 2.5 - 10% hydrofluoric acid etch and silane ) or a combination of both ( silicon oxide sandblasting ) optimize adhesion between the ceramic and the resin cement . the combination of hydrofluoric acid and silane on the internal surface of the ceramic has been the most recommended method to increase bond strength between the ceramic and the tooth substrate when using resin cements . however , it has been demonstrated that following hydrofluoric acid removal from the ceramic surface , considerable amounts of deposits are formed , which can affect bond strength . some studies have proposed cleaning techniques for ceramic surface aiming at removing the residues left by hydrofluoric acid etching , including the use of ultrasonic bath , 37% phosphoric acid smear , rinsing under running wateror a combination of 37% phosphoric acid smear and ultrasonic bath . considering the lack of an established protocol to remove residues created by hydrofluoric acid etching of the ceramic surface , the aims of this study were to evaluate : a ) the micro - shear bond strength and failure mode between the feldspathic ceramic and the adhesive system following pretreatment of the ceramic surface with different cleaning methods to remove particles generated by hydrofluoric acid etching ; b ) the feldspathic ceramic surface micromorphology after using different cleaning methods to remove hydrofluoric acid etching residues by scanning electron microscopy ( sem ) . forty six feldspathic ceramic discs ( super porcelain ex-3 , noritake kizai co. , ltd , nishi - ku , nagoya , japan , batch 011906 , shade a3,5b ) were made , 6 of which were used to assess micromorphology and 40 were used for micro - shear bond strength testing . the ceramic discs were obtained from 12.5 mm diameter by 3.6 mm thick wax patterns using lost wax casting , which gave room to the feldspathic ceramic . the ceramic powder / liquid ratio was used as instructed by the manufacturer and the mixture was placed into the plaster mold using a specific brush provided in the ceramic kit . the investment plaster and ceramic set were placed in a furnace for sintering at the temperature specified by the manufacturer . as the ceramic shrinks by approximately 20% during sintering , the final disc dimensions were close to 10 mm in diameter and 3 mm thick . forty ceramic discs were included in pvc cylinders using polyester resin ( hobby automotivo , 3 m do brasil , sumar , sp , brazil ) . the surfaces of the ceramic discs were sequentially polished using silicium carbide sandpaper ( lixa d'gua , 3 m , sumar , sp , brazil ) of grades 400 , 600 and 12005 , mounted on a running water - cooled electric polisher ( aropol 2v , arotec s / a , so paulo , sp , brazil ) . the discs were randomly divided into 5 experimental groups of 8 discs ( n=8 ) . the surface of all ceramic discs was treated with 10% hydrofluoric acid ( villevie , joinville , santa catarina , brazil ) for 2 minutes , followed by water and water spray rinse for 30 seconds . then , the discs were treated according to the experimental groups as follows : group c : no additional surface treatment ; group s : water spray + air for 1 minute , using the 3-in-1 syringe ; group us : ultrasonic bath ( usc1400 , unique ind . ltda , so paulo , sp , brazil ) with distilled water for 5 minutes ; group f : 37% phosphoric acid smear ( acid gel , villevie , joinville , sc , brazil ) , using a disposable brush ( disposable micro - applicators , microbrush , rio de janeiro , rj , brazil ) for 1 minute , followed by rinsing under running water for 1 minute ; group f+us : 37% phosphoric acid smear ( acid gel , villevie , joinville , sc , brazil ) , using a disposable brush ( disposable micro - applicators , microbrush , rio de janeiro , rj , brazil ) for 1 minute , followed by rinsing under running water for 1 minute , combined with ultrasonic bath ( usc1400 , unique ind e com de prod elet . ltda , so paulo , sp , brazil ) with distilled water for 5 minutes . five 0.8 mm diameter x 4 mm high composite resin ( filtek z100 , 3 m espe , st . therefore , a layer of silane ( dentsply , petrpolis , rj , brazil ) was previously applied to bond the resin onto the ceramic . a layer of adhesive ( adper scotchbond - adhesive , 3 m espe , st . paul , mn , usa ) was applied and light - cured using halogen light ( demetron , lc kerr corporation , orange , ca , usa ) for 10 seconds . then , silicone tubes 4 mm high x 0.8 mm diameter were placed on the ceramic surface to be filled with resin composite . after filling the tubes , the resin cylinders were light - cured for 40 seconds ( demetron , lc kerr corporation , orange , ca , usa ) with the light source placed at the upper end of the cylinders , thus light - curing all of them simultaneously . the light - curing equipment was set at 499 mw / cm ( from 491 mw / cm to 507 mw / cm ) with the aid of a radiometer ( radimetro digital , newdent , ribeiro preto , sp , brazil ) and reviewed after every five light - curing cycles . immediately after light - curing , the silicone tubes were cut using a scalpel blade 12 and disposed of in order to obtain 4 mm high x 0.8 mm diameter composite resin cylinders bonded to the ceramic surface , with a bonding area of 0.5 mm . the specimens were stored in a bacteriological incubator ( ecb 1.3 digital , odontobrs ind . ltda , ribeiro preto , sp , brazil ) at 37c for 24 hours until the bond strength tests were performed . the ceramic discs were fixed to a device attached to the universal testing machine ( dl2000 , emic ind e com ltda , so jos dos pinhais , pr , brazil ) . a 0.3 mm diameter stainless steel wire looped around both the extremity of the loading cell and the composite resin cylinder , simultaneously . the wire maintained contact with the inferior semicircle of the cylinders , as close as possible to the area bonded to the ceramic . the test was carried out at a speed of 0.5 mm / min , until bonding failure , for each cylinder sequentially , that is , one at a time . the failure mode was assessed using a stereoscopic lens ( ek3st model , eikonal , so paulo , so paulo , brazil ) at 20x magnification . the failure modes were classified as adhesive failure , ceramic or resin cohesion failure , or mixed . cohesion failure was defined as a fracture in the body of the ceramic or the resin , adhesive failure when there was a fault at the junction between the ceramic and the resin , and mixed when there was both adhesive and cohesion failure , simultaneously . one of the six ceramic discs used to evaluate surface micromorphology did not receive any surface treatment , including hydrofluoric acid . the specimens were then stored in an incubator at 50c for 3 hours to remove any water particles , 24 hours prior to sem assessment . the samples were then placed in airtight glass jars containing silica gel and sealed to ensure moisture elimination . the discs were positioned in the sem ( jsm 5600lv , jeol , tokyo , japan ) and images were obtained at 500x and 2,500x magnification . for each ceramic disc , the mean micro - shear bond strength value for the resin cylinders was considered . after exploratory analysis , the data were tested using one - way analysis of variance ( p<0.05 ) on sas statistical software ( release 9.2 , 2008 , institute inc . , forty ceramic discs were included in pvc cylinders using polyester resin ( hobby automotivo , 3 m do brasil , sumar , sp , brazil ) . the surfaces of the ceramic discs were sequentially polished using silicium carbide sandpaper ( lixa d'gua , 3 m , sumar , sp , brazil ) of grades 400 , 600 and 12005 , mounted on a running water - cooled electric polisher ( aropol 2v , arotec s / a , so paulo , sp , brazil ) . the discs were randomly divided into 5 experimental groups of 8 discs ( n=8 ) . the surface of all ceramic discs was treated with 10% hydrofluoric acid ( villevie , joinville , santa catarina , brazil ) for 2 minutes , followed by water and water spray rinse for 30 seconds . then , the discs were treated according to the experimental groups as follows : group c : no additional surface treatment ; group s : water spray + air for 1 minute , using the 3-in-1 syringe ; group us : ultrasonic bath ( usc1400 , unique ind . ltda , so paulo , sp , brazil ) with distilled water for 5 minutes ; group f : 37% phosphoric acid smear ( acid gel , villevie , joinville , sc , brazil ) , using a disposable brush ( disposable micro - applicators , microbrush , rio de janeiro , rj , brazil ) for 1 minute , followed by rinsing under running water for 1 minute ; group f+us : 37% phosphoric acid smear ( acid gel , villevie , joinville , sc , brazil ) , using a disposable brush ( disposable micro - applicators , microbrush , rio de janeiro , rj , brazil ) for 1 minute , followed by rinsing under running water for 1 minute , combined with ultrasonic bath ( usc1400 , unique ind e com de prod elet . ltda , so paulo , sp , brazil ) with distilled water for 5 minutes . five 0.8 mm diameter x 4 mm high composite resin ( filtek z100 , 3 m espe , st . therefore , a layer of silane ( dentsply , petrpolis , rj , brazil ) was previously applied to bond the resin onto the ceramic . a layer of adhesive ( adper scotchbond - adhesive , 3 m espe , st . paul , mn , usa ) was applied and light - cured using halogen light ( demetron , lc kerr corporation , orange , ca , usa ) for 10 seconds . then , silicone tubes 4 mm high x 0.8 mm diameter were placed on the ceramic surface to be filled with resin composite . after filling the tubes , the resin cylinders were light - cured for 40 seconds ( demetron , lc kerr corporation , orange , ca , usa ) with the light source placed at the upper end of the cylinders , thus light - curing all of them simultaneously . the light - curing equipment was set at 499 mw / cm ( from 491 mw / cm to 507 mw / cm ) with the aid of a radiometer ( radimetro digital , newdent , ribeiro preto , sp , brazil ) and reviewed after every five light - curing cycles . immediately after light - curing , the silicone tubes were cut using a scalpel blade 12 and disposed of in order to obtain 4 mm high x 0.8 mm diameter composite resin cylinders bonded to the ceramic surface , with a bonding area of 0.5 mm . the specimens were stored in a bacteriological incubator ( ecb 1.3 digital , odontobrs ind . ltda , ribeiro preto , sp , brazil ) at 37c for 24 hours until the bond strength tests were performed . the ceramic discs were fixed to a device attached to the universal testing machine ( dl2000 , emic ind e com ltda , so jos dos pinhais , pr , brazil ) . a 0.3 mm diameter stainless steel wire looped around both the extremity of the loading cell and the composite resin cylinder , simultaneously . the wire maintained contact with the inferior semicircle of the cylinders , as close as possible to the area bonded to the ceramic . the test was carried out at a speed of 0.5 mm / min , until bonding failure , for each cylinder sequentially , that is , one at a time . the failure mode was assessed using a stereoscopic lens ( ek3st model , eikonal , so paulo , so paulo , brazil ) at 20x magnification . the failure modes were classified as adhesive failure , ceramic or resin cohesion failure , or mixed . cohesion failure was defined as a fracture in the body of the ceramic or the resin , adhesive failure when there was a fault at the junction between the ceramic and the resin , and mixed when there was both adhesive and cohesion failure , simultaneously . one of the six ceramic discs used to evaluate surface micromorphology did not receive any surface treatment , including hydrofluoric acid . the specimens were then stored in an incubator at 50c for 3 hours to remove any water particles , 24 hours prior to sem assessment . the samples were then placed in airtight glass jars containing silica gel and sealed to ensure moisture elimination . the discs were positioned in the sem ( jsm 5600lv , jeol , tokyo , japan ) and images were obtained at 500x and 2,500x magnification . for each ceramic disc , the mean micro - shear bond strength value for the resin cylinders was considered . after exploratory analysis , the data were tested using one - way analysis of variance ( p<0.05 ) on sas statistical software ( release 9.2 , 2008 , institute inc . , cary , nc , usa ) . table 1 describes the mean values of micro - shear bond strength and shows no significant difference between the surface treatment methods ( p=0.3197 ) . table 2 shows the frequency of failure modes following micro - shear bond strength testing . mean and standard deviation for bond strength ( mpa ) for the different ceramic surface treatments ( p=0.3197 ) mean followed by same letter indicates no significant difference failure mode for the different ceramic surface treatments when inspecting the sem images , the ceramic surface that received no treatment appeared smooth without porosity , as a result of the polish obtained when the ceramic disc was prepared ( figure 1a ) . regarding the control group ( c ) and the spray group ( s ) , which had the hydrofluoric acid rinsed with water and water - air spray , respectively , irregular micrometric structures were observed , which probably consisted of products from the acid etch step ( figures 1b and 1c ) . such structures were not encountered on the surfaces of the other groups . regarding groups us , f and f+us , no significant difference was observed in terms of surface micromorphology ( figures 1d , 1e and 1f ) . photomicrographs of the ceramic surfaces as seen using a scanning electron microscope ( sem ) at 500x magnification . a - unetched ceramic surface ; b - ceramic surface of group c ; c - ceramic surface of group s ; d ceramic surface of group f ; e - ceramic surface of group us ; f - ceramic surface of group f+us . the arrows show residues left following cleaning at higher magnification ( 2,500x ) , groups us and f+us ( figures 2e and 2f ) showed no debris or acid etch residue , whereas , in the two groups in which no ultrasound was used , some debris were still present . a curious observation was noticed when the phosphoric acid smear was used . at lower magnification , some darkened areas were observed , which could have been remains of the phosphoric acid itself , which was not completely removed under running water only . at higher magnification , it is obvious that this surface is different from the other groups . it is likely that some form of over - etching may have occurred on the ceramic surface , when phosphoric acid was applied . as no ultrasonic treatment was used in this group , the products from over - etching were not efficiently removed . photomicrographs of the ceramic surfaces as seen using a scanning electron microscope ( sem ) at 2,500x magnification . a - unetched ceramic surface ; b - ceramic surface of group c ; c - ceramic surface of group s ; d - ceramic surface of group f ; e ceramic surface of group us ; f ceramic surface of group f+us . resin cements and internal ceramic surface treatment are used to promote bonding between ceramic surface and tooth . such treatments are necessary to modify the surface of these structures , because they are usually smooth and with low surface energy , which prevents the penetration of the bonding agents that create satisfactory chemical or micromechanical retention . dental ceramics have a crystalline microstructure containing large amounts of loose crystals or granules , which form their microtopography . feldspathic ceramic is composed of feldspath ( 65% ) , quartz ( 25% ) and metal oxides ( 10% ) . sodium and alkaline earth oxides are added as bivalent glass modifiers to improve its translucent properties . potassium oxide ( k2o ) is one of the main components of feldspathic ceramics , which is used to provide the material with a coefficient of thermal dilation as similar as possible to the metal alloys used in metal - ceramic restorations . the large quantity of glass matrix optimizes the materials vulnerability to acid etching , which allows the formation of microporosities that improve adhesion . aluminium oxide sandblasting and hydrofluoric acid etching promote an increase of ceramic surface area , creating an irregular topography and increasing surface energy for micromechanical retention of the resin cement . hydrofluoric acid etching is widely used to pretreat feldspathic ceramic surfaces , because it acts selectively on the silica glass matrix , forming hexafluorosilicates , thus creating a porous surface to facilitate micromechanical interlocking with the resin cement . for feldspathic ceramics , surface etching with hydrofluoric acid was used at a concentration of 9% to 10% , and the etching time ranged between one and two minutes . however , studies comparing etching times reported no difference among the times used , as long as the minimum etching time was one minute . following etching of silica - based ceramics , it has been observed that hydrofluoric acid leads to the formation of insoluble byproducts , such as silica and fluoride salts , which may remain on the ceramic surface . these particles may interfere with the bond strength between the ceramic and the substrate onto which it is cemented . in the present study , the control groups sem images ( which only underwent etching with hydrofluoric acid ) , revealed large quantities of residues when compared to the untreated ceramic and to the groups that received some form of surface cleaning . however , phark , et al.(2009 ) reported that shear bond strength is not affected by contamination or cleaning procedures , as shown in this study . in order to remove byproducts following hydrofluoric acid etching , some authors used ultrasonic washing , 37% phosphoric acid smear , rinsing with running water , or a combination of 37% phosphoric acid smear and ultrasonic washing . the present study demonstrated no significant difference in bond strength using different methods of ceramic surface cleaning , despite the presence of byproducts observed by sem following surface cleaning with water spray , unlike in groups us and f+us , in which ultrasonic bath was used . at 2,500x magnification , ultrasonic washing improved the removal of debris , when compared with the group in which phosphoric acid was used , where residue was observed after rinsing under running water . however , the longterm effect of this interference has not yet been evaluated . in this study , the main failure mode was resin cohesion followed by adhesive failure , thus showing that the hydrofluoric acid residue did not affect bond strength . ( 2009 ) also observed no influence on bond strength from the contaminants left by the etching agent on the ceramic surface . in addition , fabianelli , et al.(2010 ) demonstrated that the silane application technique is more important than hydrofluoric acid etching . other studies also have shown increase in bond strength following silane application , regardless of the surface etching technique . therefore , the residue does not seem to interfere with bond strength , as shown in this study . regardless of the cleaning technique used on the ceramic surface , the presence or absence of residue following hydrofluoric acid etching did not influence the bond strength between ceramic and resin . the post - hydrofluoric acid etching cleaning method may simply consist of spraying with water for 90 seconds . the micro - shear bond strength between the feldspathic ceramic and adhesive system was not influenced by different ceramic cleaning techniques , following hydrofluoric acid etching . micromorphological evaluation of the ceramic surface revealed little to no residue following hydrofluoric acid etching , when combined with ultrasonic washing .

objective the aim of this study was to evaluate the effect of feldspathic ceramic surface cleaning on micro - shear bond strength and ceramic surface morphology . material and methods forty discs of feldspathic ceramic were prepared and etched with 10% hydrofluoric acid for 2 minutes . the discs were randomly distributed into five groups ( n=8 ) : c : no treatment , s : water spray + air drying for 1 minute , us : immersion in ultrasonic bath for 5 minutes , f : etching with 37% phosphoric acid for 1 minute , followed by 1-minute rinse , f+us : etching with 37% phosphoric acid for 1 minute , 1-minute rinse and ultrasonic bath for 5 minutes . composite cylinders were bonded to the discs following application of silane and hydrophobic adhesive for micro - shear bond strength testing in a universal testing machine at 0.5 mm / min crosshead speed until failure . stereomicroscopy was used to classify failure type . surface micromorphology of each treatment type was evaluated by scanning electron microscopy at 500 and 2,500 times magnification . results one - way anova test showed no significant difference between treatments ( p=0.3197 ) and the most common failure types were cohesive resin cohesion followed by adhesive failure . micro - shear bond strength of the feldspathic ceramic substrate to the adhesive system was not influenced by the different surface cleaning techniques . absence of or less residue was observed after etching with hydrofluoric acid for the groups us and f+us . conclusions combining ceramic cleaning techniques with hydrofluoric acid etching did not affect ceramic bond strength , whereas , when cleaning was associated with ultrasound , less residue was observed .

INTRODUCTION MATERIAL AND METHODS Micro-shear bond strength test Micromorphological analysis Statistical analysis RESULTS DISCUSSION CONCLUSION

PMC3977575

rna molecules are not merely simple carriers of genetic information but can assemble into complex tertiary structures and even catalyze reactions . in fact , the existence of catalytic rna molecules ( ribozymes ) has led to the proposition of the rna world hypothesis . in modern cells , rna molecules catalyze just two classes of chemical reactions : modifications of phosphodiester bonds ( dna and rna cleavage , rna splicing ) and peptide bond formation . artificially designed ribozymes , however , are known to catalyze a wide range of chemical reactions . in some ribozymes , the slow opening and closing of tertiary structure ( rna breathing ) this latter process is hierarchical , first proceeding on the secondary structure level via formation of fairly stable watson crick base pairs . subsequently , secondary structure elements fold into a compact , three - dimensional structure . the folding of rna into the native tertiary fold may proceed via a complex sequence of secondary structures . formation of compact tertiary structures may require the presence of counterions , particularly divalent cations such as mg , which screen the intrinsic negative charges on the rna phosphate groups and , thereby , stabilize certain tertiary motifs . even small modifications of single nucleotides may result in different tertiary structures and hence different energy landscapes . indeed , rna sequence , structure , and function interact in a complex , not yet fully understood fashion , and the characterization of rna folding kinetics , including the pathways of secondary and tertiary structure changes , remains an intricate problem . in this work , we have investigated the conformational equilibrium and the folding pathway of the 49mer single - stranded rna ribozyme diels alderase ( dase ) using a novel hidden markov model ( hmm ) analysis of single - molecule fret data . dase catalyzes a diels alder reaction , i.e. , the [ 4 + 2 ] cycloaddition reaction between anthracene dienes and maleimide dienophiles . dase is a true multiple - turnover catalyst and shows remarkable enantioselectivity ( > 95% enantiomeric excess ) . it has a well - defined folded structure , as revealed by x - ray crystallography . the folded state consists of three helices arranged around a pseudoknot region , in which the catalytic pocket of the ribozyme is located ( figure 1b ) . a continuous sequence of stacking interactions runs from the bottom of helix ii to the top of helix iii and has been termed the spine of the folded structure . the tertiary fold is held together by a pseudoknot , in which the 5-g1-g2-a3-g4 segment bridges the unpaired strands of the asymmetric bulge ( figure 1a ) . the precise hydrogen - bond pattern in the pseudoknot region is known to be crucial both for thermal stability of the overall fold as well as for the shape of the catalytic pocket . recent experimental and computational evidence showed that cations specifically bind to certain sites that stabilize the tertiary fold , without interfering with the catalytic reaction . low mg concentrations were found to destabilize the folded conformation and to dramatically decrease the catalytic activity of the ribozyme . diels solid lines , secondary structure base pairs ; dotted lines , tertiary structure base pairs . attachment sites of the fret labels are marked by green ( donor dye cy3 at u6 in construct i and at the 5 end in construct ii ) and orange ( acceptor dye cy5 at u42 in construct i and at u30 in construct ii ) arrows . color - coding of the secondary structure elements as in panel a. attachment sites of the fret labels are indicated by green ( cy3 , donor ) and red ( cy5 , acceptor ) spheres . ( the figure has been adapted from figure 1 in ref ( 9 ) . ) single - molecule frster resonance energy transfer ( smfret ) is a powerful tool to follow conformational fluctuations of biomolecules on length scales of a few nanometers in real time . smfret measurements with surface - immobilized molecules revealed that dase is highly dynamic and can exist in substantially different conformations , which were found to interconvert on time scales of hundreds of milliseconds . the concentration of mg influences the shape or population of the accessible conformational states , as indicated by the mg dependence of the fret efficiency histograms and the apparent folding rates . consistent with conformational fluctuations , a poor resolution of dase spectra was found in subsequent nmr studies . the mg - dependent fret efficiency histograms revealed at least two conformational ensembles : ( i ) a high fret state , attributed to the folded conformation , whose population increases with increasing mg concentration , and ( ii ) a distribution of intermediate fret efficiencies , whose population decreases with increasing mg concentration . the intermediates were observed to spread out over a wide range of fret efficiency values and , presumably , comprise multiple conformations with different secondary and tertiary structures . in practice , only two or three states with significantly different fret efficiencies can be distinguished in a histogram - based analysis . consequently , stochastic fluctuations of the number of photons within a time bin ( shot noise ) significantly contribute to the widths of the fret distributions and prevent the separation of states with similar mean fret efficiencies . it completely neglects the time sequence of events in the single - molecule trajectories and , thus , discards a substantial part of the available information . in contrast , hidden markov models can distinguish states in the data by using both the differences in fret efficiency and the time sequence of events , and , thus , can decompose states with similar fret efficiencies but different kinetic properties . recent studies on single - molecule protein and rna data sets have demonstrated the power of hmms to resolve a multitude of states . hmm analysis has its intrinsic challenges , however , because ( i ) the results depend on the number of states used , ( ii ) the hmm optimization may get stuck in local minima , ( iii ) models with many states are difficult to validate , and ( iv ) the quality of the model depends crucially on the validity of the underlying likelihood function ( i.e. , the stochastic model of the measured process ) . here , we present an hmm analysis scheme that addresses these problems . at the core of this scheme is the idea that the number of states required to describe the kinetics in a hierarchical energy landscape is not fixed but depends on the time scales of interest ( figure 2 ) . directly estimating hmms with a few states often yields wrong kinetics , as they tend to prefer models whose states have clearly different fret efficiency . however , in real data , distinct and slowly interconverting conformations may have strongly overlapping fret efficiency distributions , which are difficult to separate . therefore , we construct an initial hmm with many states ( corresponding to a fine discretization of conformational space ) . the initial number of states is determined by a validation scheme , which tests reproducibility and consistency of the model with the underlying data set . the initial states are subsequently coarse - grained on the basis of their kinetics . this approach allows us to model ( coarse ) states even when they strongly overlap in their fret efficiencies and have very irregular ( e.g. , non - gaussian ) fret distributions . our hmm uses a poissonian likelihood function to model the physical process of photon emission . this approach is preferable over using gaussian likelihood functions of the fret efficiency . for a detailed discussion , see the supporting information . in addition , we have developed an approach to account for the trace - specific background noise . ( a ) hierarchical free energy landscape with various minima ( conformations ) interconverting on different time scales . ( b ) a fret efficiency versus distance curve , with the five conformations in panel a assigned to certain fret efficiencies ( distances ) . conformations with suitably long lifetimes can be distinguished by hmm analysis of fret traces but may have overlapping fret efficiencies even when they are distinct . ( c ) probability density function of finding the system at a certain value of the distance parameter . ( d ) the states found in the hmm analysis are depicted as disks located in a two - dimensional space of efficiency ( abcissa ) and lifetime ( ordinate ) . ( e , f ) some states kinetically merge on longer observation time scales indicated by the blue and red areas in panels e ( = 10 ms ) and f ( = 100 ms ) . for example , state pairs ( i , ii ) and ( iv , v ) each merge into a single apparent state for times longer than 10 ms . independent hmm analyses were carried out on two differently labeled dase constructs , referred to as constructs i and ii . altogether , four different data sets were analyzed ( dase construct i at mg concentrations of 0.0 , 5.0 , and 40.0 mm and dase construct ii at mg concentration 5.0 mm ) , yielding hmms with seven to nine conformational states . these hmms provide comprehensive models of the dynamics on millisecond time scales . we also determined relaxation times , identified the associated conformational transitions by an eigenvector / eigenvalue analysis of the transition matrix , and computed the ensemble of rna folding pathways . on the basis of their kinetics , the original states were lumped together to effective five - state ( on time scales of tens of milliseconds ) and three- or four - state models ( on time scales of hundreds of milliseconds ) . most notably , we identified consistent , characteristic features of the kinetic network of dase in all four data sets . to the best of our knowledge , these results represent the most detailed rna folding models obtained from single - molecule measurements to date . furthermore , they reveal that the transition rates in this landscape change substantially as the mg concentration is varied , while the general topology of the landscape ( position of minima , relative height of energy barriers ) is not affected . at all mg concentrations , the observed kinetic processes can be attributed to either secondary or tertiary structure rearrangements . by using a combinatorial strategy , we had earlier synthesized a set of nine dase fret constructs with dyes attached at different nucleotide positions . construct i was chosen for in - depth studies because it showed the most pronounced changes in its fret histogram with varying mg concentration . here we have also performed surface - immobilized measurements on a second variant , construct ii , because ( 1 ) its fret histogram was multimodal , suggesting that multiple states could be distinguished by the hmm , and ( 2 ) it was not too different from construct i and , therefore , could serve for validation ( see below ) . single - molecule fluorescence time traces of surface - immobilized dase were obtained for construct i ( cy3 at u6 and cy5 at u42 ) at mg concentrations of 0 , 5 , and 40 mm and for construct ii ( cy3 at the 5 end and cy5 at u30 ) at a mg concentration of 5 mm . details on the data , the experimental procedures , and the effects of surface immobilization are included in the supporting information , tables s1 and s2 and figures s2 and s3 . for each trace , the rates of the background noise , ka , bg an kd , bg , in the acceptor and donor channel , respectively , as well as the amount of spectral crosstalk , , from the donor into the acceptor channel were estimated , as described in the supporting information . we have developed an hmm analysis and associated optimization algorithms for single - molecule fret . the hmm analysis scheme has the following features:the hmm works with discrete photon counts , which are assumed to obey poissonian statistics ( figure s4 , supporting information).background noise levels of measured photon traces are taken into account explicitly by employing an appropriate emission probability.the reproducibility of the hmms is tested.the number of states of the hmm is maximized under a number of constraints , which ensures that the model reproduces physically and chemically relevant quantities.the final hmm represents a fine discretization into states that , depending on the time scale , are lumped into larger states according to kinetic proximity . the hmm works with discrete photon counts , which are assumed to obey poissonian statistics ( figure s4 , supporting information ) . background noise levels of measured photon traces are taken into account explicitly by employing an appropriate emission probability . the number of states of the hmm is maximized under a number of constraints , which ensures that the model reproduces physically and chemically relevant quantities . the final hmm represents a fine discretization into states that , depending on the time scale , are lumped into larger states according to kinetic proximity . a workflow diagram of the hmm analysis scheme is shown in figure 3 . the algorithms are described in full detail in the supporting information , and the salient characteristics of the workflow are discussed in the following sections . workflow diagram used for our hmm analysis of single - molecule fret data . consider the hypothetical energy landscape with five minima in panel a in figure 2 . each minimum corresponds to a conformational state and is associated with a mean fret efficiency , ei ( figure 2b ) , and a fractional population in equilibrium , i ( figure 2c ) , where i denotes the number of the state . using hmm analysis , these five states can be extracted from smfret traces of a molecule diffusing in this free energy landscape . we represent the main characteristics of the states of the hmms by scatter plots ( figure 2d ) : each state is marked by a disc , the position of which encodes the mean fret efficiency of the state and its lifetime , i . the area of the disc is proportional to the stationary probability i of the state , as computed from the hmm . the hmm transition matrix has eigenvalues corresponding to time scales of transitions and eigenvectors denoting states that interconvert on these time scales . , states i and ii interconvert on time scales of 10 ms ( figure 2a ) . thus , when computing a fret histogram with an averaging window much longer than 10 ms , these two states merge into a single apparent state . likewise , states iv and v kinetically merge for time scales longer than 10 ms , as depicted by the red and blue regions in figure 2e . states i and ii kinetically merge with state iii for time scales above 100 ms ( figure 2f ) . complete equilibration occurs for times longer than 1 s. in a high - dimensional energy landscape , kinetic merging may not necessarily involve only neighboring states along the fret efficiency axis . in fact , high fret efficiency states can merge kinetically with low fret efficiency states even if there are states with intermediate fret efficiencies in between . hidden markov models ( hmms ) are stochastic models , = ( t , e ) , of the observed ( measured ) trace , o = ( o1 , ... , on ) , with oi = ( na , i , nd , i ) containing the number of photons observed in the acceptor and donor channels at each time step i. in the construction of hmms , it is assumed that the observation is generated by a hidden markov chain with transition matrix t , whose states represent regions in the conformational space of the molecule . at every time step in the markov chain , an additional stochastic process , the emission probability , , describes the conditional probability of observing the signal , oi , given that the molecule is currently in conformation ( hidden state ) si . one typically chooses the same functional form of for all hidden states but uses a parameter ej to adapt it to a specific hidden state . the parameters ej form a vector e and are part of the model . the hmm optimization problem maximizes the likelihood ( i.e. , the conditional probability of observing the measured trace o , given that the molecule is accurately described by the model = ( t , e)):1over all values of ( t , e ) and all possible hidden paths . for a given number of states , n , the model consists of an n n transition matrix , t , and of a vector of observation parameters e , of length n. hmm classes differ by the way that the hidden process and the measurement process are modeled and by the way that the corresponding parameters are optimized . it is crucial to choose an emission probability , , that models the measurement process as accurately as possible . the arrival times of the photons are assumed to obey poissonian statistics , which is validated in figure s4 ( supporting information ) . n ) is a poisson distribution of variable n with rate coefficient k. the acceptor and donor photon count rates , ka and kd , are given as3where ei is the apparent fret efficiency of the current hidden state si and kmol is the detection rate of photons emitted by the labeled molecule ( through either the donor or the acceptor ) . a problem inherent in the experimental data is the presence of trace - dependent background noise , which may cause identical conformational states to display different apparent fret efficiencies in different time traces . the trace specific background rates , ka , bg and kd , bg , can be estimated from the bleached phase of the measured photon traces . given these rates , we derive a likelihood of observing ( na , nd ) photons during a time step , t , in the acceptor and donor channels , respectively ( see the supporting information ) . the emission probability has the functional form given in eq 2 , but the photon count rates are now given as4we assume that background noise may vary from trace to trace but that all other measurement errors , including spectral cross - talk and differences in the quantum yield of the chromophores , depend on the conformational state but are identical for different traces . then , e contains the apparent fret efficiencies ( without background noise ) of the hidden states . these apparent fret efficiencies can be corrected for spectral cross - talk a posteriori to obtain the true fret efficiencies ( see the supporting information ) . hmm optimization is done by using the expectation - maximization algorithm , which finds a local maximum of from an initial guess of the parameters ( t , e ) . to facilitate finding the global optimum , the hmms presented here are obtained by first running 100 explorations that optimize random starting values of ( t , e ) for a few steps only . subsequently , the parameter set with the largest likelihood is optimized to full convergence . nonetheless , the hmm algorithm might find different local maxima for different initial parameters . hence , for each mg concentration , we compute 10 hmms in the described way to test for reproducibility . two hmms are accepted as identical if their log - likelihoods differ by less than 1.0 . by a heuristic criterion , an hmm optimization is reproducible if identical maximum likelihood hmms are found in at least 2 out of the 10 trials . the number of states , n , is an input parameter for the hmm optimization algorithm . as argued in the supporting information , information - criteria - based choices of the number of states are inadequate for the present data . to determine the number of hidden states , we instead adopt a viewpoint for the construction of direct markov models that is well established in the community : rather than finding the ideal number of states to statistically classify the data consequently , the resulting discretization of state space will be fine enough that the hmms can reproduce the stationary and long - time kinetic behavior of the data . the resulting states can subsequently be grouped according to kinetic connectivity given by t , as described in refs ( 31 and 32 ) and illustrated in figure 2 . following this approach , we build hmms for a varying number of states , n = 2 , 3 , ... , and choose the largest number of states for which hmms can be constructed reproducibly . different tests were used to check whether the hmms are consistent with the data set from which they were parametrized , and whether the hidden paths obtained from the hmms are consistent with markovian dynamics . the consistency of the hmm with the underlying data set was tested by comparing fret efficiency histograms obtained from the data with histograms estimated from the hmms . for this test , we used time windows between 10 and 100 ms . as previously discussed , this approach tests both the stationary and kinetic properties of the model . the data - based distributions were obtained using the likelihood from eq 2 , as described in the supporting information . the hmm - based distributions were obtained by sampling hidden trajectories of the time window length from an equilibrium distribution , and then generating artificial photon counts using poisson statistics with the appropriate output rates ( figure 4a and figures s6a , s7a , and s8a , supporting information ) . the markovianity of individual states was tested by inspecting their lifetime distributions , which can be computed from the maximum - likelihood hidden paths , (t ) , of the hmm . a single exponential decay in these distributions is consistent with markovian dynamics ( figure 4b ) . states that failed this test were split using a newly developed bayesian model selection algorithm ( supporting information ) . the overall markovianity of the hmms was tested using the implied time scales test that is frequently used for simulation - based markov state models . to this end , the relaxation time scales , tihmm = t / ln ihmm , were computed , where ihmm are the eigenvalues of the hmm transition matrix t. these are compared to the implied time scales of a markov model t( ) constructed from the maximum likelihood hidden paths , (t ) , for different lag times . if the overall dynamics is markovian , these time scales should be independent of the lag time used to compute them , hence yielding constant functions in figure 4c . as an additional test , they should agree with the hmm time scales , tihmm . figure s9 ( supporting information ) shows fret traces colored according to the hidden states in the final model . ( a ) dependence of the fret efficiency histograms on the lengths of the time windows ( 10 , 50 , and 100 ms ) . dashed colored lines , prediction from the hidden markov model ; gray areas / dotted black lines , estimation from the smfret data set ( bootstrapping mean/95% confidence interval ) . ( b ) lifetime distributions of the individual states calculated from the maximum - likelihood paths . ( c ) implied time scales , indicating that the long - time kinetics of the hidden paths is markovian and converges to time scales similar to those found in the hmm . the divergence of the shortest time scales at larger lag times is expected and due to numerical problems . we analyzed three sets of smfret traces of dase construct i ( chromophores attached to residues 6 and 42 , see figure 1 ) , measured at different mg concentrations , 0.0 , 5.0 , and 40.0 mm . background - corrected fret efficiency distributions were calculated from these data sets by using the likelihood ( eq 2 ) and a bootstrapping procedure to estimate the uncertainty in the data ( dotted gray lines and gray areas in figure 4a ) . two ensembles of states can be visually distinguished : a broad intermediate state in the fret efficiency range 0.40.8 and a putative native state at efficiency values of 0.91.0 . with increasing mg concentration , the populations shift to states with high fret efficiency . in ref ( 9 ) , it was already hypothesized that the broad ensemble at intermediate fret efficiencies may consist of multiple conformational states with overlapping fret efficiency distributions . hmms were constructed for the smfret data sets as described in the materials and methods section . the largest number of states for which hmms could be reproducibly obtained was eight ( 0 mm mg ) , eight ( 5 mm mg ) , and seven ( 40 mm mg ) states , where we used the optimization protocol described in the materials and methods section . the eight - state models for 0 and 5 mm mg passed the validation test ( figure 4 ) . a single , weakly populated state with fret efficiency e 0 , which was assigned to an acceptor blinking state , was removed from these models a posteriori . the seven - state model at 40 mm mg required an intermediate step , in which non - markovian states were split and regrouped according to kinetic proximity , yielding a nine - state model . ( see the supporting information for a detailed description of the protocol employed . ) to test whether the remaining nonexponentiality came from an actual non - markovianity of the discrete state dynamics or just from spurious transitions generated from the estimation of the maximum likelihood , we conducted the implied time scale test as described in the materials and methods section . the results shown in figure 4c demonstrate that the maximum likelihood hidden paths , (t ) , are non - markovian in all models at short time scales but then converge to approximately constant time scale estimates at lag times of 1030 ms . the time scales agree with the time scales estimated from the hmm transition matrix , indicating that the kinetics of all three hmms are consistent with the data . note that the hmms for the three different mg concentrations were constructed independently of each other . therefore , when similar or consistent features are found across all three mg concentrations , this is a two - fold validation of an observation . the scatter plots in figure 5a ( upper row ) show the main characteristics of the ( hidden ) states of the hmms : each state is represented by a disc whose position indicates the mean fret efficiency of the state and its lifetime i = t / ln tii , where t is the time step of the hmm transition matrix and tii are the diagonal elements of this matrix . the area of the disc is proportional to the stationary probability i of the state as computed from the hmm . the states that consistently appear in construct i at different mg concentrations are depicted in the same color ( i.e. , black , blue , red , and green states ) . the purple state at 0.0 mm mg could not be matched to any state at higher mg concentrations . conformational states and subensembles found by the hmm analysis of construct i and construct ii . ( a ) first row : state parameters of the hidden markov models which are for each state i : the fret efficiency ei ( abcissa ) , the state lifetime i ( ordinate ) , and the equilibrium population i ( disc size ) . second and third rows : state decomposition for time scales of 10 and > 100 ms . ( b ) fret histograms of the subensembles of the states shown in the second row of panel a. a feature found for all mg concentrations is the black high - fret efficiency state . it has a relatively small stationary probability but a long lifetime at all mg conditions . the region of intermediate fret efficiencies is populated mostly by short - lived states ( blue , red ) , and a few long - lived states with low fret efficiencies ( green ) . remarkably , the states appearing at multiple mg concentrations show only rather subtle changes . there are two cooperative effects upon mg increase : ( i ) all states shift to slightly higher fret efficiencies , indicating that mg causes these conformations to become more compact , ( ii ) the intermediate - efficiency purple state is depopulated with increasing mg , while some substates with higher fret efficiencies ( light red state , which is split into an orange and a dark red state at 40 mm mg , as well as the dark blue state ) become more populated at high mg concentrations . the populations of the other red and blue states , as well as the black state , show surprisingly little dependence on the mg concentration , indicating that the associated conformations do not experience stabilization by mg ions . to better understand the nature of the conformational states of the hmms , we have investigated their kinetics . detailed information is presented by the networks plotted in figures s10s13 ( supporting information ) . alternatively , an eigenvector / eigenvalue analysis of the transition matrix t allows conformational states interconverting faster than the time scale of interest to be grouped ( figures s10s13 , supporting information ) . the second row of figure 5a shows a striking feature found independently for the hmms at all mg concentrations : at a few tens of milliseconds , the substates of the red subensemble as well as the substates of the blue subensemble interconvert . consequently , kinetic proximitiy and proximity on the fret axis are , in general , unrelated properties . this finding is emphasized by the fret efficiency histograms of the corresponding subsembles in figure 5b , which were constructed by partitioning the photon traces according to the associated hidden states . the blue and red subensembles are doubly peaked because they are composed of multiple hidden states . in addition , these subensembles overlap strongly , clearly showing why the present single - molecule fret data were difficult to model kinetically , and emphasizing the usefulness of a detailed hmm analysis for dissecting them . for all mg concentrations , the high - efficiency peak in the fret histograms of the blue subensemble overlaps with the high - efficiency black state , indicating that the high - fret - efficiency peak identified in ref ( 9 ) consists of two states , one of which rapidly interconverts with a state of intermediate fret efficiency ( blue ) and is stabilized by mg , and a long - lived high - efficiency state ( black ) , which is insensitive to mg . in figure 5a , the third row shows that , on time scales of a few hundred ms , the long - lived state ( black ) interconverts with the blue subsemble . the mixing time for all subensembles is on the order of seconds ( see figure 6 ) . these results indicate the presence of a hierarchical energy landscape , with different processes occurring on very different time scales , ranging from a few milliseconds to 1 s. free energy landscape and folding pathways . states are indicated by bars or discs with the same colors used in figure 5 . gray bullets indicate transition states facilitating that states or sets of states kinetically merge at longer time scales . ( b ) the complete ensemble of folding pathways from the least compact states ( green / yellow ) to the most compact state ( black ) . the states are positioned depending on their mean fret efficiency ( abcissa ) and the probability of folding ( committor , q , ordinate ) . the thickness of an arrow is proportional to the probability that a green / yellow state will fold along this pathway . on the basis of the processes depicted in figure 5 , we find fast interconversion between the open ( e 0.5 ) and closed ( e > 0.7 ) states within the blue and red subensembles , while the exchange dynamics between these subensembles happens much slower . we propose that the states within each subensemble ( with a given color in figure 5 ) have similar secondary structures yet different tertiary structures , interconverting rapidly without breaking large strands of watson crick base pairs . this proposition is supported by the fact that , at high mg concentrations , the compact parts of the red and blue subensemble are stabilized . different subensembles are proposed to correspond to different secondary structures because they are long - lived , suggesting that the stable watson crick base pairs need to be broken in order to transit to another subensemble . the connectivity between different subensembles ( and , thus , presumably different secondary structures ) is similar at all mg concentrations . the high - efficiency ( black ) state is connected to the blue subensemble in the presence of mg ( 5 and 40 mm ) directly and , at 0 mm mg , via the purple intermediate . figure 6a illustrates this connectivity , and the free energies of these conformations as well as the transition states ( see the materials and methods section ) . this connectivity suggests an ordering of subensembles from the least compact ( lowest fret efficiencies ) to the most compact ( highest fret efficiencies ) which can be found at all mg concentrations : ( 1 ) green , ( 2 ) red , ( 3 ) blue , and ( 4 ) black . the fact that they have high lifetimes and fret efficiencies that are much greater than zero suggests that they still have some secondary structure , although probably not the native one . they are therefore called misfolded . this ordering suggests to study the transition pathways from the misfolded states ( green ) to the most compact state ( black ) . transition path theory provides the basis for calculating the pathways between two subensembles . we use the protocol and equations described in ref ( 36 ) employing the implementation in the emma software . a transition pathway is defined as a series of transitions that lead from the misfolded to the native state without returning to the misfolded state . figure 6b locates the states by their fret efficiency , and by the committor value ( vertical axis ) , i.e. , the probability of the system , when being in this state , to move forward and fold toward the black state , rather than misfold back to the green state . the committor value q = 0.5 designates states in which the molecule is equally likely to go either way . note that there is a continuous shift of these transition states with increasing mg concentration . at 0 mm mg , the transition state lies between the green and red subensembles . once a molecule has reached the red subensemble , it is likely to continue folding to the black state . with increasing mg concentration , the red and blue subensembles become more and more kinetic intermediates , and lie at committor values around 0.5 for 40 mm mg . figure 6b shows the probability fluxes of transition pathways from misfolded states to the folded state . the size of the arrows indicates the probability flux , which is related to the folding rate . without mg , the folding rate kab is about 0.09 s , and increases to 0.28 s for 5 mm and 0.17 s for 40 mm mg . the strong increase in folding rate from 0 to 5 mm mg is mainly due to a lowering of the transition state energy , while the decrease in folding rate from 5 to 40 mm mg is mainly due to an increased stability of the dark blue intermediate state ( compare figure 6a and b ) . moreover , it is apparent that addition of mg increases the number of accessible pathways , making the folding process more parallel . two main mechanisms are observed at all mg concentrations : a compact folding mechanism , in which the green misfolded state refolds via the higher fret efficiency substates of red and blue toward the black state , and an close open close mechanism , in which the green state folds via the open substates , or via successive closing , opening , and closing , i.e. , involving tertiary unfolded states . both types of pathways have similar weights , with some preference for close open close pathways at low mg concentrations and a slight preference for compact pathways at high mg concentrations . to further confirm our findings , we performed a fourth independent measurement on a dase ( construct ii ) with a different set of label positions . if they do not introduce major energetic conflicts , the state probabilities , time scales , and the kinetic connectivity should remain comparable . single - molecule fret data were recorded , and an hmm was computed using the same approach as above . a seven - state model was found to pass the validation test ( see figure s14a and s14b , supporting information ) . open states at efficiencies of 0.40.6 and high - fret , closed states at efficiencies above 0.8 . as for construct i , two pairs of rapidly interconverting states , each with a low- and a high - fret state , were found . additionally , a single stable state with high efficiency was also identified . consequently , the red , blue , and black subensembles of construct ii match the corresponding subensembles in construct i and , thus , can be identified in all experimental data with high confidence ( see figure 5a ) . moreover , the time scales found in constructs i and ii are in qualitative agreement ( see figure s14c in the supporting information ) . open and closed states of the red and blue subensembles interconvert at time scales of a few milliseconds ( 10 ms in construct i , 3 ms in construct ii ) . at time scales of 100 ms to seconds , ( i ) the blue ensemble merges with the black state and ( ii ) the red and blue ensembles kinetically merge . at low mg concentrations , the blue black interconversion is several 100 ms faster than the blue red interconversion , while , at 40 mm mg , the two processes happen at about the same time scales ( figure s14c , supporting information ) . the gray states in construct ii and the green / yellow states in construct i do not have clear corresponding states in the other construct . for example , the presence of a label in a particular position may prevent certain structures from forming . in the following discussion , we will thus concentrate on those states that can be safely matched across all data sets ( red , blue , and black ) . note that , due to the reduced state lifetimes in construct ii , the partitioning of the photon traces resulted in subtraces which were too short for a histogram analysis . hence , the subensemble fret histograms could not be generated ( see figure 5b ) . a kinetic pattern is found consistently for different mg ion concentrations and for different attachment points of the chromophores : ( i ) a long - lived , high - fret - efficiency state ( black ) , ( ii ) two ensembles of states ( red , blue ) comprising rapidly interconverting open and closed states , the ratio of which depends on mg , and ( iii ) a linear connection between the three subensembles ( red , blue , black ) . the long interconversion times along this linear connection suggest that these transitions involve breaking and reforming of watson crick base pairs . to investigate whether there are secondary structures consistent with this pattern , minimum energy secondary structures of the dase were calculated using the vienna rna webserver ( see the supporting information ) . the algorithm correctly identified the secondary structure of the known folded state ( excluding the pseudoknot connectivity ) as the lowest free - energy structure . two alternative secondary structures with low free energies ( g < 1.4 kj / mol above native , i.e. , accessible at room temperature ) were also identified . these structures ( labeled 2 and 3 ) are shown along with the secondary structure of the folded state ( labeled 1 ) in figure 7 . although they are very close in energy and structurally very similar to each other , structures 2 and 3 differ from structure 1 in that helix ii is broken and helix i is prolonged by two base pairs . all other secondary structures identified by the algorithm had estimated free energy differences of g > 9.5 kj / mol with respect to structure 1 . proposed folding mechanism . the red set of states has a non - native secondary structure but includes both open ( low - fret ) tertiary structures and compact ( high - fret ) tertiary structures . the blue set of states has the native secondary structure but also includes both open and compact tertiary structures . in contrast to the compact blue state , it is additionally stabilized by the tertiary watson crick pairs that form the pseudoknot . in the absence of stabilizing tertiary interactions , secondary structures 1 , 2 , and 3 facilitate transitions between open and compact states , associated with large fluctuations in the donor therefore , they have properties matching those found for the blue and red subensembles in the hmm analysis . the black state displays exclusively high fret efficiencies in all constructs under all conditions and its long lifetime and the fact that its population does not vary strongly with the mg concentration suggest that it is stabilized by base - pairing rather than mg ions . therefore , we propose that the black state represents the tertiary folded structure including the pseudoknot topology . the pseudoknot base pairs ( g1c26 , g2c25 , a3u45 , g4c44 ; see figure 1 ) are consistent with stable interactions that do not depend on mg . their formation stabilizes an already compact structure with the correct secondary fold so as to acquire a well - defined tertiary structure . this proposal is supported by computer simulations which show that the active site of dase is distorted if mg is removed ( explaining the loss in catalytic activity ) but the overall lambda - shaped tertiary structure stays intact . since the blue subensemble acts as a precursor to the black tertiary folded structure ( linearly connected folding path , figure 6 ) , it is only logical to match the blue state with the secondary structure of the folded state ( structure 1 ) . the native secondary structure still facilitates extended and compact states . like the fully native black state , the high - efficiency blue states are compact and possess the correct native secondary structure , but in contrast to the black state , they lack the pseudoknot base pairs , which stabilize the native tertiary fold . consistently , the probability of extended versus compact blue states depends on the concentration of mg ions that are required to stabilize the compact state in the absence of tertiary base pairs . consequently , the red ensemble contains structures 2 and/or 3 , i.e. , extended and compact states with non - native secondary structure . the proposed assignment is consistent not only with the kinetic connectivity and the mg - dependent equilibrium populations but also with the observed time scales . the fluctuation between open and compact conformations within the blue and red ensembles involves no or little secondary structure change , consistent with relatively short transition time scales ( figures 5 and 6 ) . in contrast , a transition from the red to the blue subensemble involves rupture of watson crick pairs , which is consistent with slower transition time scales of hundreds of milliseconds ( figures 5 and 6 ) . likewise , the change of tertiary base - pairing is consistent with long transition time scales between the blue and black states and the long lifetime of the black native state . the kinetic model found here and our proposed folding mechanism exhibits a number of features consistent with previous findings or hypotheses for other rna systems . in particular , secondary and tertiary structure formation has been proposed to be kinetically decoupled , such that secondary structure elements can exist without further stabilization by specific tertiary interactions . for the tetrahymena thermophila ribosome , metastable structures with a partially misfolded secondary structure have been described , lending credibility to the present assignment of the red subensemble to structures 2 and/or 3 . in addition , other rnas have been proposed to fold via multiple parallel pathways . to the best of our knowledge , we have presented the most detailed experimentally derived model of an rna folding mechanism , providing a kinetic model connecting different secondary and tertiary stabilized structures , and showing how they are orchestrated during the folding pathways . the multitude of time scales found in the data provide direct evidence that the rna folding landscape is hierarchical and that secondary and tertiary structure formation occur on different time scales . the techniques described here also facilitate detailed kinetic models to be derived for other macromolecular systems . as yet , the field is still lacking an experiment that could simultaneously resolve kinetics and the structures of the individual states in detail . unfortunately , computational approaches can not step in here . with folding times on the order of seconds , the dynamics are as yet out of reach for direct molecular dynamics ( md ) simulation . over time , however , enhanced sampling strategies may help access these processes . however , molecular modeling and md simulation may be useful for exploring the local dynamics within individual states , and by using new biophysical techniques , the distribution of measurable fret values can be computed and compared to the subensemble distributions shown in figure 5b . on the experimental side , using multicolor - fret or the systematic reconciliation of multiple dual - color - fret experiments may provide distance constraints to resolve the structures in more detail . finally , the combination of fret and site - specific fluorescence quenching may also be employed to disentangle the tertiary dynamics from secondary structure formation .

we have developed a hidden markov model and optimization procedure for photon - based single - molecule fret data , which takes into account the trace - dependent background intensities . this analysis technique reveals an unprecedented amount of detail in the folding kinetics of the diels alderase ribozyme . we find a multitude of extended ( low - fret ) and compact ( high - fret ) states . five states were consistently and independently identified in two fret constructs and at three mg2 + concentrations . structures generally tend to become more compact upon addition of mg2 + . some compact structures are observed to significantly depend on mg2 + concentration , suggesting a tertiary fold stabilized by mg2 + ions . one compact structure was observed to be mg2 + -independent , consistent with stabilization by tertiary watson crick base pairing found in the folded diels alderase structure . a hierarchy of time scales was discovered , including dynamics of 10 ms or faster , likely due to tertiary structure fluctuations , and slow dynamics on the seconds time scale , presumably associated with significant changes in secondary structure . the folding pathways proceed through a series of intermediate secondary structures . there exist both compact pathways and more complex ones , which display tertiary unfolding , then secondary refolding , and , subsequently , again tertiary refolding .

Introduction Materials and Methods Results and Discussion Discussion

PMC3507314

primary disorders of tendons are common and account for a high proportion of referrals to rheumatologists and orthopaedic surgeons . the most commonly involved tendons are the rotator cuff ( particularly supraspinatus ) in the shoulder , the forearm extensor ( tennis elbow ) and flexor tendons ( golfers elbow ) in the forearm , the patella tendon in the knee , the achilles tendon in the lower leg , and the tibialis posterior tendon in the ankle and foot . the intrinsic pathogenetic mechanisms underlying the development of tendinopathies are largely unknown however proinflammatory cytokines , apoptosis , and mechanical stress have recently been functionally implicated in several model systems [ 2 , 3 ] . increasing evidence is emerging that repetitive tissue trauma and its associated damage in stromal tissues are recognized at the cell level via receptor - mediated detection of intracellular proteins released by necrotic cells . the term alarmin is proposed to categorise such endogenous molecules that function to mobilise and activate immune cells after interaction with their specific receptors during host defence and tissue repair . heat shock proteins ( hsps ) , a type of stress molecules involved in protein folding , are implicated as important tissue alarmins . hsp activation can directly affect both innate and adaptive immunity , although controversial studies and opinions exist in the field [ 68 ] . the innate immune responses induced by hsps include cytokine and chemokine release and activation of nk cells . their expression in response to stress also has an important function in protection against apoptosis and in regulation of apoptotic cell signaling . thus , their evolutionary conservation and the upregulation during stress and binding to pattern recognition proteins make it logical that hsps can act directly as danger signals in tendinopathy . in this paper we summarize recent findings of heat shock proteins in inflammatory and tendon disease and highlight our key findings which may be important in understanding the pathogenesis of primary tendon diseases . hsps are expressed both constitutively ( cognate proteins ) and under stressful conditions ( inducible forms ) . additionally , a variety of stressful situations including environmental , pathological , or physiological stimuli induce a marked increase in hsp synthesis , known as the stress response and upon necrotic cell death , hsps are leaked into the extracellular compartment . in addition , hsps can be released extracellularly independent of necrosis in response to a number of stressful conditions [ 13 , 14 ] . hsps are present in the circulation of normal individuals , and their circulating levels decrease with age and increase in a number of pathological conditions such as hypertension , atherosclerosis , and rheumatoid arthritis . the principal hsps range in molecular mass from ~15 to 110 kda and are divided into groups based on both size and function . they are present in the cytosol , mitochondria , endoplasmic reticulum , and nucleus , although these locations vary depending on the particular protein . the most well - studied and understood hsps in mammals are those with molecular masses of ~60 , 70 , 90 , and 110 kda . these hsps are expressed at normal body temperatures ( ~37c ) and in conditions of stress . the primary function of the hsps appears to serve as molecular chaperones in which they recognize and bind to nascent polypeptide chains and partially folded intermediates of proteins , preventing their aggregation and misfolding , or as molecules that directly mediate protein folding [ 22 , 23 ] . several important cytoprotective functions ( the folding and unfolding of proteins , translocation of proteins across membranes , and the prevention of protein aggregation have been attributed to hsps , in particular , the hsp70 family [ 2426 ] . interestingly , it has also been noted that hsps can play a role in apoptosis with hsp27 , hsp70 , and hsp90 proteins predominantly antiapoptotic , while hsp60 is proapoptotic . moreover , it appears that these hsps function at multiple points in the apoptotic signalling pathway to elicit this response . thus their relevance to tendon disease is made all the more important due to the strong association of apoptosis in human tendon pathology [ 28 , 29 ] . although the primary focus of research on hsps has been directed toward their functions and accumulation inside the cell in response to a physiological stress , there is emerging recognition that hsps serve as key modulating signals for immune and inflammatory responses . one area of investigation pertinent to the topic of stress tolerance has dealt with the potential role of hsps in cytokine production . elevations in intracellular hsp levels have been shown to improve cell tolerance to inflammatory cytokines such as tnf- and interleukin-1 [ 23 , 31 ] . hsp accumulation within a cell produces both transcriptional inhibition and a decrease in tnf- and interleukin-1 secretion . . demonstrated that heat conditioning and the resultant increase in intracellular hsp70 levels protected animals from an endotoxin dose that was lethal in unconditioned rats . moreover , this paradigm was associated with a decrease in serum tnf- levels after administration of endotoxin in the heat - conditioned animals . these results suggest that intracellular hsp accumulation may contribute to a reduction in inflammatory cytokine production with cellular challenge . hsps have become increasingly associated with rheumatic disease . in animal studies using freund 's adjuvant transfer of an autoreactive t cell clone recognising a determinant on the mycobacterial 65 kda antigen was arthritogenic while prior immunisation with the 65 kda heat shock protein abrogated this effect . human investigations have revealed that 49% of patients with ankylosing spondylitis have antibodies against hsp63 while patients with systemic lupus erythematosus have serum igg antibodies to hsp90 and both igm and igg to hsp70 . more recently various groups have highlighted elevated levels of hsps in rheumatoid arthritis [ 19 , 3840 ] with the rheumatological community considering their merit as small molecular targets . thus hsps released in response to tissue injury / stress seem capable of straddling the divide between tissue survival versus tissue death mechanisms in inflammatory diseases ( figure 1 ) . overuse tendon injuries , namely , tendinopathies pose a significant , highly prevalent problem in musculoskeletal medicine with shoulder tendon injuries alone amounting to an annual cost of $ 3 billion to the us healthcare system . the intrinsic pathogenetic mechanisms underlying the development of tendinopathies are largely unknown however excessive cellular load and repetitive stress have been shown to be functionally important . thus the pathological process of repetitive microtrauma / stress lends itself well to the investigation of heat shock proteins which are so inextricably linked to tissue stress . tendinopathy is an overuse injury characterized by pain with movement , local tenderness , weakness , and decreased mobility at the injured site . these symptoms are the result of deviation from the tendon 's normal physiology . in healthy tendon , 95% of tendon tissue is collagen i , residing within fibroblast - like tenocytes , glycoproteins , and glycosaminoglycans . collagen iii is mainly produced during tendon healing and remodelling and is biomechanically weaker then type i collagen . in addition to mucoid , hyaline , hypoxic , or fibrinoid degeneration , collagen iii is observed in symptomatic tendons at a higher percentage than uninjured tendons . microscopically , collagen fibrils are disorganized with decreased tropocollagen cross - linking , glycosaminoglycan production is increased , both of which contribute to increased water retention and ultimate decrease in tensile strength ( table 1 ) . this increased neural volume is posed to cause pain in tendinopathy [ 48 , 49 ] . one of the major limitations of human studies is that tendon biopsies are usually obtained when patients are symptomatic and therefore biopsy material likely represents chronic , rather than early phase disease . medical intervention at this early stage may offer considerable therapeutic advantage over later surgical approaches . we previously demonstrated that matched subscapularis tendon from patients with full thickness rotator cuff tears represents a model of early human tendinopathy based on histological appearances and significantly increased levels of cytokines and apoptotic markers in these tissues ( figure 2(a ) ) . these studies established a human model of early tendinopathy for the first time and have been confirmed by an independent group . this model has now not only allowed us to elucidate a role for hsps in tendinopathy but also has finally allowed the targeted mechanistic investigation into key molecular events in early tendon disease . animal investigations have provided helpful insight . pan and halper described the effects of increased temperature , mechanical stress , and growth factors on hsp47 and their data showed that transforming growth factor 1 ( tgf-1 ) was a key regulator of hsp47 expression as the addition of tgf-1 led to a moderate increase in the expression of hsp47 mrna . induction of hsp47 protein expression by heat shock , mechanical stress and tgf-1 was likely achieved through activation and translocation of heat shock transcription factor 1 into the nucleus . jagodzinski et al . examined the expression of hsp72 in tendon fibroblasts subjected to mechanical stress . they showed that hsp72 accumulates in the nucleus with an associated transient upregulation after cyclic longitudinal stretching suggesting a role as a tissue repair mechanism . investigated the influence of repetitive cyclic longitudinal stress patterns on proliferation , apoptosis , and expression of hsp72 in tenocytes . stress patterns applied during two days resulted in a reduced proliferation and apoptosis rate whereas the expression of hsp72 showed a significant increase . this study suggested that inhibition of proliferation and apoptosis occurred through increased hsp72 activity and may implicate it in tendon tissue reparation and tissue engineering . based on reports of excessive apoptosis in torn supraspinatus tendon and mechanically loaded tendon cells , we hypothesized heat shock proteins may be present in rodent and human models of tendinopathy due to their central role in caspase - dependent apoptotic cell signaling . we utilized a running rat supraspinatus tendinopathy overuse model with custom microarrays to investigate the process at a genetic level . additionally torn supraspinatus tendon and matched intact subscapularis tendon samples ( early pathology ) were collected from patients undergoing arthroscopic shoulder surgery . overall , 91 genes were found to be significantly upregulated , and 37 significantly downregulated . the differential expression of apoptotic - related genes represented 6% ( 5 genes ) of the significantly upregulated genes and 8% ( 3 genes ) of significantly downregulated genes . upregulation ( p < 0.01 ) of hsp27 ( 3.4 ) and 70 ( 2.5 ) , cflip ( 2.2 ) receptor and caspase 8 ( 3.1 ) occurred in degenerative rat supraspinatus tendon subjected to daily treadmill running for 4weeks . we further confirmed increased levels of heat shock protein and apoptotic regulatory genes in human supraspinatus and subscapularis tendon at the rna and protein level ( figures 2(b ) and 2(c ) ) . overexpression of hsp27 is essential in preventing cells from undergoing apoptosis , a switch that may be redox - regulated . hsp27 inhibits specifically the cytochrome c and atp - triggered activity of caspase 9 on the apoptotic pathway . furthermore , hsp27 indirectly interferes with cell death because of its ability to modulate intracellular glutathione , a parameter that is also regulated by exercise . cytochrome c also triggers the oligomerization of apaf-1 , which in turn recruits pro - caspase 9 and pro - caspase 3 into the apoptosome ( the caspase activation multiprotein complex ) . hsp70 also protects cells from heat stress , from the cytotoxic effects of tnf , and from nitric oxide . based on these observations it would appear that heat shock proteins act as a check rein to apoptotic cell damage in tendinopathy . in conclusion , heat shock proteins are components of nature 's immune response that can act in a positive or negative way to the host immune system during the course of disease . these molecules seem to act as early regulators of the decision of a tissue / cell towards a reparative versus degenerative / inflammatory pathological process in joint related diseases . repetitive microtrauma / stresses are now considered as one of the main pathophysiological causes of tendinopathy . our investigations in early tendon damage have revealed a role for a heat shock proteins along with other investigators . we propose that when these molecules are released from stressed tenocytes they act as orchestrators of both the tissue healing response and subsequent inflammatory reaction with a fine balance between reparative versus degenerative change ( figure 3 ) . further work is ongoing within our institute to further elucidate their mechanistic role and possible therapeutic targeting .

tendon disorders tendinopathies are the primary reason for musculoskeletal consultation in primary care and account for up to 30% of rheumatological consultations . whilst the molecular pathophysiology of tendinopathy remains difficult to interpret the disease process involving repetitive stress , and cellular load provides important mechanistic insight into the area of heat shock proteins which spans many disease processes in the autoimmune community . heat shock proteins , also called damage - associated molecular patterns ( damps ) , are rapidly released following nonprogrammed cell death , are key effectors of the innate immune system , and critically restore homeostasis by promoting the reconstruction of the effected tissue . our investigations have highlighted a key role for hsps in tendion disease which may ultimately affect tissue rescue mechanisms in tendon pathology . this paper aims to provide an overview of the biology of heat shock proteins in soft tissue and how these mediators may be important regulators of inflammatory mediators and matrix regulation in tendinopathy .

1. Introduction 2. Heat Shock Proteins 3. Heat Shock Proteins and Inflammatory Disease 4. Tendinopathy 5. A Human Model of Early Tendinopathy 6. Heat Shock Proteins in Tendon 7. Summary

PMC5172470

the study of k - string composition of long dna sequences including complete genomes is a natural extension of g + c content , i.e. , k = 1 , analysis . using k values greater than 1 takes into account short - range ( up to k 1 ) correlations of nucleotides and enhances species - specific features in the sequence . visualization of the k - string distribution on a computer screen using a crude color code is essentially a kind of coarse - graining that helps to highlight some prominent feature of the dna sequence . for example , short palindromic strings of a certain type are avoided or under - represented in some bacterial genomes , leading to quite specific 2d histograms , while in mammalian genomic sequences the lower content of the dinucleotide cg as compared to gc dominates the picture 1 . , 2 .. two - dimensional portraits of the human chromosome 22 and the genomes of three bacteria are given in figure 1 . the similar patterns in the portraits of escherichia coli and shigella flexneri are caused by the under - representation of strings that contain ctag as substrings . the species - specific avoidance signature of complete bacterial genomes has eventually led to a new way of inferring phylogenetic relationship of prokaryotes without sequence alignment 3 . , 4 .. studies of the 1d histograms of extant complete genomes in contrast to their random counterparts have revealed the existence of universal length in complete genomes that can be explained by a simple universal model for genome growth and evolution 5 . , 6 .. in order to see that an observed feature does not occur in a random sequence , it is desirable to have randomization function built - in . a seemingly surprising effect is the appearance of fine structures in some randomized prokaryotic genomes with significant g + c bias . in figure 2 , the 1d histograms for k = 4 to 9 are shown for mycobacterium tuberculosis whose g and c make 65.6% of the genome . at fixed k , a total of k + 1 peaks can be seen in the histogram . this phenomenon has been fully understood . in particular , each peak may be well approximated by a poisson distribution ( 7 ) . the visualization tool used by us to obtain the aforementioned results in the cited papers has been improved over the years . from a unix command line tool for 2d histograms based on xlib and xtoolkit ( the old code is available at request to the corresponding author ) , it has evolved into a linux software using the gtk+ package with a user - friendly graphic interface . in order to count the frequency of occurrence of k - strings , a total of 4 counters are needed . to visualize the frequency distribution , these counters are allocated in a 2 2 square matrix and a color code is used to show the range of counting . the allocation of counters is realized by taking the direct product of k copies of the 2 2 matrix ( 1)(gcat).we put g and c in the first row to make the effect of g+c content readily visible . what we obtain is a 2d histogram or a portrait of the dna sequence . a 1d histogram is constructed by putting the counts along the abscissa from a minimal ( may be zero ) to a maximal count and the number of string types within a narrow range ( a bin ) of counts along the ordinate . to provide a reference for comparison , the program can randomize the input sequence , keeping the number of each type of nucleotides unchanged . the program takes one or more dna sequences in either genbank or fasta format as input . the user may form a list of sequences and then work with them to conduct comparative studies . seedna displays 2d as well as 1d histograms of the designated sequence or its reverse - conjugate or both ( by concatenating them ) using the original input or its randomized counterpart . using k greater than 9 would extend the picture beyond the screen of most present - day computers . the cursor will cause the designated string and its count ( 2d ) or the count range and the number of string types whose counts fall in that range ( 1d ) to be displayed . the 2d histograms of closely related species show strong similarities in k - string composition . this is clearly seen in the two lower portraits of figure 1 and the portrait of figure 3c , as e. coli , s. flexneri and s. typhi all belong to the same family enterobacteriaceae . therefore , it makes sense to display the difference of counts for each string type . to put the comparison on equal footing , then the two counts c1 and c2 for the same string type are used to calculate ( c1 c2)/(c1 + c2 ) . the last ratio is displayed using seven colors for the ranges ( 0.01 , 0.01 ) , (0.01 , 0.1 ) , (0.1 , 0.5 ) , and (0.5 , 1 ) . the string and its counts c1 and c2 are shown interactively at the bottom of the graph . this comparison feature is experimental for the time being and the way of showing the difference of portraits will be improved as more applications are implemented . for example , the user may change the color code or determine how many times the randomization procedure would be applied to the sequence before it is treated . at the users choice a screen figure may be exported to a gif file under a separate name for later manipulation . besides our old unix code , a very limited version of the 2d histogram was implemented at the european bioinformatics institute ( ebi ; http://industry.ebi.ac.uk/openbsa/bsa_viewers/ ) and the national institute for standard and technology ( nist ; http://math.nist.gov/~fhunt/genpatterns/ ) . these implementations did not provide built - in randomization , 1d histogram and comparison of 2d histograms . our full - fledged seedna program is written in c language using the gtk+ graphic package . a user - friendly interface makes the choice or combination of features a matter of clicking on buttons . the 2d histograms , if shown only in black / white , look similar to the chaos game representation ( cgr ) of dna sequence 8 . , 9 .. the chaos program in the free emboss package ( 10 ) implements the cgr algorithm . the consistency of the limiting measure of cgr and seedna algorithms has been analyzed by tin ( 11 ) . however , the seedna realizes the visualization of density in one pass , keeping the resolution k fixed . this can not be done in cgr without changing the algorithm and program . moreover , due to finite resolution of the computer screen the actual k is out of control and it varies along different directions in the cgr . therefore , seedna may replace chaos entirely with many new features added ( 1d histogram , randomization , comparison of 2d histograms , etc . ) . the source code of seedna is freely available under the gnu general public license at the authors website ( www.itp.ac.cn/~hao/seedna.tar.gz ; http://tlife.fudan.edu.cn/seedna.tar.gz ) . installation and running information as well as references are included as separate files in the above release package . since the definition of direct product of matrices applies to rectangular matrices as well , the idea of using direct product of matrices to represent k - strings may also be extended to protein sequences . we may define a 4 5 matrix ( 12)x=(acdefghiklmnpqrstvwy),where the matrix elements are the one - letter abbreviation of the amino acids . however , a similar visualization scheme would only work for k 4 if one does not scroll the picture behind the screen . furthermore , as protein sequences are much shorter than nucleic acids , the highlight of visualization must come from those strings that are present instead of those missing .

an interactive tool to visualize the k - string composition of long dna sequences including bacterial complete genomes is described . it is especially useful for exploring short palindromic structures in the sequences . the seedna program runs on red hat linux with gtk+ support . it displays two - dimensional ( 2d ) or one - dimensional ( 1d ) histograms of the k - string distribution of a given sequence and/or its randomized counterpart . it is also capable of showing the difference of k - string distributions between two sequences . the c source code using the gtk+ package is freely available .

Introduction Algorithm and Features Implementation and Availability

PMC3659633

throughout the 70 s of the 20th century , investigations on a new class of pharmacologically active substances actoprotectors ( aids for improving human s physical and mental efficiency ) were guided by professor vladimir vinogradov . these investigations resulted from the development of the first and the most commonly used actoprotector , bemitil ( chemical structure 2-ethylbenzimidazole hydrobromide ) ( fig . 1 ) . later , other actoprotectors were created , such as bromantane . for the last 20 yr , people synthesized and studied other compounds with actoprotecive properties belonging to different chemical classes : thiazoloindole derivatives , 3-hydroxypyridine derivatives , nicotinic acid derivatives , 1-oxa-4-aza-2-silacyclanes etc [ 3 - 5 ] . at the same time , experimental studies and analyses of pharmacological properties of certain herbs proved that some phytochemicals , while having a very low toxicity , are also aids that improve human s physical and mental efficiency . among such herbs , there are interesting plant adaptogens : panax ginseng and other species from genus panax , eleutherococcus senticosus , pfaffia paniculata , withania somnifera , schisandra chinensis , gynostemma pentaphyllum , rhodiola rosea , etc . actoprotectors are preparations that enhance the body stability against physical loads without increasing oxygen consumption or heat production , increasing the efficiency factor . actoprotectors fall under the metabolic drugs of non - consumptive class of action and possibly possessing antihypoxic characteristics at either higher or lower extent . the agents differ from antihypoxants since actoprotectors primarily stimulate protein synthesis and increase working capacity . moreover , the preparations exert an antihypoxic effect under hypoxic conditions , which may advance as a result of mitochondrion - decreased ability to oxidize substrates under higher physical loads . however , this is not the case in hypoxic conditions of other etiology . the principal difference of actoprotectors and psychostimulants ( caffeine , sydnocarb , phenamine , methylphenidate , modafinil , adrafinil etc . ) is that actoprotectors are agents of non - exhaustive actions . in actions of actoprotectors , there is no increase in oxygen consumption or heat production ; differing with nootropic agents actoprotectors increase not only mental , but physical work capacity as well . vinogradov presumed that actoprotectors did not have enough theoretical background to be labeled as a new class of pharmacological compounds . this separation appeared as a result of development of military medicine to improve physical strength . our opinion about this connection is that the actoprotectors are considered as synthetic ( and possibly , natural origin ) adaptogens with strong positive influence on physical work capacity . it means , for the convenience of pharmacological classification , some synthetic adaptogens that highly increase the physical performance can be determined as for example , benzimidazole derivatives dibazol ( bendazol ) , levamisole and afobazol are all regarded in scientific literature as adaptogens . dibazol s adaptogenic action was initially realized in adaptation in difficult environment conditions through immune mechanisms [ 18 - 27 ] . levamisole s adaptogenic activity is also connected primarily with adaptive changes in the immune system [ 28 - 31 ] . afobazol has neuroprotective properties established in vitro on survival of ht-22 neurons in the model of oxidative stress and glutamate toxicity , and its adaptogenic action was determined through central nervous system adaptation [ 33 - 35 ] . since benzimidazole derivatives have adaptogenic properties , these compounds are similar to bemitil , but their influence on physical work capacity is either absent or minimal . we think that briefly , actoprotectors can be mentioned as synthetic or natural origin adaptogens with significant capacity to increase physical and mental performance. the classic reference actoprotector is bemitil ; in its chemical structure , bemitil is 2-ethylthiobenzimidazole hydrobromide ( fig . currently , only two compounds , among all actoprotectors , are permitted for medical administration : bemitil ( commercial name antihot , certified in ukraine as dietary supplement ) and bromantane ( commercial name ladasten , certified in russia as a drug ) ( fig . 1 ) . adaptogenic herbs are more available all over the world . in many countries it becomes more convenient in the usage for people with active lifestyle or whose professional activity is related with heavy physical and/or mental loads ( athletes , military service men , firefighters , crew members , computer operators , night shift doctors and nurses , etc . ) . therefore , adaptogenic herbs as potential actoprotectors have not just only theoretical importance for the understanding of mechanisms of their pharmacological action , but also practical applications in sport and occupational medicine . a primary analysis of actoprotector s effect under heavy physical loads observed its influence on carbohydrates and energy metabolism : slight decreases of glycogen and creatine phosphate content in the liver and muscles , and of glucose in the blood and lower accumulation of lactates in the tissues and blood , and lower increases in heat production and oxygen consumption . after the period of exertion ended , rehabilitation of the factors under study was accelerated , and indeed , some factors showed super compensation . 2 illustrates the influence of benzimidazole actoprotector on glycogen content in rat liver during recovery . interestingly , the 4 hour time - point showed a reversed pattern for all of the parameters . the authors assumed that at this point of the recovery period , the energy resources are expended on different biosynthetic processes , leaving the energy supply for muscular activity is severely limited . thus , in this situation , switching of intracellular metabolism to the anabolic reactions , rather than catabolic predominantly , occurs . this is why the work capacity settles at a relatively low level and can not be increased using pharmacological agents . noted the increase of the heart and not the liver glycogen content by the 4th hour of the recovery period , and drew conclusions about the importance of the liver glycogen content to the capacity for work . however , this phenomenon reflects a more complex mechanism that can not be explained on the aforementioned basis alone and requires further research . the significant decrease of total rna content in the 4th hour of the recovery period in liver under benzimidazole actoprotector administration is believed to be the evidence behind this reasoning ( fig . it has been established that the therapeutic effect of actoprotectors ( bemitil , as an example ) is a function of its complex mechanism entailing cell genome activation , optimization of mitochondrial oxidation , oxidative stress reduction , and stimulation of cellular immune response . recently p. ginseng ( known also as ginseng and korean ginseng ) is one of the most well - known and studied adaptogens . it is grown in china , korea , japan , and russia while having a long - time ( some thousands years ) history of its administration in oriental medicine . nowadays , p. ginseng as a dietary and medicinal custom is not only in asia ( especially korea and china ) , but is also used world - wide . ginseng is available in many forms : whole root , root powder ( white ginseng ) , steamed root powder ( red ginseng ) , heat processed root powder ( sun ginseng ) , steamed and dried roots for 5 d and 9 times respectively ( black ginseng ) , teas , tinctures , and standardized root extracts containing known and reproducible amounts of ginseng saponins in every batch [ 37 - 40 ] . in some countries , other species from genus panax ( p. bipinnatifidus , p. japonicus , p. notoginseng , p. pseudoginseng , p. quinquefolius , p. stipuleanatus , p. trifolius , p. vietnamensis , p. wangianus , p. zingiberensis ) are not as well - known as p. ginseng , but they are also used in oriental medicine . among the diverse constituents of ginseng , steroid - like phytochemicals with adaptogenic properties named ginsenosides have been found to be major components responsible for their biological and pharmacological actions . ginsenosides are a special group of triterpenoid saponins that can be classified into two main groups by the skeleton of their aglycones : panaxadiol group ( rb1 , rb2 , rb3 , rc , rd , rg3 , rh2 , and rs1 ) and panaxatriol group ( re , rf , rg1 , rg2 , and rh1 ) ( fig . more than 150 naturally occurring ginsenosides have been isolated from roots , leaves / stems , fruits , and/or flower buds of ginseng . ginsenosides have been the target to many researches as they are believed to be the main active components behind the claims of ginseng s efficacy . these steroid - like phytochemicals , which are known to counter the negative influence of stress , are beneficial for health property . the glycosides act on the adrenal glands , helping to prevent adrenal hypertrophy and excess corticosteroid production in response to stress . ginseng stimulates the formation of blood vessels and improves blood circulation in the brain , thereby improving memory and cognitive abilities . ginseng is also used as treatments of diabetes , migraines , infections , cancer , radiation and chemotherapy protection , sleep aid , and appetite stimulation [ 42 - 44 ] . the ginsenosides content in ginseng preparations can vary depending on the species , the age , portion of the plant , the preservation method , the season of harvest , and the extraction method [ 45 - 47 ] . for example , comparative study on the ginsenosides of 47 samples of ginseng products derived from different panax species was conducted using a reverse - phase hplc method . the results showed that the ginsenoside compositions in ginseng products of different origins were considerably variable . chikusetsu - ninjin derived from p. japonicus ( japan ) was found to have the highest content ( 192.80 - 296.18 mg / g ) and a product from p. ginseng to be the lowest ( 5.78 - 15.63 mg / g ) . two main groups suggested by phytochemical data were clearly observed : group i mainly containing dammarane saponins consisted of p. ginseng , p. quinquefolius , p. notoginseng , p. vietnamensis and p. vietnamensis var . fuscidiscus and group ii containing a large amount of oleanolic acid saponins was composed of p. japonicus ( japan ) , p. zingiberensis , p. japonicus ( china ) , p. japonicus var . the ratios of the subtotal of dammarane saponins to that of oleanolic acid saponins were found to be > 1.9 and < 0.25 for groups i and ii , respectively . the product samples derived from the same botanical origin revealed similar constituent patterns , in other words , each panax taxon showed its own characteristic chromatographic profile , which appeared in the specific shape of an 11-direction radar graph constructed on the basis of the result of quantitative analysis . similarities of chemical constitution were seen among the closely phylogenetically - related taxa , including p. ginseng and p. quinquefolius , p. vietnamensis and p. vietnamensis var . fuscidiscus , p. japonicus ( china ) and its varieties , except for p. japonicus ( japan ) and p. zingiberensis . as mentioned above , except for ginsenosides , plants from genus panax contain other active compounds ( carbohydrates including polysaccharides , vitamins , alkaloids , fat soluble components , organic acids , microelements and macroelements , etc . ) which make important contributions in their pharmacological activity , but the quantity and composition of these compounds differ among the different species [ 49 - 53 ] . all together these facts explain why pharmacological properties of plants from genus panax are similar , but not the same ; moreover , pharmacological activity of different preparations from the same part ( roots , leaves etc . ) of the same species can be different depending on the season of harvest and the extraction method . finally , differences between different ginseng preparations can have an influence not just only in their potency , but also in kinds of their pharmacological activity . recently , the most widely standardized ginseng extracts , both commercially and for research purposes , are g115 and products of korea ginseng corporation ( seoul , korea ) , concentrated aqueous extracts from p. ginseng root , which are standardized to contain a certain amount of ginsenosides . poor standardization can cause difficulties in evaluation of data received from the animal experiments and human studies related with pharmacological activity of adaptogens including ginseng . it means that sometimes data from different laboratories connected with pharmacological properties ( including neurocognitive and actoprotective activity ) of preparations from the same species can not be compared ( or , at least , their comparison is very complicated ) . in conclusion , data on pharmacological activity of preparations received from different species from genus panax should be evaluated separately . analyses of scientific literature connected with influence of preparations from plants of genus panax on physical work capacity is more complicated when compared with that of their influence on cognitive functions . similar to the situation with memory and attention , most studies are connected with preparations from p. ginseng . results of many animal experiments attest that p. ginseng preparations can significantly increase physical work capacity . administration preparations with different qualities from this plant in different dosages increase exhaustion time for swimming in mice [ 55 - 58 ] and rats and exhaustion time for treadmill running in rats [ 60 - 62 ] . short - term ( 4 d ) , although not acute , treatment with complex of ginseng saponins ( 10 and 20 mg / kg / d ) significantly prolongs the aerobic endurance of non - trained rats exercising at approximately 70% vo2max . wang et al . established that pec ( the oral liquid which consists of p. quinquefolius , epimedium brevicornum , s. chinensis bail and cervus eplaphus ) administration could prolong swimming duration of mice in water tank and increase the tolerant ability against oxygen - deficiency . however at the same time , results of some other experimental studies show no significant influence of ginseng on physical work capacity . martinez and staba established that saponin extracts from different kinds of p. ginseng ( korean red , shiu - chi red , kirin red and sanchi ginseng ) and p. quinquefolius ( canadian , american white and american red ginseng ) ergogenic stems from the greek roots ergon and genes, meaning work and any means of enhancing energy production or utilization may be described as an ergogenic aid . it defines ergogenic aids as substances , foods , or training methods that enhance energy production , in use or recovery , while providing athletes with a competitive advantage . regarding to herbs currently being used to enhance physical performance , bucci subscribed that they can have different reasons for use including their adaptogenic properties , testosterone - like ( anabolic ) effect , stimulating effect on central nervous system , effect on capacity to increase endogenous testosterone production ( testosterone booster ) , and alpha - adrenergic agonist properties etc . a wide understanding of ergogenic aid makes this term a practical definition in the field of sport science and sport medicine but not pharmacology . according to modern pharmacological classification , p. ginseng and most other herbs from genus panax should be definite adaptogens due to their ability to increase physical work capacity in a healthy person , this being one of the important components of adaptogens action . however on the other hand , different adaptogens have different capacities to increase physical performance . that is why adaptogens with the strongest potency to increase physical work capacity are referred to actoprotecors . according to conventional wisdom , it is reasonable to refer agents from actoprotectors class to synthetic adaptogens and to regard their strong actoprotective effect as one of their components of adaptogenic action . it should be stressed that the focus of this study is on the ability to enhance physical work capacity and not on the adaptogens origins , synthetic or natural . according to this point of view , adaptogens of natural origin also can be referred to actoprotectors if they have potent influence on physical work capacity . some mechanisms of antifatigue action can be included into adaptogen action ; but other mechanisms can be different . as we see regarding to ginseng , its actoprotective properties are very discussible , but antifatigue properties have even more evidence supporting this along with proofs . from the results of experimental and human studies on the influence of ginseng preparations on physical performance and restoration after loads , there are many other controversies besides those mentioned about critical points of protocols and experimental techniques , including administration of different quality and composition ginseng supplements . detail analyses of experimental results demonstrated no influence by ginseng based on the swimming exhaustion time of rats and mice indicating critical points . for example , in one study some experimental groups included very few animals ( 3 - 4 ) . in this study , results of the swimming test generally showed variable swimming time ( 205 - 592 min ) . it can not be excluded that such various individual results can possibly be connected with methodological defects because of the difficulties in managing swimming test ( water of room temperature was preferred to avoid gas sorption on hair but could not use fresh tap water ) along with insufficient quantity of animals in some experimental groups . in jung et al.s study , very high doses of ginseng extract were used ( 500 mg / kg / d ) during a long time period ( 4 wk ) , although it is well - known that too high of dose of adaptogens can lead to inverse effects . results of human studies with plants from genus panax on physical performance ( adapted with modifications from ) # , data not listed or unavailable ; co , crossover ; db , double - blind ; dmae , dimethylaminoethanol ; f , female ; fsh , follicle stimulating hormone ; lh , luteinizing hormone ; m , male ; nc , not controlled ; panas , positive and negative affect schedule ; pc , placebo - controlled ; poms , profile of mood survey ; r , randomized ; rer , respiratory exchange ratio ; rpe , ratings of perceived exertion ; sb , single - blind ; ve , expiratory ventilation . results from numerous human studies , which are summarized in table 1 , are also controversial . similar to animal experiments , ( cited in ) demonstrated that ginseng supplementation had no effect but had a very small sample size ( 5 athletes in experimental group and 6 in placebo group ) . ziemba et al . established that ginseng administration does improve psychomotor performance during exercise without affecting exercise capacity in their study with soccer players , but used non - specific method for this kind of sport test ( incremental bicycle ergometer exercise test ) . . demonstrated substantial ergogenic effects but for ginseng combined with dimethylaminoethanol bitartrate , vitamins , minerals , and trace element . established that ginseng supplementation does not exert an ergogenic property on aerobic fitness enhancement in well - fit individuals with 60 young men ( 30 in experimental and 30 in control group ) , but used non - standardized 100% ginseng instead of any standardized ginseng preparation such as g115 , products of korea ginseng corporation . or any other standardized extract . two separate studies forgo et al , 1981 and 1982 respectively ( both cited in ) , showed significant change in aerobic capacity , lactate level , and heart rate under ginseng administration but failed to show either placebo or control conditions . forgo did , however , extend these studies with a double blind placebo - controlled investigation into the effects of 9 weeks administration of g115 , g115 plus tocopherol , or placebo , on physiological and hormonal measures ( luteinizing hormone , testosterone , and cortisol ) in athletes . he reported the significant increase in oxygen uptake and significant decreases in both exercise blood lactates and heart rate , but no change in hormone levels for both of the active treatments in comparison to placebo . this was followed by a further double blind study investigating the duration of the effects of 9 weeks administration of g115 ( 100 mg twice daily ) during exercise . results reported a significant increase in oxygen uptake and forced expiratory volume and significant decrease in heart rate and visual reaction times . some of these differences persisted at 3 weeks at the end of administration of g115 ( forgo and schimert , cited in ) . liang et al . reported that 30 d administration of p. notoginseng improves endurance time of exhaustion , and lowers mean blood pressure and vo2 during endurance exercise in healthy untrained adults . other studies do not support the view on ginseng as supplementation increases physical work capacity . . reported that acute p. ginseng supplementation does not affect the endurance of running performance of the heat - adapted male recreational runners in the heat . morris et al . found that 1week administration of two different doses of ginseng does not show better effect on any of the physiological indices under investigation ( oxygen , free fatty acids , lactate , and glucose ) than placebo in a placebo - controlled , cross - over study . allen et al . reported , in a randomized double - blind , placebo - controlled study involving 28 healthy young adults , that the administration of 200 mg ginseng extract for 21 d did not significantly affect heart rate or perceived exertion at 150 and 200 w ergometric exercise and claimed that it did not affect vo2 , exercise time , workload , plasma lactate , or hematocrit at peak levels of exercise . did not establish any increasing of physical work capacity after its course administration in a many - years series of ergogenic properties of g115 . thus , engels and wirth failed to demonstrate any effect of 8-weeks administration of ginseng on o2 consumption , respiratory exchange ratio , minute ventilation , blood lactic acid levels , heart rate , or perceived effort in a randomized double blind placebo - controlled g115 trial involving 36 healthy men . engels et al . found no effect of 400 mg / d g115 for 8 wk on supramaximal exercise performance and postexercise heart rate in 19 healthy women . engels et al . failed to demonstrate improvement of physical performance and heart rate recovery of individuals undergoing repeated bouts of exhausting exercise of 8-weeks administration of ginseng ( g115 , 400 mg / d ) in a double - blind , placebo - controlled , randomized study involving 38 active healthy women . morris et al . found that no influence of short ( 7 d ) administration of p. quinquefolius water - ethanol extract ( 8 or 16 mg / kg / d ) on cycle time to exhaustion and physiologic responses for loads in healthy people . direct increasing of physical work capacity after acute or course administration ( actoprotective properties ) should be separated from increasing of recovery speed after previous physical or other heavy loads for the convenience of administration of ergogenic aids in practice of sport preparedness as well as for pharmacological study of their mechanism of action . the property of increasing of recovery speed after previous physical or other heavy loads can be defined as drugs and dietary supplements with antifatigue properties are very important in some types of sports with demand for repeated performance after short intervals ( academic rowing , kayak and canoe paddling , different styles of wrestling , etc . ) . positive influence of p. ginseng on parameters of recovery after exhaustive physical loads has been proved by numerous animal experiments since the 1970 s . the first article published in 1974 studied the effects of extracts obtained from p. ginseng root ( 50 - 200 mg / kg intraperitoneal , immediately following the exercise ) on recovery from exhaustion ( four hour oscillation movements ) using six methods : exploratory movement ( em ) , hole cross ( hc ) , rotating rod ( rr ) , sliding angle ( sa ) , spring balance ( sb ) and rectal temperature ( rt ) tests . water extract significantly accelerated the recovery after em , increased motor activity index in em test , and elevated rectal temperature . however , water extract decreased the index in hc test and grip tone in sb test . lipophilic fraction significantly sped up the recovery from fatigue in em , rr , rt , and sb tests while delaying recovery in hc and sa tests . later , banerjee and izquierdo studied antistress and antifatigue properties of p. ginseng preparation on swiss albino mice which were exposed to various experimental models of stress in comparison with piracetam . both ginseng and piracetam were administered chronically in drinking water for 16 to 18 d as well as acutely 30 to 60 min prior to the experiments by injection . reactivity of the mice , loss of body weight , amount of feces , length of endurance , and incidence of mortality were graded and measured . both piracetam and ginseng treatment provided good protection against electroshock stress when compared to untreated mice . fighting scores , incidence of tonic convulsion , and mortality were significantly less in the treated groups . in the heat stress experiments , both piracetam and ginseng provided significant protection to the heat - exposed mice . in the fatigue stress of forced swim test , ginseng treatment was provided to be effective on adaptation to fatigue and ginseng also increased the endurance in both male and female mice . piracetam , on the other hand , only showed some antifatigue effects in the male mice . in the locomotor activity tests , ginseng did not depress motility , while piracetam did as described in the later part of the tests . from human studies on healthy people and clinical studies on patients who suffered from fatigue , the antifatigue property of p. ginseng preparations can be confirmed . ( cited in ) reported that administration of ginseng extract during 12-weeks improves postexercise recovery . later , postexercise recovery under ginseng administration was confirmed by ng and ng ( cited in ) , mcnaughton et al . evaluated the effect of the traditional chinese medicine tongxinluo and ginseng which had a good effect on the excess fatigue rats using metabolomics approach . a metabolomics study was performed on the excess fatigue rats treated with traditional tongxinluo or ginseng based on ultra - fast liquid chromatography coupled with ion trap - time of flight mass spectrometry . the plasma metabolic profiling data of the control rats , excess fatigue rats , and excess fatigue rats treated with tongxinluo or ginseng were acquired . the orthogonal partial least squares analysis was applied for the multivariate statistics , leading to the discovery of important differential metabolites distinguishing the excess fatigue rats treated with tongxinluo or ginseng from the control rats and excess fatigue rats . the results showed that tryptophan , bile acid , and lysophosphatidylcholine metabolism were disturbed in the excess fatigue rats . the metabolic pattern including the related metabolic pathways of the rats , after being treated with tongxinluo or ginseng , was adjusted towards the normal state . clinical studies of p. ginseng administration in treatment of unexplained chronic fatigue of unknown etiology demonstrated as effective in 56% cases ; p. ginseng and p. quinquefolius preparations can be regarded also as perspective compounds to improve cancer - related fatigue . in the last 20 yr of scientific literature , has been widely discussed [ 67,97 - 101 ] . based on modern understanding of pharmacology terms and definitions , it seems logical to concretize this debate by dividing it into two questions : is p. ginseng an actoprotector and is p. ginseng an antifatigue supplementation ? such division has a principal character and needs separate explanation . analyses of data from properly controlled studies on p. ginseng preparations ( table 1 ) allow us to make the preliminary conclusion that the controversies related to this study are connected with different doses , duration of courses being used in different studies , and physical condition of subjects who participated in these studies . enhancement of physical work capacity was only observed in studies when : 1 ) higher doses of ginseng supplementation ( over 200 mg / kg / d of standardized extract ) were used , 2 ) there were longer durations of study ( not less than 8 wk ) , 3 ) had larger subjects number , indicating greater statistical power , and 4 ) subjects were in relatively poor physical condition . one of the explanations that ginseng supplementation has no positive influence on physical performance was made by ferrando et al . . in their experiments , the effects of the ginseng extract on various biochemical and hematological parameters in male wistar rats subjected to a treadmill exercise protocol were studied for 12 wk . the increase was the largest for the animals treated with the extract during the third month of the study . the exercise alone also led to the increase in these parameters , while the combination of both exercise and ginseng extract produced smaller increase . this study shows a clear physiological response due to the ginseng extract administration that reproduces many of the effects obtained after long - term exercise . authors made conclusion that the combination of exercise and treatments seems to support the theory that there is no clear synergic effect when compared with the performance of exercise . in other work , the same authors established that treatment with p. ginseng increases the capillary density and the mitochondrial content of the red gastrocnemius muscle of rats . the results suggest that prolonged treatment with p. ginseng increases the capillary density and the oxidative capacity of the muscles with greater aerobic potential in a manner similar to the performance of physical exercise . this explanation can be accepted as one possibility , but it generally can not be a foundation for the conclusion that any effects of physical training and ginseng supplementation may be obtained separately but not when combined . the facts mentioned above allow us to conclude that p. ginseng ( and other plants from genus panax , possibly ) can be regarded as a potential actoprotector and antifatigue preparation with further research of its influence on physical work capacity , endurance and restoration after exhaustive physical loads in comparing with reference actoprotector bemitil . possible biochemical mechanisms for increasing the endurance after ginseng administration are connected with its influence on 1 ) carbohydrate and fat metabolism [ 57,63,104 - 106 ] , 2 ) immune and endocrine mechanisms , and 3 ) regulation of oxidative balance [ 106 - 111 ] . it seems possible , that in realization of the actoprotective and antifatigue properties of ginseng preparations , saponins are not the only components that have big importance . wang et al . established that ginseng polysaccharides have anti - fatigue activity , reflecting in the effects on the physiological markers for fatigue . it seems logical that such controversies are connected with different doses , duration of courses used in different studies , as well as with administration of different quality and composition of ginseng supplements . such a conclusion also allows ginseng to be regarded as potential actoprotector and allows for further research on its influence on physical work capacity , endurance and restoration after exhaustive physical loads in being compared to reference actoprotector , bemitil . numerous animal experiments and clinical studies about the influence of ginseng preparations ( mainly preparations from p. ginseng ) on cognitive functions show less controversial results compared to studies connected with exercise performance . several of them show memory improvement after ginseng administration . various memory - impairment models ( aged animals , scopolamine - induced memory deficit , ethanol - induced memory deficit , electroconvulsive shock - induced memory disturbances , muscarinic - induced memory deficit , dopamine - induced memory deficit , brain ischemia , cerebral infarct , sham or medial prefrontal cortex lesions , reserpine - induced orofacial dyskinesia , beta - amyloid - induced amnesia model , etc . ) have been used to evaluate the effects of ginseng and its active ingredients on a person s learning and memory . results of experimental studies on normal animals and on mentioned above animal models are summarized in table 2 . results of animal experiments with separated ginsenosides and preparations of plants from genus panax on cognitive functions # , data not listed or unavailable ; i.p . , intraperitoneal ; p.o . , per os ; wsf , water - soluble fractions ; lsf , lipid - soluble fractions ; pd , protopanaxadiol ; pt , protopanaxatriol ; pnb , p. notoginseng burk ; bdnf , brain derivative neurotrophic factor ; pns , p. notoginseng saponins ; samp8 , senescence accelerated mouse prone 8 . in most studies , independent of experimental model used , course administration of p. ginseng preparations had as a final result with significant antiamnaestic effect . in addition , positive results for cognitive functions enhancement were received from preparations of p. quinquefolium , p. notoginseng and p. pseudoginseng . however , for example , saito et al . found that extracts of ginseng inhibits conditioned avoidance response and discrimination behavior on pole climbing and shuttle box tests . similarly , petkov and mosharrof found that high doses of g115 impaired , rather than improved , conditioned reflex activity , and takagi et al . and takagi et al . demonstrated decreasing exploratory activity and a specific blocking action of conditioned responses following the administration of a crude ginsenoside fraction . some experiments were provided with isolated components of ginseng preparations , first with ginsenosides rg1 and rb1 . in passive avoidance test , rg1 improved learning and memory acquisition , consolidation , and retrieval , indicating that rg1 can improve all stages of memory . later , to study the effect of rg1 on one s learning and memory loss induced by -amyloid , passive avoidance and performance in the morris water maze were assayed after the final treatment . ginsenoside rg1 significantly decreased the latency and swimming distance , improved corresponding changes in search strategies in the morris water maze , and increased the step - through latency . in other studies , rg1 significantly improved memory deficits in aged rats , ovariectomized rats , and cerebral ischemia - reperfusion rats ( qiu et al . these results showed that ginseng extract and ginsenosides rg1 and rb1 facilitated acquisition and retrieval of memory . moreover , ginsenosides rb1 and rg1 also antagonized memory loss and cognitive deficit under various pathological conditions , such as cerebral ischemia and dementia ( qiu et al . , cited in ) . at the same time , other ginsenosides like rg3(s ) , rg5/rk1 , and rh2 and even nonsapongin fraction of red ginseng also have antiamnaestic properties and may have some importance on prevention of memory impairment or treatment of memory disorders . results of most human studies , connected with evaluation of influence of ginseng supplementation on cognitive functions , are summarized in table 3 . some of these studies show positive effects of ginseng preparations even at acute [ 148 - 150 ] or short course administration . poor standardization of different preparations unfortunately does not allow for the comparing of results from different studies . in this connection , it is necessary to put attention on row of studies provided with g115 [ 152 - 160 ] . effects of acute administration of g115 on cognition in young healthy individuals were evaluated in randomized double blind placebo - controlled studies [ 152,156 - 158 ] where ginseng differentially improved scores on a secondary memory factor ( a composite of four memory tasks ) . in the first study , secondary memory was found following 400 mg at four post - dose testing sessions while the lower and higher dosage reduced performance on the speed of attention factor . secondary memory. a later study assessed the effects of 200 and 400 mg ginseng during sustained cognitive demand - repeated cycles of serial threes , serial sevens , and the bakan rapid visual information processing ( rvip ) task . serial sevens performance was improved by the 200 mg dose . in a follow - up study , it appears that p. ginseng or its constituents are capable of producing tangible cognitive enhancing effects and that 200 to 400 mg appears to be the optimal dose range for young healthy adults when administered acutely prior to a cognitive test . course administration of ginseng preparations in most studies also showed positive effect on cognitive functions ( table 3 ) . results of human studies with plants from genus panax on neurocognitive function ( adapted with modifications from ) # , data not listed or unavailable ; co , crossover ; db , double - blind ; dmae , dimethylaminoethanol ; f , female ; fsh , follicle stimulating hormone ; grs , ginseng - rhizome saponin ; lh , luteinizing hormone ; m , male ; nc , not controlled ; panas , positive and negative affect schedule ; pc , placebo - controlled ; poms , profile of mood survey ; r , randomized ; rer , respiratory exchange ratio ; rpe , ratings of perceived exertion ; sb , single - blind ; ve , expiratory ventilation . detail analyses of mechanisms to determine underlying the positive impact on cognition under ginseng administration is out of the focus of this article . they are already described in numerous review articles published in recent literature [ 146,156,171 - 180 ] . it is necessary to subscribe that among these mechanisms is the capacity of ginsenosides to potentiate the cholinergic system in central nervous system . other important neurotransmitters for learning , memory , and cognitive functions involved in mechanism action of ginsenosides are glutamate and 5-ht . similar to studies connected with physical performance , it seems logical that such inconsistencies in the results of different studies are connected with different doses , duration of courses used in different studies , as well as with administration of different quality and composition ginseng supplements . despite that , most studies show positive influences of ginseng supplementation on intellectual work capacity in normal subjects and those of decreased cognitive functions . such conclusion also allows ginseng to be regarded as potential actoprotector and opens the way for further research of its influence on mental work capacity and cognitive functions in comparing with reference actoprotector , bemitil , and reference nootropic drug , piracetam . related to the capacity of many plant adaptogens to increase physical and mental performance , the question that arose about actoprotectors class including not just only synthesized but also natural origin compounds has importance for fundamental theory of pharmacological science and practical administration of many phytochemicals . the ginsenosides content in ginseng preparations can vary depending on the species , the age and part of the plant , the preservation method , the season of harvest , and the extraction method . in this connection , poor standardization can cause some difficulties in the evaluation of data received from animal experiments and human studies about the pharmacological activity of adaptogens including ginseng . despite these difficulties , large quantities of data received from animal experiments and human studies allow for making some preliminary conclusions about potential actoprotective properties of ginseng preparations : 1 ) results of some animal experiments and human studies attest that p. ginseng ( administered as extract ) can significantly increase physical and intellectual work capacity and the data allow ginseng to be referred as an actoprotector of natural origin ; 2 ) results related to the influence of ginseng on physical performance are more controversial than those connected with its influence on intellectual work capacity ; and 3 ) ginseng preparations can be regarded as potential actoprotectors and allow for further research of its influence on physical and mental work capacity , endurance , and restoration after exhaustive physical loads in comparing with reference actoprotectors ( bemitil ) and nootropic drugs ( piracetam ) . pharmacological activity of preparations received from different species from genus panax should be evaluated separately , but extract preparation should be standardized . comparison of ginseng preparations with preparations of other adaptogenic herbs will allow the decision of the most effective herbal actoprotectors . composition based on additive effects of herbal actoprotectors and synthesized ones can become the more useful in clinical and preventive medicine .

actoprotectors are preparations that increase the mental performance and enhance body stability against physical loads without increasing oxygen consumption . actoprotectors are regarded as a subclass of adaptogens that hold a significant capacity to increase physical performance . the focus of this article is studying adaptogen herbs of genus panax ( p. ginseng in particular ) and their capabilities as actoprotectors . some animal experiments and human studies about actoprotective properties of genus panax attest that p. ginseng ( administered as an extract ) significantly increased the physical and intellectual work capacities , and the data provided suggests that ginseng is a natural source of actoprotectors . preparations of ginseng can be regarded as potential actoprotectors which give way to further research of its influence on physical and mental work capacity , endurance and restoration after exhaustive physical loads while compared with reference actoprotectors .

INTRODUCTION DEFINITION AND CLASSIFICATION OF ACTOPROTECTORS COMPOSITION OF GINSENG PREPARATIONS AND THEIR STANDARDIZATION EFFECT ON EXERCISE PERFORMANCE EFFECTS OF GINSENG ON COGNITIVE FUNCTIONS CONCLUSION

PMC3334262

malrotation of the midgut is an abnormality in embryological development of gastrointestinal tract . by the fourth intrauterine week the gastrointestinal tract is in the form of an endoderm lined tube divided into fore , mid- and hindgut . mid- and hindgut defined by their blood supply the superior and inferior mesenteric arteries respectively . by the fifth week of life the midgut begins a process of rapid enlargement , physiological herniation and rotation . with rapid expansion of liver and kidneys , expansion of the midgut intestinal loop can not be contained within the abdominal cavity ; this results in temporary physiological midgut herniation through the umbilical cord with superior mesenteric artery forming the axis . this process forms c of the duodenum and places it behind the superior mesenteric vessels . hernial reduction occurs by week 10 with the jejunum reducing first and lying to the left and subsequent distal portions lying progressively to the right . the ceacum descends from position in the right upper quadrant forming the descending colon with its mesentery gradually disappearing . a 17-year - old male was seen in the emergency department with 10-year history of abdominal colic which is relieved by vomiting , along with dehydration . there was no history of jaundice , fever , steatorrhea , or bleeding per rectum . the patient had been treated with proton pump inhibitors , prokinetic agents by general practitioners , without any relief . his ultrasound examination and standing x - ray of abdomen physical examination was normal except minimal abdominal tenderness in epigastric , right hypochondriac region along with mild dehydration . his liver function tests , renal function tests , amylase , hemogram , and urinalysis were normal . duodenum appeared connected to small intestine seen on right side giving a whirlpool appearance due to rotation of gut around the superior mesenteric artery . superior mesenteric vein was seen on left of superior mesenteric artery [ figures 1a and b ] . stomach and duodenum appear connected to small intestine seen on the right side giving a whirlpool appearance due to rotation of gut around the superior mesenteric artery . superior mesenteric vein is seen on the left of superior mesenteric artery surgical referral was made ; he was treated with four - port laparoscopic ladd 's procedure . the peritoneum to the right of the ascending colon and caecum was incised and the anteriorly situated bands were stripped to free the duodenum . he was discharged within 2 days eating a normal diet and made a good postoperative recovery . at 3 months he was gaining weight and had no further vomiting . midgut mal and nonrotation refers to failure in counter clockwise rotation of the midgut which results in misplacement of the duodeno jejunal junction to the right of the midline ; in addition the small bowel mesentery has narrow vertical posterior attachment which is prone to volvulus . other anatomical abnormalities include peritoneal ( ladd 's ) bands running from the right colon to the lateral abdominal wall and an extensively mobile ceacum that fails to descend . acute presentation is with volvulus of midgut or ileoceacum occurring most frequently in neonate with likelihood decreasing with age . in most of the reported cases of this presentation , patients present with bilious vomiting in the first month of life because of duodenal obstruction or a volvulus . pathophysiology of these chronic symptoms may relate to the compressive effects of peritoneal bands running from ceacum and ascending colon to the right lateral wall . the short mesentery allows the small bowel to twist around the narrowed sma pedicle to create a distinctive whirlpool appearance . surgical management of intestinal malrotation was first described by ladd in 1936 and remains mainstay of management today . it involves reduction of vovulus if present , division of abnormal peritoneal bands ( duodeno colic , dodenojejunal - ileocolic ) , and placement of the small bowel to the right of the abdomen and ceacum to the left . increasingly laparoscopic ladd 's procedures are being performed and have been shown to be effective where there is no acute volvulus.[79 ] this minimally invasive approach allows for earlier oral intake and discharge from hospital .

abnormalities in midgut rotation occur during the physiological herniation of midgut between the 5th and 10th week of gestation . the most significant abnormality is narrow small bowel mesentery which is prone to volvulus . this occurs most frequently in the neonatal period , less commonly midgut malrotation presents in adulthood with either acute volvulus or chronic abdominal symptoms . it is the latter group that represents a diagnostic challenge . we report a case of a 17-year - old male patient who presented with 10-year history of nonspecific gastro - intestinal symptoms . after extensive investigation the patient was diagnosed with midgut malrotation following computed tomography of abdomen . the patient was treated with a laparoscopic ladd 's procedure and at 3 months he was gaining weight and had stopped vomiting . a laparoscopic ladd 's procedure is an acceptable alternative to the open technique in treating symptomatic malrotation in adults . midgut malrotation is a rare congenital anomaly which may present as chronic abdominal pain . abdominal ct is helpful for diagnosis .

Introduction Case Report Discussion

PMC4576365

attention - deficit and hyperactivity - disorder ( adhd ) affects 58% of the worldwide childhood population , it has high comorbidity and it shares symptoms with other behavioral and emotional disorders ( boyle et al . , 2011 ; larson et al . , 2011 ) . adhd prevalence rates vary significantly between and within countries , and depend on the ascertainment method and criteria utilized . of particular concern is the marked variability in diagnostic rates of adhd in developed countries ( getahun et al . , 2013 ) . this may reflect increased awareness of teachers and parents to symptoms of adhd in communities with a focus on education and adequate health care . however , even within communities there is marked variability in rates of adhd due to the subjective nature of primarily descriptive ( symptom - based ) diagnostic procedures ( asherson et al . , 2012 ; polanczyk et al . , 2014 ; see also recent reports by the us center for disease control and prevention ) . such diagnostic heterogeneity and its poor reliability have hampered efforts to determine the pathophysiology of adhd ( morgan et al . , 2013 ) . attempts have been made to find neuroimaging - based biomarkers of adhd using structural magnetic resonance imaging ( johnston et al . 2013 ) , resting - state functional mri ( hoekzema et al . , 2014 ; tomasi and volkow , 2014 ) , fmri data acquired during a single cognitive task ( hale et al . , 2015 ; hart et al . , 2014a ; hart et al . , 2014b ) , or combinations of some of these techniques ( anderson et al . , 2014 ; these studies provide mixed insights regarding the possible underlying neurocognitive deficit of adhd , and they report moderate classification accuracies ( rarely exceeding 80% ) , insufficient for clinical diagnosis . adhd is characterized by behavioral symptoms and multiple cognitive deficits associated with context dependent abnormal patterns of neural activity distributed across multiple brain regions . consequently , adhd is unlikely to be characterized by localized structural brain abnormalities robust enough to be detected using available structural imaging techniques . it is also unlikely that significant functional brain abnormalities characterizing adhd would be evident regardless of the mental state of the test subject , during the scan ( castellanos and proal , 2012 ; hammer et al . , 2015 ) . key characteristics of adhd include poor working memory , greater reliance on external feedback , and abnormal reward processing . these are associated with altered patterns of activity in distinct brain networks : ( i ) attention and working memory , comprising the dorsolateral prefrontal cortex , parietal cortex and temporal cortices ( burgess et al . , 2010 ; ehlis et al . , 2008 ; vance et al . , 2007 ) ; ( ii ) executive functions and cognitive control ( including feedback processing and response selection ) , comprising the dorsal , medial and ventral frontal cortices ( booth et al . , 2005 ; clark et al . , 2007 ; sonuga - barke and fairchild , 2012 ) ; and ( iii ) reward - processing , comprising the orbitofrontal cortex , anterior cingulate cortex and basal ganglia ( del campo et al . , 2013 ; plichta et al . notably , there are substantial individual differences in reactions to reward and feedback manipulation in adhd ( demurie et al . , 2011 ; , 2015 ; hammer et al . , 2015 ; plichta and scheres , 2014 ; van der schaaf et al . , accordingly , we hypothesized that using fmri data from an ensemble of visuospatial working memory ( vswm ) tasks that differ in the motivational context ( determined by the availability of reward and feedback ) would increase the odds that abnormal patterns of brain activity would be reliably detected in a larger proportion of adhd cases . we expected this to enable more accurate detection of adhd cases than the accuracies obtained by using fmri data acquired from any single vswm task , or the accuracy based on the participant 's respective behavioral performance . given the central role of vswm in adhd , we compared fmri data from four distinct vswm tasks in boys with adhd and typically developed ( td ) boys . tasks differed in the availability of trial - by - trial feedback ( feedback versus no - feedback ) and the participant 's expectation for significant monetary reward ( large versus small ) . all tasks required tracking the spatial location of letters while ignoring the letters ' identity , and executing timely responses . the manipulation of feedback and reward provided different motivational contexts , each requiring somewhat distinct executive skills found to be impaired in children with adhd . we used a multimodal analysis based on relatively few brain regions of interest , discovered using an independent univariate analysis ( hammer et al . , 2015 ; morris et al . , 2012 ; mulligan et al . , 2011 ; thoma and henson , 2011 ) . using a sparse principal component analysis , we further reduced the number of variables that were provided as input to the classifier . this substantially limited the odds of discovering an overfitted adhd classification model , and simplified the interpretation of the discovered model . we used a logistic regression ( lr ) classifier , which directly models the class conditional probabilities for each case ( i.e. , calculating the predicted probability that a given child has adhd ) by attempting to find a model allowing a decisive classification of as many cases as possible . this attribute is important in clinical settings where we aim to find a model that allows classification with confidence ( nouretdinov et al . , 2011 ) . twenty boys with a diagnosis of adhd combined - type ( mean age in years = 10.42 sd = 0.80 ) and 20 typically developed ( td ) boys ( 10.96 0.91 ) participated in the experiment . participants gave their informed consent ( and parental consent ) in accordance with the policies of the institutional review board ( irb ) at northwestern university . at the time of the fmri scanning session , adhd youth were weaned off stimulant medication for at least 24 h ( 12 participants used prescribed stimulants on a regular basis during the time they participated in this study ) . adhd diagnoses were based on exceeding the clinical cut offs on the adhd rating scale ( dupaul et al . , 1998 ) and the semi - structured diagnostic interview , the kiddie schedule for affective disorders and schizophrenia for school aged children : present and lifetime ( k - sads - pl ) version ( kaufman et al . , 1997 ) . mean total adhd score was higher in the adhd group than in the td group ( supplemental table 1 ) . participants were excluded if they had been diagnosed with a neurological disorder or were treated medically for a comorbid psychiatric disorder . all participants were right - handed native english speakers with normal or corrected to normal vision . mean full - scale iq scores were within normal range in both groups , however the average iq in the adhd group was lower than the average iq in the td group ( supplemental table 1 ) , which is not uncommon in adhd studies ( e.g. , hart et al . , 2014a ) . each task was 48 trials long . before and after each 2-back task the participant performed a fixation task where he was asked to press a key whenever the fixation - cross changed its color ( which happened in 4/12 of the trials ) . e - prime 2.0 ( psychology software tools , inc . ) was used for stimuli presentation and for recording participants ' responses ( for more details about the experimental design , see hammer et al . , 2015 ) . the two independent factors in the study were reward size ( large - reward versus small - reward ) and presence of trial - by - trial feedback ( no - feedback versus feedback ) . in an earlier practice session , and at the beginning of the scanning session , each participant was instructed that the reward for each correct decision in the large - reward task was 10 times larger than in the small - reward task . in the trial - by - trial feedback task , each key - press was followed by either a green square ( indicating a correct decision ) or a red square ( indicating an incorrect decision ) presented in the center of the screen . in the no - feedback task , the participant was informed about his overall performance only after concluding the task ( fig . 1 ) . imaging data were acquired on a 3.0 tesla siemens tim trio scanner using a 12-channel head coil . a susceptibility weighted single - shot epi ( echo planar imaging ) method with bold ( blood oxygenation level - dependent ) was used for functional image acquisition with the following scan parameters : tr = 2000 ms , te = 20 ms , flip angle = 80 , matrix size = 128 120 , field of view = 220 206.3 mm , slice thickness = 3 mm ( 0.48 mm gap ) , and number of slices = 32 ( an effective functional voxel size of 2 2 4 mm ) . a total of 145 images ( trs ) a high resolution , t1 weighted 3d image was also acquired with the following parameters : tr = 2300 ms , te = 3.36 ms , flip angle = 9 matrix size = 256 256 , field of view = 256 mm , slice thickness = 1 mm , and number of slices = 160 . the acquisition of the anatomical scan took approximately 9 min . prior to the scanning session children this enabled confirming that the participant is capable of keeping his head still for the duration of the scanning session . to minimize head movements in the scanner , gaps between the participant 's head and the head - coil data analysis was performed using mathworks matlab , spm8 ( statistical parametric mapping , wellcome trust centre for neuroimaging , london , uk ) , and ibm spss . preprocessing involved : ( i ) slice timing ; ( ii ) realignment of all functional images to the 24th image . ( iii ) co - registration of the functional and anatomical images ; ( iv ) normalization of the t1 image to the mni305 template image , which is most commonly used also for analyzing fmri data of pediatric populations ( e.g. , burgund et al . ( v ) 4 4 8 mm full width half maximum ( fwhm ) gaussian kernel smoothing . ( vi ) we confirmed that movement was kept below 4 mm ( in any of the x , y , or z dimensions ) within a scan using the artrepair software . images ( up to 9 per scan ) were realigned in artrepair , using interpolated values from the two adjacent non - outlier images . for subsequent general linear model ( glm ) analyses , the excluded noisy images were deweighted . as reported in supplemental table 2 , the two groups did not differ in patterns of head movements , and the replacement of outlier images primarily enabled reducing the signal to noise ratio in the fmri data of both groups . in order to further reduce within scan variability in neural activity , only trials in which the participant responded correctly ( hit and correct rejection ) were modeled , with onset time - locked to the beginning of each trial ( calhoun et al . ( vii ) a high pass filter with a cut - off of 256 s was applied . structural and functional brain images were inspected and found not to have significant image artifacts . we confirmed that for each participant maximal head displacement ( the distance between the two most distant fmri images within a scan ) in all translational axes was no larger than the size of a voxel ( i.e. , 4 mm ) . there were no significant between - groups differences in any head translation or rotation axes , all p > 0.25 ( supplemental table 2 ) . we also confirmed that it is unlikely that head movements underlie the adhd classification results we got based on the brain activity data ( supplemental fig . 1 ) . feature detection was based on a univariate glm analysis , intended to identify functional brain regions of interest ( frois ) that showed significant activation or deactivation in all four vswm tasks , contrasted with all the fixation tasks , using the brain activity data of the adhd boys and td boys combined ( total of 40 participants ; see chu et al . , 2012 ; friston , 2012 for discussions regarding optimal sample size ) . we found eight frois showing significant activation ( vswm > fixation ) and eight frois showing significant deactivation ( fixation > vswm ) . we used a voxel threshold of p < 0.01 ( family wise error [ fwe ] corrected ) , and voxel cluster threshold of p < 0.01 ( minimum cluster size of 50 voxels ; fwe ) , where each cluster had a single significant peak at p < 0.05 ( fwe ) . an anatomical gray - matter mask ( using the talairach daemon brain atlas gray - matter mask , with dilate = 3 ) , and a sphere mask with a radius of 15 mm from each froi peak voxel , constrained the froi volume . these 16 rois likely reflect the vswm network in a broad childhood population , in varying contexts ( fig . for each participant we calculated the difference in mean beta values between each of the four vswm 2-back tasks and the mean of all fixation tasks in each of the 16 frois ( 2-back fixation ; fixation2-back ) . overall , given four vswm tasks and 16 frois , the initial number of features , characterizing each participant , was 64 ( see supplemental fig . 2 for activation profiles ) . given the relatively large initial number of features , and the high correlations between some of the features ( see supplemental fig . 3 for the correlation matrix ) , we reduced the dimensionality of the data by using a sparse principal component analysis ( spca ) . this resulted with a relatively small number of orthogonal principal components ( pcs ) , which together explained most of the variability in the data , recalculated based only on the few features with the highest loadings ( berthet and rigollet , 2013 ; ritchie et al . this procedure has three major advantages : ( i ) spca enabled reducing the number of variables fed to the classifier to a number substantially smaller than the number of participants ; ( ii ) recalculating the pcs based only on features with the highest loadings enabled excluding lower weight features that were likely to add mostly noise ; and ( iii ) having each pc being affected by relatively few features , where each feature affects at most one pc , enabled better determining the underlying neurocognitive mechanisms represented by each pc . we used sparse loading selection based on thresholding of the rotated loadings ( loading threshold > 0.6 ; absolute weights > 0.1 ) . loading rotation was based on the varimax rotation method ( lu and zhang , 2012 ; ma , 2013 ; qi and luo , 2015 ; sjstrand et al . , 2006 ) . the use of this threshold resulted with 39 features ( out of 64 ) affecting the first 10 pcs ( pcs with eigenvalues > 2 ) , where each feature affected a single pc ( fig . the first 10 pcs explained approximately 70% of the variability evident in the original 64 features . each of the remaining pcs explained less than 3% of the variability in the data . interestingly , we found each one of the first 10 pcs to reflect brain activity from several rois from the same vswm task , but not brain activity in a specific roi in several tasks . the exception was pc-2 , which was based on the left and right middle frontal gyri in the two vswm tasks without feedback . this supports our hypothesis that functional brain - imaging data from several distinct tasks is likely to add information useful for classification . for the learning of the adhd classification model the lr directly models the class conditional probabilities for each case , attempting to decisively classify as many cases as possible . a standard , 2013 ; ponce - alvarez et al . , 2012 ) was used for finding which of the 10 pcs significantly contributed to classification accuracy . in each cross validation iteration the lr classifier was trained based on the fmri data of 39 participants , and then the goodness of fit of the learned model was evaluated based on the correlation between the clinician diagnosis of the left - out participant ( adhd or td ) and the lr classifier predicted adhd probability for this participant . the predictive power from all iterations was averaged to determine which of the 10 pcs are with statistically significant predictive power , and for estimating the predictive power of the final model . the reporting of a classification model based only on significant pcs has two advantages : ( i ) such a model is less likely to be overfitted or biased due to being based on too many predictors ( pcs ) , and thus it provides a more conservative estimate of the classification accuracies that can be achieved with the data at hand and ( ii ) it further reduces the number of brain regions by which the two groups of interest ( i.e. , adhd vs. tds ) differ , enabling a more parsimonious characterization of the differences in neurocognitive mechanisms between the two groups ( see supplemental fig . 5 for an illustration of the data processing pipeline ) . running the lr with the leave - one - out cross validation showed a statistically significant contribution for adhd classification accuracies for only four pcs ( pc-2 , p < 0.02 ; pc-3 , p < 0.04 ; pc-4 , p < 0.04 ; pc-8 , p < 0.02 ) . excluding each one of these four pcs from the classification model substantially impaired the classification accuracies , whereas adding any of the other six pcs did not increase the classification accuracies . the classification accuracy of the model based on the four statistically significant pcs was 92.5% , with 95% sensitivity and 90% specificity . importantly , most of the classification decisions had high predicted probability values ( pp > 0.67 ) assigned to most adhd boys ( 75% ) , and low values ( pp < 0.33 ) assigned to most td boys ( 80% ; fig . 3 ) , indicating a model with an excellent fit ( omnibus test for model fit , (4 ) = 28.52 , p < 0.0001 ) . this accuracy level is not statistically different from perfect accuracy reflecting the clinician 's diagnosis ( 100% ) , p = 0.12 ( one - tailed fisher exact test ; testing the hypothesis a permutation test shows that the classification accuracies of the above - described adhd classification model are unlikely to be discovered by chance . in each permutation , labels of half the cases from each group were switched with the opposite group . the mean accuracy of 15 distinct permutations ( 62.0% ) was significantly lower than that of the adhd model ( 92.5% ) , p = 0.000 ( exact test ) ; t(14 ) = 14.97 , p < 0.0001 ( one - tailed , one - sample t - test ) . even the highest observed accuracy of a permuted - labels model was close to being significantly lower ( 15% ) than that of the adhd model , p = 0.06 ( one - tailed fisher exact test ; testing the hypothesis adhd model is better than the best permuted model ) . the mean correct decisive classification accuracies ( with pp < 0.33 or pp > 0.67 ) in the 15 permutations were 24.5% , versus 77.5% of the adhd model , p = 0.000 ( exact test ) , t(14 ) = 13.59 , p < 0.0001 ( one - tailed , one - sample t - test ; see also supplemental fig . 4 ) . notably , the four significant pcs encompassed in the adhd model are based on fmri data from all four vswm tasks : pc-2 is based on the two tasks without feedback ; pc-3 is based on the small - reward with feedback task ; pc-4 is based on the large - reward with feedback task ; and pc-8 is based on the small - reward with feedback task ( fig . 2 ) . in the following analysis , we further investigated the utility of using fmri data from multiple vswm tasks as compared with using fmri data from a single task . here we used the threshold of eigenvalue larger than one ( keeping pcs that together explained more than 70% of the variance in the data ) . adhd classification accuracies based on the fmri data from each single task ( fig . 4 ; table 1 ) were significantly lower than the classification accuracy of the model based on all four tasks ( fig . 3 ) . the exception was the classification accuracy based on the fmri data from the small - reward with feedback task , which was 80% . however , here the correct decisive classification accuracy ( with pp < 0.33 or pp > 0.67 ) based on the fmri data from the small - reward with feedback vswm task was 60% , which is significantly lower ( 17.5% ) than the four - task classification accuracy , p < 0.05 ( one - tailed fisher exact test ) . feeding the lr classifier with the behavioral data from all four tasks resulted in an overall accuracy level of 75% ( 70% sensitivity ; 80% specificity ; omnibus test for model fit , (4 ) = 12.37 , p < 0.01 ) . this overall accuracy level is significantly lower ( 17.5% ) than the accuracy based on the fmri data from the four tasks , p < 0.04 ( one - tailed fisher exact test ; testing the hypothesis fmri model is better than the behavioral model ) . moreover , here only 57.5% of the classification decisions were both accurate and decisive , with high predicted probability values of being a boy with adhd ( pp > 0.67 ) assigned to only 50% of the adhd boys , and low values ( pp < 0.33 ) assigned to only 65% of the td boys ( fig . this accuracy level is significantly lower ( 20% ) than the decisive classification accuracy based on the four significant pcs ( based on four tasks ) , p < 0.04 ( one - tailed fisher exact test ) . performance levels in the four tasks were highly correlated , where a boy that exhibited low performance in one task likely exhibited low performance in the other tasks . moreover , we found high correlations between the behavioral performances in the four vswm tasks and pc-2 ( fig . that is , the brain activity represented by pc-2 ( right and left mfg , tasks without feedback ) explains much of the observed variability in the behavioral data . on the other hand , the behavioral performances had low correlations with pc-3 , pc-4 and pc-8 , hence the additional diagnostic information provided by these pcs to the classification model . feeding the lr classifier with both the behavioral and fmri data ( 10 pcs ) resulted in exactly the same model as when feeding it with only the fmri data . we show that using a logistic regression ( lr ) classifier , which received as input brain imaging data that was acquired while children perform four distinct vswm tasks , enabled an adhd classification accuracy of 92.5% ( fig . 3 ) . classification accuracies that were based on the fmri data from all four tasks were significantly higher than those obtained using the fmri data from any single task . we found that the brain regions in which boys with adhd exhibited altered pattern of brain activity differed from one task to the other . the corresponding behavioral data from the four vswm tasks enabled an classification accuracy level of 75% ( fig . 5 ) , which is significantly lower than those obtained using the fmri data from the four tasks . participants who were misclassified based on the data from one vswm task were not necessarily misclassified when using the data from other vswm tasks ( fig . when the lr classifier was fed with the pcs calculated based on the four vswm tasks together , we observed a substantial improvement in the classification accuracy ( fig . this is consistent with the underlying theorem of ensemble - based classification ( rokach , 2010 ; klppel et al . , 2012 ) . as expected based on this theorem , classifying participants by using several measurements for each participant ( e.g. , fmri data from few distinct vswm tasks ) , where each measurement enables a better than chance classification accuracy , and where there is a substantial degree of independency between the measurements ( e.g. , distinct cognitive tasks ) , enabled higher classification accuracies compared to accuracies achieved by using the data from each single measurement . our findings are with important theoretical contribution , providing additional support to the idea that the manifestation of neurocognitive abnormalities in adhd is context dependent ( dovis et al . specifically , in the absence of trial - by - trial feedback , altered activity patterns characterizing adhd were most evident in the bilateral middle frontal gyri ( mfg ) , specifically the right - mfg ( see pc-2 weight in fig . 3 ; see feature weights in fig . the mfg is believed to play an executive role in the visuospatial working memory network , and a primary role in volitional ( top - down ) allocation of attention ( burgess et al . we found that in vswm tasks without feedback , boys with adhd exhibited lower levels of activity in the mfg as compared with td boys . this may indicate poor vswm and poor capacity in allocating attention to target stimuli in children with adhd , manifested when feedback is absent ( see cortese et al . the fmri data from the small - reward with feedback vswm task also had a considerable contribution to adhd classification ( pc-8 and pc-3 ; fig . 3 ; fig . here , boys with adhd exhibited altered brain activity in a network ( pc-8 ) that primarily included the bilateral orbitofrontal cortex ( bi - ofc ) and the left fusiform gyrus ( left - ffg ) . the ofc has been reported to play two primary roles that have potential relevance to the current task : together with the inferior frontal gyrus , the anterior insula , the superior temporal cortex and the temporoparietal junction , the ofc is part of the ventral attention network , acting as a bottom - up saliency detection system determining subjective and context - dependent susceptibility to unexpected salient stimuli ( corbetta et al . , 2008 ; the ofc was also found to be involved in the processing of reward - related information ( schoenbaum and roesch , 2005 ; pauli et al . , 2012 ) . it is suggested that the ofc is involved in monitoring which recent actions were rewarded , and predicting which future actions are most likely to be rewarded ( kahnt et al . , 2010 ) . the left - ffg was found to be involved in letter identification and reading ( mccandliss et al . , 2003 ; dehaene and cohen , 2011 ; mcnorgan et al . , 2013 here we found greater deactivation in these two brain regions in td boys , as compared to boys with adhd , primarily in the small - reward with feedback vswm task . this may indicate that boys with adhd fail in suppressing task irrelevant information ( letter identity in a task that requires monitoring the spatial location of letters , and visual feedback indicating insignificant reward ) . the features with significant loadings on pc-3 ( small - reward with feedback ) were the two frois in the right medial / superior prefrontal gyrus ( right - mefg- and right - mefg+ , fig . 2b ) , the left - mfg , the right superior temporal gyrus ( right - stg ) , the right anterior insula ( right - antins ) , and the right supramarginal gyrus . these brain regions are involved in feedback processing ( ferdinand and opitz , 2014 ) , bottom - up attention and in sensory integration ( prado et al . the loadings on pc-3 ( and pc-8 ) , indicate that in the small - reward with feedback condition children with adhd are likely to exhibit altered suppression of irrelevant visual features ( ffg ) , altered visuospatial processing ( precuneus ) , altered sensory integration ( stg and supramarginal ) , altered bottom - up attention control ( ofc ) , altered feedback processing and top - down attention control ( mefg and mfg ) , and altered synchronization between bottom - up and top - down attention control ( antins ) . the fmri data from the large - reward with feedback vswm task had the smallest contribution to adhd classification ( 57.5% accuracy , with only a trend toward a significant model fit ; fig . altered activity in adhd in this vswm task was evident in pc-4 ( fig . 3 ) , which included the right inferior parietal lobe ( right - ipl ) , the right - antins , the right - mefg ( the activated right - mefg froi ; see fig . 2 ) and the right precuneus . these frois partially overlap ( right - antins and right - mefg ) with those included in pc-3 ( small - reward with feedback ) . this implies that the right - antins and right - mefg are associated with altered feedback processing in adhd ( regardless of reward expectation ) . this is consistent with earlier findings showing that the right - antins and the right - mefg ( together with the anterior cingulate ) mediate between the central executive network and brain regions involved in risk / gain prediction , where response selection is required ( menon and udin , 2010 ; preuschoff et al . , 2008 ; early studies showed that children with adhd exhibit poor cognitive control associated with lower levels of neural activity in the anterior insula , as compared with tds ( cubillo et al . we show that using fmri data from four distinct vswm tasks enabled substantially better classification accuracies than the use of fmri data from a single vswm task . nevertheless , the current investigation was limited to the manipulation of feedback and reward in vswm tasks , where many characteristics of the four tasks were identical . it is possible that an even better adhd classification can be achieved by using other , perhaps more demanding cognitive tasks that require other cognitive skills impaired in adhd ( e.g. , tasks with specifically greater response inhibition demands ; booth et al . , 2005 ; rubia et al . , 2005 moreover , the duration of each vswm task we used here was 48 trials long ( ~100 s ) . it is possible that these tasks can be shortened ( e.g. , reduced to 32 , or even fewer trials ) without compromising classification accuracies . as part of the development of a practical diagnosis tool , future investigations should aim to identify an optimized ensemble of cognitive tasks that would yield the highest classification accuracies in the shortest scanning time possible . future investigation may also involve looking for an ensemble of tasks that enable differentiation between children with adhd from other clinical populations , with shared symptoms . the use of multi - task - based fmri data may also enable earlier diagnosis of adhd , prior to the onset of clear behavioral symptoms , which in turn may enable earlier intervention . in order to be of practical clinical use , task - based fmri diagnosis requires the scanned participants to accurately perform a cognitive task while keeping still throughout a relatively long scan . thus , a multi - task - based fmri diagnosis session may not be practical for very young children or individuals with severe cognitive deficits . however , mild adhd and moderate adhd are much more common and represents more of a diagnostic dilemma for clinicians . in contrast , severe or very early onset adhd would likely have a more distinct etiology . future developments should ideally involve an integrative use of multi - task - based fmri , resting - state fmri and structural imaging . this would likely enable an effective diagnosis of most clinical cases , and the detection of the onset of some clinical condition in early childhood . ideally , future imaging - based diagnostic tools may enable finer differentiation of adhd subgroups , assisting clinicians in customizing intervention . it is likely that similarly high ( or even higher ) classification accuracies can be achieved by using other machine - learning based classification methods . future studies should specifically explore machine - learning methods based on whole brain data , from multiple tasks , which automatically detect brain regions in which the targeted clinical population exhibits abnormal patterns of brain activity ( e.g. , ryali et al . we show that fmri data acquired while participants perform a few distinct cognitive tasks enables substantial improvement in the detection of adhd cases , as compared with the use of fmri data from a single task ( or corresponding behavioral data ) . showing that fmri data enables better classification accuracies than the corresponding behavioral data suggests that even adhd cases that exhibited normal - like vswm performance were likely to be characterized by substantially altered pattern of brain activation . this provides a proof - of - concept that scanning subjects while they perform an ensemble of distinct cognitive tasks is likely to payoff , enabling greater accuracies and higher confidence diagnosis of clinical cases . we suggest that this approach can be used for diagnosing other clinical populations , and possibly also for dissociating between distinct clinical populations who share behavioral symptoms . this would require using cognitive tasks that target the neurocognitive deficits characterizing the clinical condition of interest , or a battery of tasks that may enable dissociating between distinct neurocognitive abnormalities .

finding neurobiological markers for neurodevelopmental disorders , such as attention deficit and hyperactivity disorder ( adhd ) , is a major objective of clinicians and neuroscientists . we examined if functional magnetic resonance imaging ( fmri ) data from a few distinct visuospatial working memory ( vswm ) tasks enables accurately detecting cases with adhd . we tested 20 boys with adhd combined type and 20 typically developed ( td ) boys in four vswm tasks that differed in feedback availability ( feedback , no - feedback ) and reward size ( large , small ) . we used a multimodal analysis based on brain activity in 16 regions of interest , significantly activated or deactivated in the four vswm tasks ( based on the entire participants ' sample ) . dimensionality of the data was reduced into 10 principal components that were used as the input variables to a logistic regression classifier . fmri data from the four vswm tasks enabled a classification accuracy of 92.5% , with high predicted adhd probability values for most clinical cases , and low predicted adhd probabilities for most tds . this accuracy level was higher than those achieved by using the fmri data of any single task , or the respective behavioral data . this indicates that task - based fmri data acquired while participants perform a few distinct vswm tasks enables improved detection of clinical cases .

Introduction Materials and methods Results Discussion

PMC4633586

in rhegmatogenous retinal detachment , the neurosensory retina becomes separated from the underlying retinal pigment epithelium ( rpe ) , resulting in many biochemical and structural changes [ 14 ] . morphological studies provide evidence that the most obvious detachment - induced damage within the neurosensory retina is shortening of the outer segments and gradual death of the photoreceptor cells [ 57 ] . this triggers a complex process of photoreceptors recovery that can lead to partial or even complete restoration of vision . the animal studies have demonstrated that healing is achieved by at least partial reconstruction of the outer segments of rods and cones . the process is characterized by wide individual variation and depends strongly on the duration of detachment . although complete restoration of the retinal structure has not been achieved , a modified version of the photoreceptor - rpe complex was established with significant variations in outer segment length and orientation . studies based on animal models have also demonstrated that remodeling of the outer segments varies in intensity depending on the retinal area that is being remodeled . the restoration of photoreceptor outer segments has been recently confirmed in vivo in humans using spectral domain optical coherence tomography ( sdoct ) . the hyperreflective band corresponding to the junction of photoreceptor inner and outer segments ( is / os ) was observed in 5% of eyes at one month and in 50% of eyes 6 months after surgery . postoperative visual acuity correlated with a restored is / os line . however , the detailed dynamics of this process in relation to the particular macular areas is still unclear . the purpose of this study is to analyze topographically photoreceptor outer segments recovery process in patients after pars plana vitrectomy for retinal detachment using sdoct . the medical records of the patients who underwent pars plana vitrectomy for rhegmatogenous macula - off retinal detachment at the department of ophthalmology , nicolaus copernicus university , between january and june 2010 were reviewed . the inclusion criteria were as follows : successfully treated retinal detachment involved macula and two to four fundus quadrants , minimal number of four control visits during at least one year of follow - up . exclusion criteria were as follows : high myopia , preexisting macular pathologies ( e.g. , macular hole or epiretinal membrane ) , and proliferative vitreoretinopathy stage c involving more than two hours . the eyes with the presence of any residual subretinal fluid in postoperative sdoct exam were also excluded from the study . all patients underwent a pars plana vitrectomy with gas tamponade ( sf6 , c2f6 , or c3f8 ) . in each case the study protocol was approved by an ethics committee of nicolaus copernicus university . during follow - up visits patients underwent complete ophthalmic examination , including best - corrected visual acuity ( bcva ) and fundus biomicroscopy . the first control visit was performed between the second and fourth weeks after surgery shortly after gas absorption . the third visit was between 24 and 48 weeks and the fourth one between 48 and 72 weeks after surgery . sdoct of the macula was performed during each visit ( soct copernicus , optopol , poland , or rs-3000 nidek , japan ) . fifty - two to 64 horizontal scans of 6.0 mm length were performed , each consisting of 1024 lines . the reflectivity of the is / os line in central cross section running through foveolar center was evaluated independently by two oct experts ( jjk , bls ) according to the following subjective grading scale : grade 0 : lack of the is / os line , grade 1 : the reflectivity of the is / os line similar to the reflectivity of external limiting membrane ( elm ) , grade 2 : the reflectivity of the is / os line higher than the reflectivity of elm but lower then that of rpe ( intermediate reflectivity ) , grade 3 : the reflectivity of the is / os line similar to that of rpe . the central one was foveola recognized as a flat retinal surface of 300 m in diameter in the center of foveal depression . the region located between the border of the foveola and the margin of foveal slope from nasal side was called nasal fovea and the corresponding region from temporal side was temporal fovea . both of them have diameter of 600 m . parts of the retina located outside fovea was named nasal and temporal parafovea ( figure 1 ) . additionally , in eyes examined by the rs-3000 the en face reflectivity maps at the level of the is / os were created in automatic fashion using commercially available software . this facilitated evaluating the reflectivity of this layer throughout the entire examined area . to evaluate statistical relationship between the grade of the is / os reflectivity and the retinal topography , the follow - up time , and the type of the used intraocular gas the chi - square test was used . correlation between bcva and the is / os reflectivity was evaluated using spearman 's coefficient . statistical analysis demonstrated good agreement between observers in evaluating the degree of the is / os reflectivity . the differences of the average grade in subjective scale were not statistically significant in any follow - up period . in all eyes the gradual process of at least partial restoration of normal is / os reflectivity was observed . the average is / os reflectivity grade within five topographical regions in the central sdoct cross section in relation to the elapsed time since surgery is displayed in table 2 . at the first follow - up visit ( between the second and fourth week after surgery ) the average reflectivity of the is / os line calculated in all 5 regions together was 1,05 . the is / os line had very similar reflectivity along the entire scan in all eyes except 9 in which the increase in reflectivity to grade 2 or 3 was observed in peripheral part of central cross section . at the second follow - up visit ( between the fourth and twelfth week after surgery ) grade 0 appeared only in 3,33% of evaluated regions . the most significant increase in reflectivity was observed in temporal and nasal parafovea with average values of 2,17 and 2,22 , respectively . sdoct exam performed during the third follow - up visit ( between 24 and 48 weeks after surgery ) showed further improvement in is / os appearance with the average reflectivity grade reaching value of 2,19 . when compared to the second follow - up visit , the third region , except peripheral parts of the scan , of average is / os reflectivity higher than grade 2 appeared . it was foveola with the average reflectivity of 2,33 . at the last follow - up visit ( between 48 and 72 weeks after surgery ) in all 5 regions of the central cross section the is / os reflectivity exceeded grade 2 reaching the highest average values in nasal and temporal parafovea and foveola . statistical analysis confirmed that the grade of the is / os reflectivity during all follow - up visits except the first one was significantly related to the topographical regions of the central cross section . the process of restoration of the is / os reflectivity started in peripheral part of the central scan and was followed by the improvement of the is / os visibility in centrally located foveola with persistent areas of reduced reflectivity in nasal and temporal fovea . in majority of eyes the process of the is / os line restoration was symmetrical but in 30% of them the increase in the reflectivity was initially observed either in temporal or in nasal side of the scan . the opposite part of is / os band located between foveola and parafovea got normal reflectivity in the later follow - up or remained unchanged . three eyes during final visit had highly reflective is / os layer in parafovea , foveola , and temporal part of the fovea with area of reduced reflectivity in nasal fovea . in one eye the normal is / os reflectivity returned in nasal part of the scan ( figure 2 ) . the above described changes are also visible on en face reflectivity maps ( figure 3 ) . they show that the areas of normal is / os reflectivity initially appear in peripheral macula . another small area of normal reflectivity is usually located in the center of the fovea . the maps made six months later reveal that the size of areas with increased reflectivity enlarged . as a result , peripheral and central areas of increased reflectivity approach each other , leaving only a small part of the macula with reduced reflectivity . low grade of reflectivity at the first follow - up visit was related to an average bcva of 0.19 . eyes with partial recovery of is / os reflectivity at the second and third control visit had average bcva of 0,37 and 0,49 , respectively . the correlation between bcva and the grade of the is / os reflectivity was statistically significant ( p < 0.0001 ; spearman cc = 0,63 ) . statistical analysis revealed that the type of intraocular gas used during the surgery had no influence on the level of the is / os reflectivity . after a vitrectomy for macula - off rhegmatogenous retinal detachment that ends up with retinal reattachment the gradual change in reflectivity of the is / os line can be observed . this process typically starts independently in the peripheral and central macula and can last more than a year , often leading to a complete restoration of nearly normal is / os appearance . because of a significant difference in the refractive index present within photoreceptor cells , the is / os line is clearly visible on sdoct in healthy eyes . it is noteworthy that recent data suggest better correlation of this hyperreflective line with photoreceptor ellipsoid zone rather than the junction between the inner and outer segments . its unchanged appearance is often used in research as a marker of normal photoreceptor morphology . the abnormal is / os reflectivity was observed in patients after surgical treatment of macular holes , epiretinal membranes , and diabetic macular edema [ 1113 ] . published reports have also demonstrated photoreceptor defects in patients after surgically treated retinal detachment [ 1416 ] . the defects were present in 76% to 83.3% of eyes within 3 to 7 months postoperatively , regardless of the performed procedure . in cases in which the mean time since the surgery was 23.1 months , photoreceptor defects were present in 53.3% of eyes . some authors point out that the is / os reflectivity can change over time , which may suggest some potential for tissue regeneration . similar changes have been described in the course of the natural healing process of such macular diseases as multiple evanescent white dot syndrome as well as in patients with surgically treated full - thickness and lamellar macular holes [ 1921 ] . shimoda et al . reported a reduction of the proportion of eyes with is / os abnormalities from 55% to 17% between the first and sixth postoperative months among patients in whom vitrectomy was performed because of retinal detachment , which may suggest gradual restoration of the outer photoreceptor segments . the regeneration process of the retina has been described based on an animal model , which was used to analyze the microscopic changes that occur after retinal reattachment . the results revealed that the length of the outer photoreceptor segments gradually increases depending on the duration of retinal detachment and time since reattachment . the strongest regeneration was observed in eyes with the shortest detachment time and the longest interval between the reattachment and the morphological examination . the authors also noted large discrepancies in the degree of regeneration among specific retinal regions . moreover , the study demonstrated that the cone and rod regeneration processes differ . in cone regeneration , the topographic variability of changes in reflectivity patterns observed in our study may be associated with the differences between the cone and rod regenerative processes . the processes leading to increased reflectivity of the is / os start within the peripheral macula , where rods predominate , and gradually shift toward the center . at the same time , hyperreflective areas begin to appear within the central fovea , where only cones are present . the observed regeneration processes are very individualized and do not always lead to complete is / os restoration . they can stop at any stage , resulting in abnormal reflectivity of the is / os junction and decreased visual acuity . our study shows that sdoct can visualize gradually increasing is / os reflectivity in patients with retinal detachment that has been successfully treated with vitrectomy . this process is not random and follows different patterns within the central and peripheral macula which may be attributed to the variable regenerative potential of cones and rods .

purpose . to evaluate the spatial distribution of photoreceptor inner and outer segment junction ( is / os ) reflectivity changes after successful vitrectomy for macula - off retinal detachment ( ppv - moff ) using spectral domain optical coherence tomography ( sdoct ) . methods . twenty eyes after successful ppv - moff were included in the study . during a mean follow - up period of 15.3 months , sdoct was performed four times . to evaluate the is / os reflectivity a four - grade scale was used . results . at the first follow - up visit the is / os had very similar reflectivity in entire length of the central scan with total average value of 1,05 . at the second visit the most significant increase of the reflectivity was observed in temporal and nasal parafovea with average values of 2,17 and 2,22 , respectively . the third region of increased reflectivity of an average value of 2,33 appeared during the third follow - up visit and was located in the foveola . at the last follow - up visit in entire central cross section the is / os reflectivity exceeded grade 2 reaching the highest average values in nasal and temporal parafovea and foveola . conclusions . a gradual increase of the is / os reflectivity was observed in eyes after ppv - moff . the process is not random and starts independently in the peripheral and central part of the macula which may be attributed to the variable regenerative potential of cones and rods .

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

PMC4831851

the back squat ( bs ) is a fundamental exercise prescribed for both athletes and non - athletes for developing lower - body strength . the resulting leg , hip , and back strength from the prescription of systematic squat resistance training reportedly improves athletic performance when included in a training program ( 20 , 23 ) . recently , it has been suggested in the lay media that the rear leg elevated split squat ( rless ) places less compressive force on the back , while placing higher stress on the legs , hips and stabilizer muscles ( 6 ) . since bs may be contraindicated in persons with lower back pain , it may be beneficial to examine different variations of squat exercises to determine the benefits of each , as it may have implications for athletic populations . for instance , there appears to be differences between variations of the squat for lifting heavy loads ( 3 ) , and how trunk position affects the joints and muscles involved ( 18 ) . additionally , gender differences in mechanics have been demonstrated when performing squats ( 2 , 8 , 25 ) . electromyographic ( emg ) analyses are commonly conducted to quantify electrical activity of muscles during weight training . while the majority of research examining emg activity and unilateral squats has focused on rehabilitation ( i.e. no external load ) and general populations ( 1 , 4 , 5 , 17 , 26 ) , one study ( 21 ) compared emg activity levels of the biceps femoris , rectus femoris , and gluteus medius in elite female athletes while performing both loaded back squats and loaded rless . they concluded that the rless produced greater biceps femoris and gluteus medius emg activity when compared to the traditional bilateral squat . in addition , rless produced a greater knee valgus angle , which may produce greater hamstring activity in an attempt to better stabilize the knee ( 21 ) . however , a direct comparison with other types of unilateral squat was not conducted in that study . in addition to kinematics and emg , the ground reaction forces of bilateral squatting movements have been examined ( 7 , 9 , 10 , 16 , 19 ) , in both loaded ( 10 , 16 , 19 ) , and unloaded conditions ( 7 , 9 ) . similar variables have also been examined during unilateral squatting motions ( 9 , 11 , 18 , 22 , 27 ) , however , these studies focused on unloaded single - leg squats for rehabilitation purposes . additionally , only two studies used a unilateral squat with a knee range of motion similar to a bilateral squat ( 9 , 11 ) . to our knowledge , this was also the only study that compared the kinetics of a unilateral and bilateral squat , but it focused on patellofemoral force differences and did not report any comparisons of ground reaction forces ( 9 ) . in one investigation that compared different squat techniques , peak force and peak power appeared to be similar ( 19 ) , however , this study compared the box squat and traditional bs , not unilateral and bilateral squats . in an effort to further understand the biomechanical aspects of bilateral and unilateral squat exercises , specifically bs , rless , and split squat ( ss ) , this investigation was designed to examine the vertical displacement , muscular activity and unilateral ground reaction forces of these three exercises . we hypothesized that the vertical displacement would be similar in the three types of squat . additionally , it was expected that most of the thigh and hip musculature would be similarly active in all three exercises with exception being biceps femoris , which was expected to be more active during the unilateral exercises as suggested previously ( 21 ) . further , the ankle and knee stabilizer muscles were expected to be more active in the rless and ss than the bs , and that the vertical grf will be similar between the exercises , suggesting similar demands are placed upon the prime mover musculature . nine healthy men ( ages 24 to 36 ; 26.1 3.8 years ) were recruited to participate . all participants had been participating in a heavy - resistance training program that included squatting exercises for at least the previous six months . all participants completed a health history questionnaire to screen for any pre - existing health conditions and injuries that would prevent participation . the study s purpose , procedures , and possible risks and benefits were explained to participants both orally and in written form , followed by the signing of informed consent documents . the study procedures were approved by the local university institutional review board prior to beginning research . this study used a repeated measures design to compare the biomechanical differences between a bs , ss , and rless . all three exercises were performed with one leg on the force platform and video recorded while muscle activity was monitored via emg . the independent and dependent variables were selected based on the existing literature , and we have attempted to increase the internal validity by carefully assigning loads based on bs 1-rm . all participants reported for two sessions : one informed consent and practice session , and one data collection session . after paperwork was completed during the first session , the participants completed a five - minute , self - paced general warm - up on a cycle ergometer , followed by the instruction and the practice of all three squat exercises . in addition , at the end of this session , bs one - repetition maximum ( 1-rm ) testing was completed following protocol described by harman et al . , participants completed the same cycle ergometer warm - up followed by seven warm - up sets of bilateral squats . the warm - up sets were performed as follows : six repetitions at 10% , 20% , and 30% 1-rm , three repetitions at 40% and 50% 1-rm , and one repetition at 60% and 70% 1-rm , with a rest period of exactly two minutes between sets . subsequent to warm - up , single - repetition bs , ss and rless were completed in stratified random order . for all lifts , the participant removed the weighted barbell from a rack with a high bar position on the upper back . bs were performed at 85% of the participants 1-rm with their feet shoulder - width apart and only the left foot on the force plate . this load was used because it represents a normal training load for the participants in our study . participants were instructed to squat to the lowest level possible and then complete the lift by returning to the starting position . this load was chosen for internal validity reasons , and for the fact that the participants were less trained in using rless and ss and thus 1-rm testing for those exercises was not feasible . for the rless , the participant was positioned with their left foot on the force platform under their hips , with their right foot elevated behind them with the anterior portion of the ankle on a 40-cm high stand designed for single - leg squats . the participants descended to a position where the knee of the right leg ( elevated ) touched the ground , and then returned to the starting position . the distance between the center of the stand and the tip of the 1 phalange of left foot was determined for each participant by calculating 85% of leg length ( from asis to floor ) . for the ss , each participant was positioned with their left foot in the center of the force platform and right foot behind with a stance at the same length as rless ( 1 phalange to 1 phalange ) . participants then descended until the right knee touched the ground , and then returned to the starting position . a rest period of two minutes between the different squat exercises was provided . a single video camera ( panasonic digital video camcorder , pv - dv203 ) captured the two dimensional ( 2-d ) motion for analysis . the camera was interfaced with a pc and analyzed with datapac 5 software ( run technologies ; mission viejo , ca ) . the shutter speed was set to 1/125 sec and the iris was set to + 18 db . data were sampled at 60 hz and filtered with a 4th order , low - pass butterworth filter at 20 hz . analog and kinematic data was synchronized with an analog spike ( light - emitting diode ( led ) placed in camera s view ) to serve as a signal for acquisition . a frame ( 1.8 m ) with active led markers on each corner markers ( active led ) were placed on the left end of the barbell and on the left midaxial line at the level of the anterior superior iliac spine ( asis ) . vertical displacement was determined from the barbell marker , and eccentric and concentric phases of the lifts were determined using the vertical displacement of the asis marker . muscle activity was measured for the gluteus maximus ( gmx ) , biceps femoris ( bf ) , semitendinosus ( st ) , rectus femoris ( rf ) , vastus lateralis ( vl ) , vastus medialis ( vm ) , tibialis anterior ( ta ) , and medial gastrocnemius ( mgas ) of the left leg . this leg was the non - dominant leg for all participants , and was the front leg for the unilateral squat variations in this study . the non - dominant leg as chosen because generally , balance is better in the non - dominant leg . this was thought to improve the likelihood that the participants would not lose their balance during unilateral squats . prior to electrode application , the area was shaved to remove any hair ; the skin was then gently abraded with fine sandpaper to remove any other debris and the area was cleansed with alcohol . the electrodes were placed parallel to the estimated resting pennation angle so that the same muscle fibers intersected both electrodes . electrodes ( ambu inc . ; glen burnie , md ) were 2-cm round ag / agcl with an inter - electrode distance of two cm , and the ground electrode was placed on the anterior aspect of the patella for signal noise reduction . ( run technologies ; mission viejo , ca ) with eight dual - lead channels . the system has a common mode rejection of 90db , a band pass filter ( 10450hz ) , and input impedance of 10m. gain was set at 1000 . synchronized data were collected at 2khz ( datapac 5 ; run technologies ; mission viejo , ca ) and channeled through a 12-bit analog - to - digital converter ( das1200jr ; measurement computing , middleboro , ma ) . data were quantified by computing a root mean square ( rms ) , 125ms time constant running average of the raw signal over the eccentric and concentric rom . vertical ground reaction force data ( n ) were acquired with an amti bp600900 ( watertown , ma ) force platform amplified with an amti msa-6 mini amp ( watertown , ma ) at a sampling rate of 2400hz using a das1200jr 12-bit analog to digital converter board ( measurement computing ; norton , ma ) and analyzed using datapac 5 ( run technologies ; mission viejo , ca ) . data was filtered with a 4 order low pass butterworth digital filter at 20hz . means and standard deviations were calculated for all variables of interest . each dependent variable was compared with a 1 3 repeated measures anova ( p < 0.05 ) to determine if any significant differences existed between squatting modalities . bonferroni post - hoc adjustments to dependent t - tests were used to determine where pairwise differences existed and cohen s d effect sizes ( es ) were calculated to quantify the magnitude of those differences ( 15 ) , with corrections for repeated measures . anova and bonferroni adjustments were calculated with spss v. 20 ( ibm ; armonk , ny ) . nine healthy men ( ages 24 to 36 ; 26.1 3.8 years ) were recruited to participate . all participants had been participating in a heavy - resistance training program that included squatting exercises for at least the previous six months . all participants completed a health history questionnaire to screen for any pre - existing health conditions and injuries that would prevent participation . the study s purpose , procedures , and possible risks and benefits were explained to participants both orally and in written form , followed by the signing of informed consent documents . the study procedures were approved by the local university institutional review board prior to beginning research . this study used a repeated measures design to compare the biomechanical differences between a bs , ss , and rless . all three exercises were performed with one leg on the force platform and video recorded while muscle activity was monitored via emg . the independent and dependent variables were selected based on the existing literature , and we have attempted to increase the internal validity by carefully assigning loads based on bs 1-rm . all participants reported for two sessions : one informed consent and practice session , and one data collection session . after paperwork was completed during the first session , the participants completed a five - minute , self - paced general warm - up on a cycle ergometer , followed by the instruction and the practice of all three squat exercises . in addition , at the end of this session , bs one - repetition maximum ( 1-rm ) testing was completed following protocol described by harman et al . , participants completed the same cycle ergometer warm - up followed by seven warm - up sets of bilateral squats . the warm - up sets were performed as follows : six repetitions at 10% , 20% , and 30% 1-rm , three repetitions at 40% and 50% 1-rm , and one repetition at 60% and 70% 1-rm , with a rest period of exactly two minutes between sets . subsequent to warm - up , single - repetition bs , ss and rless were completed in stratified random order . for all lifts , the participant removed the weighted barbell from a rack with a high bar position on the upper back . bs were performed at 85% of the participants 1-rm with their feet shoulder - width apart and only the left foot on the force plate . this load was used because it represents a normal training load for the participants in our study . participants were instructed to squat to the lowest level possible and then complete the lift by returning to the starting position . this load was chosen for internal validity reasons , and for the fact that the participants were less trained in using rless and ss and thus 1-rm testing for those exercises was not feasible . for the rless , the participant was positioned with their left foot on the force platform under their hips , with their right foot elevated behind them with the anterior portion of the ankle on a 40-cm high stand designed for single - leg squats . the participants descended to a position where the knee of the right leg ( elevated ) touched the ground , and then returned to the starting position . the distance between the center of the stand and the tip of the 1 phalange of left foot was determined for each participant by calculating 85% of leg length ( from asis to floor ) . for the ss , each participant was positioned with their left foot in the center of the force platform and right foot behind with a stance at the same length as rless ( 1 phalange to 1 phalange ) . participants then descended until the right knee touched the ground , and then returned to the starting position . a rest period of two minutes between the different squat exercises was provided . a single video camera ( panasonic digital video camcorder , pv - dv203 ) captured the two dimensional ( 2-d ) motion for analysis . the camera was interfaced with a pc and analyzed with datapac 5 software ( run technologies ; mission viejo , ca ) . the shutter speed was set to 1/125 sec and the iris was set to + 18 db . data were sampled at 60 hz and filtered with a 4th order , low - pass butterworth filter at 20 hz . analog and kinematic data was synchronized with an analog spike ( light - emitting diode ( led ) placed in camera s view ) to serve as a signal for acquisition . a frame ( 1.8 m ) with active led markers on each corner was used to calibrate the space for the motion analysis . markers ( active led ) were placed on the left end of the barbell and on the left midaxial line at the level of the anterior superior iliac spine ( asis ) . vertical displacement was determined from the barbell marker , and eccentric and concentric phases of the lifts were determined using the vertical displacement of the asis marker . muscle activity was measured for the gluteus maximus ( gmx ) , biceps femoris ( bf ) , semitendinosus ( st ) , rectus femoris ( rf ) , vastus lateralis ( vl ) , vastus medialis ( vm ) , tibialis anterior ( ta ) , and medial gastrocnemius ( mgas ) of the left leg . this leg was the non - dominant leg for all participants , and was the front leg for the unilateral squat variations in this study . the non - dominant leg as chosen because generally , balance is better in the non - dominant leg . this was thought to improve the likelihood that the participants would not lose their balance during unilateral squats . prior to electrode application , the area was shaved to remove any hair ; the skin was then gently abraded with fine sandpaper to remove any other debris and the area was cleansed with alcohol . the electrodes were placed parallel to the estimated resting pennation angle so that the same muscle fibers intersected both electrodes . glen burnie , md ) were 2-cm round ag / agcl with an inter - electrode distance of two cm , and the ground electrode was placed on the anterior aspect of the patella for signal noise reduction . ( run technologies ; mission viejo , ca ) with eight dual - lead channels . the system has a common mode rejection of 90db , a band pass filter ( 10450hz ) , and input impedance of 10m. gain was set at 1000 . synchronized data were collected at 2khz ( datapac 5 ; run technologies ; mission viejo , ca ) and channeled through a 12-bit analog - to - digital converter ( das1200jr ; measurement computing , middleboro , ma ) . data were quantified by computing a root mean square ( rms ) , 125ms time constant running average of the raw signal over the eccentric and concentric rom . vertical ground reaction force data ( n ) were acquired with an amti bp600900 ( watertown , ma ) force platform amplified with an amti msa-6 mini amp ( watertown , ma ) at a sampling rate of 2400hz using a das1200jr 12-bit analog to digital converter board ( measurement computing ; norton , ma ) and analyzed using datapac 5 ( run technologies ; mission viejo , ca ) . was compared with a 1 3 repeated measures anova ( p < 0.05 ) to determine if any significant differences existed between squatting modalities . bonferroni post - hoc adjustments to dependent t - tests were used to determine where pairwise differences existed and cohen s d effect sizes ( es ) were calculated to quantify the magnitude of those differences ( 15 ) , with corrections for repeated measures . anova and bonferroni adjustments were calculated with spss v. 20 ( ibm ; armonk , ny ) . all nine participants completed the study and their descriptive statistics are presented as mean sd ( table 1 ) . muscle activity was only significantly greater for the bf during rless and bs than ss during the concentric phase ( rless / ss - es= 2.11 , p=0.008 ; bs / ss - es=1.78 , p=0.029 ) , and significantly greater during rless than ss during the eccentric phase ( es= 2.13 , p=0.012 ; figures 1 and 2 ) . maximum vertical force ( n ) was also significantly greater during rless than ss ( es= 3.03 , p=0.001 ; figure 3 ) and tended to be greater during bs than ss but the trend did not reach significance ( es = 1.42 ; p=0.058 ) . this study was designed to compare two different single leg squat techniques and bilateral back squats with respect to vertical range of motion , muscle activity , and vertical ground reaction force . our data show significantly greater biceps femoris activity during rless and bs than ss , similarities in vertical displacement between the three lifts , and some differences in grf between the different variations of the squat . our data support contentions that similar lower body muscle activity can be achieved using the rless with half the load of bs . this likely would result in less compressive force on the back , however compressive force on the back is beyond the scope of the present study . our emg data support the contention that similar stimulus can be achieved with single leg squats of several types and bs . the only exception was bf activity , where rless and bs had a significantly higher activity than the split squat ( es = 2.11 and 1.78 respectively ) during the concentric phase of the lifts . rless also had significantly higher bf activity during the eccentric phase than split squats ( es = 2.19 ) . contrary to previous findings ( 21 , 24 ) , neither the rless nor the split squat had greater bf or rf activity than the bilateral squat . this may in part be due to differences in load calculation or sample population . one previous study ( 21 ) used 85% of the participants three - repetition maximum ( 3rm ) for each lift , where we used 85% of bs 1-rm for the bs and half that load for the single - leg lifts . they also used a sample of female athletes and we used a sample of resistance - trained men . the other study ( 24 ) used a 50 lb barbell for both bilateral and single leg squats , and a sample of healthy men . it would be expected that muscle activity would be higher during a single leg squat if the same load were used . however , there are always limitations to calculating relative loads between different weight training exercises . peak vertical grf were similar between the bs and rless , suggesting that we adjusted the load sufficiently for loading the leg unilaterally . the rless had significantly larger peak ground reaction force than the ss ( es=3.03 ) at the same load . peak vertical forces during the bs ( 1414.81 250.98n ) were larger than the ss ( 1198.56 187.88n ) , however failed to reach significance ( es = 1.42 ; p=0.058 ) . previous researchers reported that the rear leg supported between 25% and 45% of the load during a split squat ( 13 ) suggesting that the smaller peak forces during the split squat are likely due to a larger portion of the load being supported by the rear leg . however , this is beyond the scope of the present study , as we were only able to collect data from the lead leg within the limitations of our experimental setup . as was expected , vertical bar displacement was similar between squats , suggesting similar depth of squat . this variable has not been compared between these lifts previously , therefore comparisons with other studies is impossible . we acknowledge that similar bar displacement does not equate to similar joint ranges of motion . one study ( 21 ) reported larger trunk inclination during bilateral squats than during rless , which likely would translate to greater hip flexion and rom . since joint rom was not examined herein , it is unclear if this was true in the current study ; however , for trunk inclination to be different , lower extremity joint rom would likely be different between squats to maintain the overall vertical displacement . the findings of the current study combined with those previously reported ( 21 ) suggest further examination of joint rom in these three squat types . due to the inherent limitations in using 2d video analysis for joint motion , in addition , we acknowledge the limitation that both legs were not monitored for emg activity and grf . while adding 3d analysis and additional emg channels may be feasible , instrumenting multiple force platforms so that force information can be collected from both legs may be challenging , especially instrumenting a stand for the rear leg in the rless . in spite of these limitations , our data are an important contribution to the relative scarcity of data on these types of squat . additionally , future studies may consider basing the prescribed loads off of the 1-rm for each individual squat type . performing multiple repetition sets , including those designed to induce considerable fatigue , may also be of interest to the practitioner . rear leg elevated split squats ( rless ) activate the lower body musculature similar to bilateral back squats while using half the load , but increased bf activity was seen for rless . therefore , if additional bf activity is desired , rless may be more appropriate than ss or bs . future research should consider additional measures of force and time such as impulse to clarify the potential differences in bilateral and unilateral squatting . additionally , comparisons of the dominant and non - dominant leg in unilateral squats may be of interest .

muscular activity , vertical displacement and ground reaction forces of back squats ( bs ) , rear - leg elevated split squats ( rless ) and split squats ( ss ) were examined . nine resistance - trained men reported for two sessions . the first session consisted of the consent process , practice , and bs 1-repetition maximum testing . in the second session , participants performed the three exercises while emg , displacment and ground reaction force data ( one leg on plate ) were collected . emg data were collected from the gluteus maximus ( gmx ) , biceps femoris ( bf ) , semitendinosus ( st ) , rectus femoris ( rf ) , vastus lateralis ( vl ) , vastus medialis ( vm ) , tibialis anterior ( ta ) , and medial gastrocnemius ( mgas ) of the left leg ( non - dominant , front leg for unilateral squats ) . load for bs was 85% one repetition maximum , and rless and ss were performed at 50% of bs load . repeated measures anova was used to compare all variables for the three exercises , with bonferroni adjustments for post hoc multiple comparisons , in addition to calculation of standardized mean differences ( es ) . muscle activity was similar between exercises except for biceps femoris , which was significantly higher during rless than ss during both concentric and eccentric phases ( es = 2.11 ; p=0.012 and es= 2.19 ; p=0.008 ) , and significantly higher during bs than the ss during the concentric phase ( es = 1.78 ; p=0.029 ) . vertical displacement was similar between all exercises . peak vertical force was similar between bs and rless and significantly greater during rless than ss ( es = 3.03 ; p=0.001 ) . these findings may be helpful in designing resistance training programs by using rless if greater biceps femoris activity is desired .

INTRODUCTION METHODS Participants Protocol Statistical Analyses RESULTS DISCUSSION

PMC4030086

diabetes is a growing epidemic affecting more than 346 million people worldwide ( 13 ) . the metabolic abnormalities associated with diabetes lead to altered platelet , endothelial cell , and smooth muscle cell function ( 47 ) . together , these changes promote systemic atherosclerosis and lead to the traditional micro- and macrovascular complications commonly observed in diabetes . morbidity and mortality in the population with diabetes are largely due to complications of atherosclerosis . furthermore , vascular disease in one arterial bed increases the risk for concomitant vascular disease in other arterial territories ( 8,9 ) . vascular disease in the peripheral arteries presents a significant burden to health care expenditure and represents a major cause of myocardial infarction , stroke , and limb morbidity ( 1012 ) . in addition , both diabetes and disease in the peripheral vasculature are associated with increased long - term mortality ( 1316 ) . however , the peripheral neuropathy associated with diabetes masks symptoms of atherosclerosis , making the true prevalence of disease in the peripheral arteries difficult to ascertain without additional testing and presenting a challenge to early identification and prevention of disease progression ( 12,17 ) . given the known clustering of traditional cardiovascular risk factors and coronary heart disease with diabetes , this study sought to investigate the independent association between diabetes and the presence and severity of vascular disease in the peripheral arteries . while several studies have evaluated the association between diabetes and lower extremity peripheral artery disease ( pad ) , few studies have explored carotid artery stenosis ( cas ) or abdominal aortic aneurysms ( aaa ) in populations with and without diabetes , and no study to date has evaluated the association between diabetes and all three major vascular disease subtypes in a large unselected population . in this study , we aim to evaluate the association between diabetes and the presence and severity of pad , cas , and aaa in more than 3 million subjects . a total of 3,696,778 primarily self - referred people participated in the life line screening ( lls ; independence , ohio ) vascular survey at more than 20,000 screening sites , representing all 50 states and broad geographical and socioeconomic characteristics , between 2003 and 2008 . as noted previously , the prevalence of different cardiovascular risk factors in this population database is similar to that of the general u.s . census data linked by the subject s zip code , we categorized individuals based on income and education and found fairly good representation across different levels of socioeconomic status ( 19 ) . a variety of costs was incurred by the individuals based on the package of tests purchased . all lls sites use identical protocols and are subject to a quality control program ( 18 ) . in accord with the office for human research protections , this study is exempt from review by an institutional review board ( 20 ) . participants completed a two - page questionnaire , providing their demographic information , height and weight , and medical and surgical history , including cardiovascular risk factors , physical activity , nutrition , and family history of cardiovascular disease . obesity was identified as bmi 30 kg / m , and tobacco use was defined as use of at least 100 cigarettes during their lifetime and currently smoking ( current ) or not currently smoking ( former ) . hypertension was defined as systolic blood pressure of 140 mmhg in either upper extremity or self - reported physician diagnosis or medication use . history of coronary artery disease was defined as having a history of myocardial infarction , percutaneous coronary intervention , or coronary artery bypass graft . presence of pad was assessed with the bilateral ankle - brachial pressure index ( abi ) , defined as systolic blood pressure in the left and right ankle ( measured in the posterior tibial artery or dorsalis pedis artery if a posterior tibial artery doppler signal was inaudible ) divided by the highest of the two systolic blood pressures in the left or right arm ( brachial artery ) . in the overall population , 5.6% ( n = 207,818 ) pad was defined as either left or right abi 0.9 or history of peripheral arterial intervention . pad was defined as symptomatic if participants had a prior revascularization procedure or answered yes to , do you have aching or pain in the legs that is worse with walking or running or degree of pad was categorized as normal ( abi 0.911.4 ) , mild ( abi 0.810.90 ) , moderate ( abi 0.610.80 ) , severe ( abi 0.60 ) , or unable to compress artery ( abi > 1.4 ) ( 17 ) . presence of cas was assessed using duplex ultrasound , and in the population of interest , 2.9% ( n = 107,318 ) did not have carotid ultrasound results recorded . symptomatic cas was defined as participants with a prior stroke , transient ischemic attack , or operation on the carotid arteries . the degree of cas was categorized as moderate for 5069% stenosis or severe for 70% stenosis . all participants had abdominal aortic ultrasound results recorded , and aaa was defined as presence of an infrarenal abdominal aortic diameter of 3 cm on ultrasound ( the greater of the anteroposterior or transverse measurement was used ) or prior surgical or special procedure to repair an aaa . each individual was assigned a unique identifier , and the investigators had access only to de - identified data . baseline characteristics and the presence of pad , cas , and aaa between participants with and without diabetes were compared using the test for proportions and a two - sided independent sample t test for continuous variables . odds ratios ( ors ) and 95% cis were assessed using a logistic regression model to examine the strength of association between diabetes and pad , cas , and aaa . in addition to unadjusted or , model 1 adjusted for age , sex , and race , whereas model 2 adjusted for age , sex , race , bmi , hypertension , hyperlipidemia , tobacco use , coronary artery disease , and stroke or transient ischemic attack . in sex - specific analysis , the strength of association between diabetes and pad , cas , and aaa was evaluated using the logistic regression model in model 2 . the wald test was used for testing interaction between the presence of diabetes and sex for the presence of each of the vascular disease subtypes . chicago , il ) , sas ( version 9.12 ; sas institute inc . , cary , nc ) , and the r package ( r development core team ; available from http://www.r-project.org/ ) . a total of 3,696,778 primarily self - referred people participated in the life line screening ( lls ; independence , ohio ) vascular survey at more than 20,000 screening sites , representing all 50 states and broad geographical and socioeconomic characteristics , between 2003 and 2008 . as noted previously , the prevalence of different cardiovascular risk factors in this population database is similar to that of the general u.s . census data linked by the subject s zip code , we categorized individuals based on income and education and found fairly good representation across different levels of socioeconomic status ( 19 ) . a variety of costs was incurred by the individuals based on the package of tests purchased . all lls sites use identical protocols and are subject to a quality control program ( 18 ) . in accord with the office for human research protections , this study is exempt from review by an institutional review board ( 20 ) . participants completed a two - page questionnaire , providing their demographic information , height and weight , and medical and surgical history , including cardiovascular risk factors , physical activity , nutrition , and family history of cardiovascular disease . obesity was identified as bmi 30 kg / m , and tobacco use was defined as use of at least 100 cigarettes during their lifetime and currently smoking ( current ) or not currently smoking ( former ) . hypertension was defined as systolic blood pressure of 140 mmhg in either upper extremity or self - reported physician diagnosis or medication use . history of coronary artery disease was defined as having a history of myocardial infarction , percutaneous coronary intervention , or coronary artery bypass graft . presence of pad was assessed with the bilateral ankle - brachial pressure index ( abi ) , defined as systolic blood pressure in the left and right ankle ( measured in the posterior tibial artery or dorsalis pedis artery if a posterior tibial artery doppler signal was inaudible ) divided by the highest of the two systolic blood pressures in the left or right arm ( brachial artery ) . in the overall population , 5.6% ( n = 207,818 ) pad was defined as either left or right abi 0.9 or history of peripheral arterial intervention . pad was defined as symptomatic if participants had a prior revascularization procedure or answered yes to , do you have aching or pain in the legs that is worse with walking or running or degree of pad was categorized as normal ( abi 0.911.4 ) , mild ( abi 0.810.90 ) , moderate ( abi 0.610.80 ) , severe ( abi 0.60 ) , or unable to compress artery ( abi > 1.4 ) ( 17 ) . presence of cas was assessed using duplex ultrasound , and in the population of interest , 2.9% ( n = 107,318 ) did not have carotid ultrasound results recorded . symptomatic cas was defined as participants with a prior stroke , transient ischemic attack , or operation on the carotid arteries . the degree of cas was categorized as moderate for 5069% stenosis or severe for 70% stenosis . all participants had abdominal aortic ultrasound results recorded , and aaa was defined as presence of an infrarenal abdominal aortic diameter of 3 cm on ultrasound ( the greater of the anteroposterior or transverse measurement was used ) or prior surgical or special procedure to repair an aaa . each individual was assigned a unique identifier , and the investigators had access only to de - identified data . baseline characteristics and the presence of pad , cas , and aaa between participants with and without diabetes were compared using the test for proportions and a two - sided independent sample t test for continuous variables . odds ratios ( ors ) and 95% cis were assessed using a logistic regression model to examine the strength of association between diabetes and pad , cas , and aaa . in addition to unadjusted or , model 1 adjusted for age , sex , and race , whereas model 2 adjusted for age , sex , race , bmi , hypertension , hyperlipidemia , tobacco use , coronary artery disease , and stroke or transient ischemic attack . in sex - specific analysis , the strength of association between diabetes and pad , cas , and aaa was evaluated using the logistic regression model in model 2 . the wald test was used for testing interaction between the presence of diabetes and sex for the presence of each of the vascular disease subtypes . chicago , il ) , sas ( version 9.12 ; sas institute inc . , cary , nc ) , and the r package ( r development core team ; available from http://www.r-project.org/ ) . consistent with prior observations , participants with diabetes were older , more frequently male , less frequently white , and more likely to have other cardiovascular risk factors . subjects with vascular disease had a higher prevalence of risk factors than subjects without vascular disease . subjects with aaa were more likely to be men and had a higher prevalence of heart disease and stroke than subjects with pad or cas ( supplementary table 1 ) . among the 3,696,778 participants in the study , 422,501 participants had any vascular disease ; 233,958 participants had pad , 193,734 participants had cas , and 73,443 participants had aaa . participants with diabetes had a higher prevalence of any vascular disease ( unadjusted or 1.98 [ 95% ci 1.962.00 ] ; p < 0.0001 ) ( fig . 1 ) . diabetes was associated with a higher odds of pad ( unadjusted or 1.96 [ 95% ci 1.941.98 ] ; p < 0.0001 ) , cas ( unadjusted or 2.13 [ 95% ci 2.112.16 ] ; p < 0.0001 ) , and aaa ( unadjusted or 1.40 [ 95% ci 1.371.43 ] ; p < 0.0001 ) compared with those without diabetes ( table 1 ) . vascular disease in more than one peripheral vascular bed was also more common in participants with diabetes . compared with participants without diabetes , participants with diabetes were more likely to have peripheral vascular disease in one territory ( 15.1% vs. 9.0% ; p < 0.0001 ) , two territories ( 2.9% vs. 1.2% ; p < 0.0001 ) , or three territories ( 0.7% vs. 0.4% ; p < 0.0001 ) ( supplementary fig . 1 ) . proportion of survey participants with lower extremity pad , cas , and aaa in the populations with and without diabetes ( dm ) . odds of vascular disease by degree of severity in the presence of diabetes after adjustment for age , sex , race , hypertension , hyperlipidemia , smoking status , bmi , coronary artery disease , and stroke or transient ischemic attack , diabetes was significantly associated with increased odds of any vascular disease ( adjusted or 1.41 [ 95% ci 1.391.42 ] ; p < 0.0001 ) . a significant interaction existed between diabetes and vascular disease phenotype ( p < 0.0001 ) ; diabetes was significantly associated with increased odds of pad ( adjusted or 1.42 [ 95% ci 1.411.44 ] ; p < 0.0001 ) and cas ( adjusted or 1.45 [ 95% ci 1.431.47 ] ; p < 0.0001 ) . in contrast , after multivariable adjustment , diabetes was significantly associated with lower odds of aaa ( adjusted or 0.86 [ 95% ci 0.840.88 ] ; p < 0.001 ) ( table 1 ) . the relationship between severity of vascular disease and diabetes across the spectrum of vascular phenotypes is shown in table 1 and fig . the association between diabetes and pad became more pronounced with severity of disease ( adjusted ors : mild pad , 1.37 ; moderate pad , 1.77 ; severe pad , 2.16 ; p for trend < 0.001 ) . although less striking , a similar observation was noted in cas ; the association between diabetes and cas increased with severity of disease ( adjusted or 1.53 [ 95% ci 1.501.56 ] ; p < 0.001 for moderate cas and adjusted or 1.60 [ 95% ci 1.561.63 ] ; p < 0.001 for severe cas ) . for the end point of aaa , diabetes was associated with a 20% and 25% lower odds of smaller and larger aaa , respectively ( adjusted or 0.80 [ 95% ci 0.770.83 ] ; p < 0.001 for 3- to 5-cm - diameter aneurysm and adjusted or 0.75 [ 95% ci 0.660.85 ] ; p < 0.001 for > 5-cm - diameter aneurysm ) . proportion of survey participants with lower extremity pad ( a ) , cas ( b ) , and aaa ( c ) by disease severity in the population with and without diabetes ( dm ) . 2 shows the prevalence of vascular disease stratified by symptoms in participants with and without diabetes . participants with diabetes were more likely to have both asymptomatic disease and symptomatic disease . to minimize recall bias in our analysis , we excluded subjects with symptomatic disease . results were not greatly altered , with increased odds of pad ( adjusted or 1.54 [ 95% ci 1.501.57 ] ; p < 0.0001 ) and cas ( adjusted or 1.57 [ 95% ci 1.551.60 ] ; p < 0.0001 ) and decreased odds of aaa ( adjusted or 0.80 [ 95% ci 0.770.83 ] ; p < 0.0001 ) . we further investigated the association between diabetes and different phenotypes of vascular disease in women and men ( table 2 ) . a significant association between diabetes and pad and cas was present in women and men , respectively . diabetes was associated with a 21% decreased odds of aaa in men , but no significant association between diabetes and aaa was noted among women ( adjusted or 0.99 [ 95% ci 0.961.03 ] ; p = 0.76 ) . consistent with prior observations , participants with diabetes were older , more frequently male , less frequently white , and more likely to have other cardiovascular risk factors . subjects with vascular disease had a higher prevalence of risk factors than subjects without vascular disease . subjects with aaa were more likely to be men and had a higher prevalence of heart disease and stroke than subjects with pad or cas ( supplementary table 1 ) . among the 3,696,778 participants in the study , 422,501 participants had any vascular disease ; 233,958 participants had pad , 193,734 participants had cas , and 73,443 participants had aaa . participants with diabetes had a higher prevalence of any vascular disease ( unadjusted or 1.98 [ 95% ci 1.962.00 ] ; p < 0.0001 ) ( fig . diabetes was associated with a higher odds of pad ( unadjusted or 1.96 [ 95% ci 1.941.98 ] ; p < 0.0001 ) , cas ( unadjusted or 2.13 [ 95% ci 2.112.16 ] ; p < 0.0001 ) , and aaa ( unadjusted or 1.40 [ 95% ci 1.371.43 ] ; p < 0.0001 ) compared with those without diabetes ( table 1 ) . vascular disease in more than one peripheral vascular bed was also more common in participants with diabetes . compared with participants without diabetes , participants with diabetes were more likely to have peripheral vascular disease in one territory ( 15.1% vs. 9.0% ; p < 0.0001 ) , two territories ( 2.9% vs. 1.2% ; p < 0.0001 ) , or three territories ( 0.7% vs. 0.4% ; p 1 ) . proportion of survey participants with lower extremity pad , cas , and aaa in the populations with and without diabetes ( dm ) . odds of vascular disease by degree of severity in the presence of diabetes after adjustment for age , sex , race , hypertension , hyperlipidemia , smoking status , bmi , coronary artery disease , and stroke or transient ischemic attack , diabetes was significantly associated with increased odds of any vascular disease ( adjusted or 1.41 [ 95% ci 1.391.42 ] ; p < 0.0001 ) . a significant interaction existed between diabetes and vascular disease phenotype ( p < 0.0001 ) ; diabetes was significantly associated with increased odds of pad ( adjusted or 1.42 [ 95% ci 1.411.44 ] ; p < 0.0001 ) and cas ( adjusted or 1.45 [ 95% ci 1.431.47 ] ; p < 0.0001 ) . in contrast , after multivariable adjustment , diabetes was significantly associated with lower odds of aaa ( adjusted or 0.86 [ 95% ci 0.840.88 ] ; p < 0.001 ) ( table 1 ) . the relationship between severity of vascular disease and diabetes across the spectrum of vascular phenotypes is shown in table 1 and fig . 2 . subjects with diabetes were more likely to have more severe pad than subjects without diabetes . the association between diabetes and pad became more pronounced with severity of disease ( adjusted ors : mild pad , 1.37 ; moderate pad , 1.77 ; severe pad , 2.16 ; p for trend < 0.001 ) . although less striking , a similar observation was noted in cas ; the association between diabetes and cas increased with severity of disease ( adjusted or 1.53 [ 95% ci 1.501.56 ] ; p < 0.001 for moderate cas and adjusted or 1.60 [ 95% ci 1.561.63 ] ; p < 0.001 for severe cas ) . for the end point of aaa , diabetes was associated with a 20% and 25% lower odds of smaller and larger aaa , respectively ( adjusted or 0.80 [ 95% ci 0.770.83 ] ; p < 0.001 for 3- to 5-cm - diameter aneurysm and adjusted or 0.75 [ 95% ci 0.660.85 ] ; p < 0.001 for > 5-cm - diameter aneurysm ) . proportion of survey participants with lower extremity pad ( a ) , cas ( b ) , and aaa ( c ) by disease severity in the population with and without diabetes ( dm ) . 2 shows the prevalence of vascular disease stratified by symptoms in participants with and without diabetes . participants with diabetes were more likely to have both asymptomatic disease and symptomatic disease . to minimize recall bias in our analysis , we excluded subjects with symptomatic disease . results were not greatly altered , with increased odds of pad ( adjusted or 1.54 [ 95% ci 1.501.57 ] ; p < 0.0001 ) and cas ( adjusted or 1.57 [ 95% ci 1.551.60 ] ; p < 0.0001 ) and decreased odds of aaa ( adjusted or 0.80 [ 95% ci 0.770.83 ] ; p < 0.0001 ) . we further investigated the association between diabetes and different phenotypes of vascular disease in women and men ( table 2 ) . a significant association between diabetes and pad and cas was present in women and men , respectively . diabetes was associated with a 21% decreased odds of aaa in men , but no significant association between diabetes and aaa was noted among women ( adjusted or 0.99 [ 95% ci 0.961.03 ] ; p = 0.76 ) . in this large population - based analysis , all three major forms of vascular disease were significantly higher in people with diabetes compared with those without diabetes . however , after adjustment for baseline demographics and traditional risk factors , diabetes was significantly associated with an increased odds of pad and cas but lower odds of aaa . in support of this observation , the strength of association was more pronounced with increasing severity of disease in each of the three vascular phenotypes . moreover , the positive association between diabetes and pad and cas , as well as the negative association between diabetes and aaa , remained robust in subjects with asymptomatic disease . finally , the increased association between diabetes and pad and cas was similar in women and men ; however , diabetes was associated with lower odds of aaa in men , which was not observed in women . the results of this study are consistent with a recent analysis of more than 40,000 men without a history of cardiovascular disease at baseline who were followed for 25 years in the prospective health professionals follow - up study , which demonstrated an independent association between diabetes and pad ( 21 ) . another study using national health and nutrition examination survey ( nhanes ) data demonstrated almost double the proportion of individuals with pad in the population with diabetes compared with the overall population ( 22 ) . in contrast to the current study , though , the abi in the nhanes was measured using systolic blood pressure in only the right upper extremity , and the diagnosis of pad did not include history of prior lower extremity revascularization ( 22 ) . furthermore , the authors noted an increase in the proportion of individuals with pad in the elderly , non - hispanic black , and mexican american populations but did not perform adjusted analyses ( 22 ) . our results demonstrating a significant positive association between diabetes and cas are consistent with those reported in prior smaller cohorts . one study evaluating 1,058 patients referred for doppler ultrasonography of the carotid arteries reported a significant association between diabetes and severe cas ( or 2.77 ) in a multivariate logistic regression analysis that included age , sex , and ischemic heart disease ( 23 ) . another small prospective study evaluated 782 subjects ( 14% with diabetes ) with high - resolution b - mode ultrasound and demonstrated a nonsignificant association between the presence of diabetes without cardiovascular disease and cas ( adjusted for age and sex : or 2.16 [ 95% ci 0.964.86 ] ; adjusted for age , sex , and cardiovascular risk factors : or 2.19 [ 95% ci 0.925.20 ] ) ( 24 ) . unlike pad and cas , however , the odds of aaa in participants with diabetes significantly decrease after multivariate adjustment . similar to our findings , a cross - sectional screening study of 73,451 veterans with no history of aaa demonstrated a negative association between the presence of diabetes and aaa in a multivariable model accounting for demographics and traditional cardiovascular risk factors ( or 0.68 [ 95% ci 0.600.77 ] for aaa 3.03.9 cm and or 0.54 [ 95% ci 0.440.65 ] for aaa 4.0 cm , compared with < 3.0 cm ) ( 25 ) . consistent with these results , a report of 6,142 veterans screened for aaa demonstrated that the presence of diabetes was associated with a normal aortic size ( 26 ) . another international , prospective , observational outpatient registry of 68,236 patients 45 years of age with established coronary , cerebral , or peripheral arterial disease or at least three vascular disease risk factors also demonstrated an inverse relationship between diabetes and aaa in a multivariate analysis ( or 0.59 [ 95% ci 0.520.66 ] ) ( 27 ) . although these studies demonstrate a lower or then that presented in the current study , the populations studied are different . for example , the studies from the veterans affairs system consisted almost entirely of men , and we found sex differences in the association between diabetes and aaa , whereas the international registry represents a highly selective population with established vascular disease or at high risk for vascular disease . although aaa is considered an atherosclerotic disease process ( similar to pad and cas ) , an experimental mouse model demonstrated elevated glucose concentrations to be associated with increased plasminogen activator inhibitor-1 concentrations ( concomitant with increased aneurysmal aortic pai-1 gene expression ) , reduced plasmin generation , and reduced abdominal aortic diameter , suggesting a role for the fibrinolytic pathway in aaa pathophysiology and a possible mechanism of inhibition of aaa disease in the presence of diabetes ( 28 ) . a prospective cohort study of predominately men demonstrated the development of thicker aortic walls in diabetes , a reduction in the secretion of matrix metalloproteinase , and , thereby , reduced aortic wall stress and subsequent development of aaa ( 29 ) . this is consistent with a recent report of 360 patients ( 96% men ) with small aaa ; the study demonstrated an inverse correlation between diabetes and aneurysm growth ( 30 ) . it is interesting that we note a neutral association between diabetes and aaa in women . the current study is one of the few with a large number of women ( > 2 million ) . one report of 9,342 women did demonstrate a six - times lower prevalence of aaa in women than men but did not comment on the prevalence of aaa by diabetes status ( 31 ) . in addition , one meta - analysis of more than 15,000 patients from 18 studies , the majority of which consisted of more than 80% men , demonstrated that female sex was not associated with aneurysm growth ; however , it did not comment on the association between female sex and the prevalence of aaa ( 32 ) . the underlying mechanism for the sex disparities noted in the prevalence of aaa , and particularly in subjects with diabetes , needs further elucidation . there are several limitations to this study , including those inherent to a retrospective observational study design . first , participants were provided access to the lls measures for a fee ; therefore the cohort may underrepresent people from lower socioeconomic backgrounds . second , the diagnosis of diabetes was based on participant self - report and was not verified by medical records . nonetheless , we found that the prevalence of diabetes was 10.8% , which is similar to the prevalence of diabetes noted in more representative populations ( e.g. , nhanes [ 9.7% ] and 2013 american heart association heart disease and stroke statistics [ 11.8% ] ) . third , while abi was measured during pad screening in this survey , the toe brachial pressure index could be used to establish the diagnosis of pad in people in whom pad is clinically suspected , but abi tests are unreliable because of noncompressible vessels , such as in people with long - standing diabetes or advanced age . however , this population comprises only 1.2% of the total cohort with available abi data and is therefore unlikely to have a significant effect on the differences detected . in addition , in this study , abi was measured using the posterior tibial artery ; the dorsalis pedis artery was used only when the posterior tibial artery was inaudible . the highest of the dorsalis pedis or posterior tibial artery pressures at the ankle traditionally is used to form the calculation ( 17 ) . however , a recent report compared this traditional method with an alternative method using the lower of the two ankle artery pressures , and both methods had similar diagnostic and predictive accuracy for all - cause and cardiovascular mortality , suggesting the utility of the alternate method in identifying a clinically meaningful population with pad ( 33 ) . fourth , severe pad is defined in this study as abi 0.6 , whereas some definitions use abi 0.4 to define severe pad . we did evaluate the population with abi < 0.4 and demonstrated similar results ( adjusted or 2.23 [ 95% ci 2.072.41 ] ; p < 0.001 ) . finally , while a small percentage ( 7.5% ) of individuals did not have abi calculated , did not receive carotid ultrasound screenings , or both , subjects excluded because of incomplete data were similar to the subjects included in the final analyses . despite these limitations , this is the largest evaluation of the relationship between vascular disease in the peripheral arteries and diabetes , as well as the only study to examine all three major vascular subtypes in one cohort . in a large contemporary survey , after adjustment of demographics and traditional risk factors , the presence of diabetes is associated with higher odds of pad and cas but lower odds of aaa . future studies exploring this difference in vascular subtype and assessing the risks versus benefits of screening for pad and cas in asymptomatic people with diabetes are needed . there are several limitations to this study , including those inherent to a retrospective observational study design . first , participants were provided access to the lls measures for a fee ; therefore the cohort may underrepresent people from lower socioeconomic backgrounds . second , the diagnosis of diabetes was based on participant self - report and was not verified by medical records . nonetheless , we found that the prevalence of diabetes was 10.8% , which is similar to the prevalence of diabetes noted in more representative populations ( e.g. , nhanes [ 9.7% ] and 2013 american heart association heart disease and stroke statistics [ 11.8% ] ) . third , while abi was measured during pad screening in this survey , the toe brachial pressure index could be used to establish the diagnosis of pad in people in whom pad is clinically suspected , but abi tests are unreliable because of noncompressible vessels , such as in people with long - standing diabetes or advanced age . however , this population comprises only 1.2% of the total cohort with available abi data and is therefore unlikely to have a significant effect on the differences detected . in addition , in this study , abi was measured using the posterior tibial artery ; the dorsalis pedis artery was used only when the posterior tibial artery was inaudible . the highest of the dorsalis pedis or posterior tibial artery pressures at the ankle traditionally is used to form the calculation ( 17 ) . however , a recent report compared this traditional method with an alternative method using the lower of the two ankle artery pressures , and both methods had similar diagnostic and predictive accuracy for all - cause and cardiovascular mortality , suggesting the utility of the alternate method in identifying a clinically meaningful population with pad ( 33 ) . fourth , severe pad is defined in this study as abi 0.6 , whereas some definitions use abi 0.4 to define severe pad . we did evaluate the population with abi < 0.4 and demonstrated similar results ( adjusted or 2.23 [ 95% ci 2.072.41 ] ; p < 0.001 ) . finally , while a small percentage ( 7.5% ) of individuals did not have abi calculated , did not receive carotid ultrasound screenings , or both , subjects excluded because of incomplete data were similar to the subjects included in the final analyses . despite these limitations , this is the largest evaluation of the relationship between vascular disease in the peripheral arteries and diabetes , as well as the only study to examine all three major vascular subtypes in one cohort . in a large contemporary survey , after adjustment of demographics and traditional risk factors , the presence of diabetes is associated with higher odds of pad and cas but lower odds of aaa . future studies exploring this difference in vascular subtype and assessing the risks versus benefits of screening for pad and cas in asymptomatic people with diabetes are needed .

objectivethe aim of this study was to investigate the relationship between diabetes and different phenotypes of peripheral vascular disease ( lower extremity peripheral artery disease [ pad ] , carotid artery stenosis [ cas ] , and abdominal aortic aneurysm [ aaa]).research design and methodsprevalence of vascular disease was evaluated in 3,696,778 participants of the life line screening survey between 2003 and 2008 . pad was defined as ankle - brachial pressure index < 0.90 or prior revascularization , cas as 50% stenosis or prior revascularization , and aaa as infrarenal aortic diameter 3 cm or prior repair . odds ratios ( ors ) and 95% cis were assessed using logistic regression modeling.resultsdiabetes mellitus was present in 10.8% of participants ( n = 399,884 ) . prevalence of pad , cas , and aaa was significantly higher ( p < 0.0001 ) in participants with compared with those without diabetes . after multivariate adjustment for baseline demographics and clinical risk factors , a significant interaction existed between diabetes and vascular disease phenotype ( p < 0.0001 ) . diabetes was associated with increased odds of pad ( or 1.42 [ 95% ci 1.411.4 ] ; p < 0.0001 ) and cas ( 1.45 [ 1.431.47 ] ; p < 0.0001 ) but decreased odds of aaa ( 0.86 [ 0.840.88 ] ; p < 0.0001 ) . the strength of association increased with increasing severity of disease in each vascular phenotype , and this association persisted in the population with asymptomatic vascular disease.conclusionsin a large population - based study , the association between diabetes and vascular disease differed according to vascular phenotype . future studies exploring the mechanism for these vascular - specific differences are needed .

Introduction Research Design and Methods Study Population Covariates Vascular Disease Outcomes Statistical Analysis Results Baseline Characteristics Prevalence of Vascular Disease Vascular Disease Severity Sex Differences Conclusions Limitations Conclusion

PMC3948570

secondary surgery of the neck for persistent or recurrent thyroid cancer ( tc ) is still the subject of debate . the advocated extent of the salvage neck dissection ( nd ) has not been established yet . subsequent procedure for the central compartment ( cc ) carries the risk related directly to the anatomy of this region . upper mediastinum ( level vii ) and supraclavicular fossa ( level iv ) remains challenging even in the primary approach . in patients with tc , the balance between the risk of morbidity from secondary nd and the risk of untreated cancer disease still remains an unresolved issue . previous articles have evaluated the feasibility and efficacy of reoperations in thyroid cancer ( in persistent or recurrent disease ) , but authors had focused on well differentiated thyroid cancer ( wdtc ) and surgeries limited to the central compartment [ 35 ] . in most cases , thyroid cancer recurrences in cervical lymph nodes can be visualized with ultrasound examination , what helps to arrange further therapeutic procedures . the aim of the study was ( 1 ) to assess the feasibility of secondary neck dissections ( nd ) in well differentiated thyroid cancer , medullary cancer ( mtc ) and poorly differentiated thyroid cancer ( pdtc ) , ( 2 ) to evaluate the influence of nd extent on morbidity and ( 3 ) to describe the biochemical and clinical outcomes with special regard to lower neck levels ( iv , vi , vii ) . the study group consisted of 51 consecutive patients with persistent / recurrent thyroid cancer ( 32 females and 19 males ) . all patients enrolled to the study had been previously operated for tc in 11 different general surgery units and then followed - up in the department of endocrinology , metabolism and internal medicine of poznan university of medical sciences . treatment failures had been subsequently referred to the department of otorhinolaryngology , head and neck surgery , poznan medical university . the recurrent or persistent thyroid cancer neck disease was defined as : positive imaging on ultrasound , computed tomography or radioiodine whole body scan , increased thyroglobulin ( tg ) or calcitonin levels . the age of the patients at the onset of disease ranged from 16 to 76 years ( mean 48 years ) . 46 patients had previously undergone one surgical procedure for tc and 5 patients two or more . histological appearance of the primary lesion was determined according to the world health organization histological classification and was as follows : 33 cases of wdtc , 15 cases of mtc , 3 cases of pdtc . staging was performed according to the american joint committee on cancer classification ( ajcc ) cancer staging manual revised in 2009 . the advancement of the primary tumor was as follows : t1 in 6 cases , t2 in 16 cases , t3 in 18 cases and t4 in 11 cases . nodal disease at the first presentation was n0 in 36 ( 60.8 % ) , and n+ in 15 ( 39.2 % ) patients respectively . the time between the primary surgery and reoperation ranged from 7 months to 16 years ( median 2 years ) . thyrotropin ( tsh ) stimulation had shown increased tg level ( > 2 ng / ml ) in 31/33 patients with wdtc . the values ranged from 2.32 to > 900 ng / ml . the supraphysiological thyroid hormone replacement was introduced in this group to achieve tsh suppression ( < 0.1 u / ml ) . in all patients with medullary cancer calcitonin level was increased ( > 10 pg / ml ) and ranged from 13 pg / ml to over scale measurements > 480 pg / ml . the indirect laryngoscopy and stroboscopy were performed before and after the reoperation ( on the 1st , 7th day and 6 months later if needed ) . the mean follow - up time after the neck reoperation was 3.4 2.3 years . vii neck levels . the choice of the procedure depended on rn advancement , macrometastases location and tumor histology . the radical neck dissection ( rnd ) included the lateral node compartment : jugular chain nodes ( levels i , ii , iii , iv ) , posterior neck ( level v ) , from the skull base to the subclavian vein and laterally to the trapezius muscle . in all cases the sternocleidomastoid muscle and jugular vein were dissected in strictly chosen cases ( in patients with lymph nodes metastases > 3 cm adjacent to the vessel wall ) . selective neck dissection ( snd ) most often cleared levels iia , iii and iv . dissection of the central compartment ( cc ) included level vi nodes extended vertically from the hyoid bone to the thoracic inlet and horizontally between the carotid sheaths ( prelaryngeal , pretracheal and paratracheal nodes ) . the extension of the secondary nd and patients data concerning neck relapse are presented in table 1.table 1the types of thyroid cancer , localization of the excised nodes and the complications of secondary neck dissectionno.follow up time ( years)no . of complicationsrlnhpotherwdtc332.68126medullary cancer151.3383poorly differentiated thyroid cancer31.3222neck levels i iii only62.7220neck level vi , vii only21111neck level iv only15000neck level iv , vi , vii+ other141.1685 total 51 1.8 13 22 11 rln recurrent laryngeal nerve injury ( both persistent and transient ) , hp persistent hypoparathyroidism the types of thyroid cancer , localization of the excised nodes and the complications of secondary neck dissection rln recurrent laryngeal nerve injury ( both persistent and transient ) , hp persistent hypoparathyroidism rnd was performed in 22 patients ( 43.1 % ; 15-wdtc , 6-mtc , 1-pdtc ) and snd in 29 patients ( 56.9 % ; 18-wdtc , 9-mtc , 2-pdtc ) . cc ( level vi ) was cleaned in 14 patients ( 27.5 % ; 9-wdtc , 4-mtc , 1-pdtc)in two as the main procedure and in 12 as complementary to lateral node compartment dissection . level vii ( upper mediastinal nodes ) were dissected in 10 patients ( 19.6 % ; 7-wdtc , 1-mtc , 2-pdtc ) , levels i , ii , iii , iv and v in 3 , 36 , 40 , 41 and 21 patients respectively . all together lower neck reoperation ( iv , vi , vii ) was performed in 46 cases ( 90.2 % ) . 11 patients were operated more than once ( 4 patients with wdtc , 6-mtc , 1-pdtc ) . total amount of neck dissections was 66 . in our study group the localization of recurrence ( level i iii , iv , v , vi , vii ) and the time between the primary surgery and the recurrence did not depend on patients gender , age or the type of thyroid cancer . in three patients very aggressive course of the recurrent disease was observed : in two cases gross infiltration of the larynx and esophagus requiring total laryngectomy with partial esophagectomy . the number of removed lymph nodes ranged from 1 to 42 . in the majority of patients data of the neck dissection specimens including number of metastatic nodes end extracapsular spread ( ecs ) was presented in table 2.table 2histopathological results of the neck dissection specimenneck levelno . of nodes removedno . of patientspn+ecsi / ii / iii only34680iv only1111iv , vi , vii + other16714946vi , vii only112101 ecs extra capsular spread histopathological results of the neck dissection specimen ecs extra capsular spread the number of metastatic nodes ranged from 1 to 28 . single metastatic lymph nodes were found in 11 patients ( 21.6 % ) , 2 metastatic lymph nodes in 10 patients ( 19.6 % ) , three in 1 patient ( 2 % ) , 4 in 3 patients ( 5.9 % ) and 5 or more in 25 patients ( 47.1 % ) . metastases were confirmed in all 14 cc and all 10 mediastinal specimens ; thus in levels vi and vii there were no false negative results . out of 59 lateral neck specimens metastases were found in the levels ii and iii in 37 cases ( 72 % ) and in the level iv in 35 cases ( 68 % ) . the occurrence of bilateral metastases did not depend on the type of thyroid cancer , but was often observed in younger patients ( p = 0.07 ) and in patients with primary surgery performed at younger age ( p = 0.049 ) . furthermore , these two groups more frequently required radical neck dissection ( p < 0.01 in both groups ) . in six specimens from the viith level the technical difficulties included disturbed anatomy , scars , infiltration of subclavicular vessels or of the junction between subclavian and internal jugular vein ( 5 patients ) , infiltration of the larynx or esophagus ( 2 patients ) , infiltration of trachea ( 1 patient ) . postoperative vocal cord paresis was observed in 13 ( 25.5 % ) , transient in 5 ( 9.8 % ) , permanent in 8 ( 15.7 % ) patients . in seven patients recurrent laryngeal nerve ( rln ) was injured due to direct tumor invasionin three ( mtc ) nerve was intentionally sectioned and in four ( wdtc ) nerve was detached from the tumor . in six patients rln visualization was impossible due to rigidity of the scar tissue ; paresis occurred postoperatively . six patients suffered from severe dyspnoea : in three laterofixation and in three emergency tracheotomy was indispensable . permanent tracheostomy was maintained in five ( 9.8 % ) patients : in three with vocal cord paresis and in two after laryngectomy . four patients had postoperative chyle leakage : in two cases low output , stopped after conservative treatment after 68 days , in one patient high - output leak was controlled on the 5th day by direct ligation . in the 4th case the patient suffered from chylothorax , required two reoperations , that occurred unsuccessful and the patient expired on the 36th day . massive bleeding occurred intraoperatively in three patients ( in one from common carotid artery , in two from the stump of internal jugular vein ) . one patient died due to massive bleeding from common carotid artery on the 2nd day after surgery . the postoperative pth serum level was within the normal range ( 1565 pg / ml ) in 30 ( 58.8 % ) patients . however , hypocalcaemia requiring vitamin d and/or calcium supplementation appeared in 22 patients ( 43.1 % ) . it is important to note , that 8 out of 14 patients operated on iv , vi , vii levels developed hypoparathyroidism ( 57 % ) . there was no correlation between the rate of complications and patients age , gender , time from primary surgery or cancer histology . furthermore , the number of dissected lymph nodes and the presence of bilateral metastatic changes also did not influence significantly the frequency of complications . secondary nd performed in the left subclavian or in the upper mediastinal area were connected with gross complications : 5/8 cases of rln paresis , 2 chyle leakage and 1 chylothorax and 2 massive bleedings . two of the patients had perioperative complications and expired soon after the procedure ( 2nd and 36th day)both with wdtc . out of the other patients with wdtc : one patient died of disseminated tc 2 years after neck dissection , at age of 74 years ; two patients ( 6 % ) developed new recurrence in the upper mediastinum , not qualifying for the additional nd , 5 patients ( 15.2 % ) developed lung metastases and one bone metastases ( 3 % ) . seven patients ( 21.2 % ) indicated biochemical cure at the first control examination after the reoperative surgery ( tg level < 2 ng / ml evaluated on thyrotropin stimulation ) , in ten patients tg was < 2 ng / ml in the follow - up ( 30.3 % ) , in six patients ( 18.2 % ) tg level decreased after operation . seven patients ( 21 % ) still presented with notably elevated tg levels > 30 ng / ml . none of the patients with mtc achieved calcitonin level lower than 10 pg / ml . nine patients developed distant metastases in 1016 months time ( lung metastases in 7 patients , bone metastases in 3 patients ) . none of the patients with poorly differentiated thyroid cancer achieved tg level < 2 ng / ml ) . one patient developed disseminated lung and bone metastases and is still under control of our clinic ( 4 years after neck dissection ) . distant metastases were observed more frequently in patients with mtc and pdtc ( p = 0.007 ) . the number of removed lymph nodes ranged from 1 to 42 . in the majority of patients metastatic tc was present in multiple nodes . data of the neck dissection specimens including number of metastatic nodes end extracapsular spread ( ecs ) was presented in table 2.table 2histopathological results of the neck dissection specimenneck levelno . of nodes removedno . of patientspn+ecsi / ii / iii only34680iv only1111iv , vi , vii + other16714946vi , vii only112101 ecs extra capsular spread histopathological results of the neck dissection specimen ecs extra capsular spread the number of metastatic nodes ranged from 1 to 28 . single metastatic lymph nodes were found in 11 patients ( 21.6 % ) , 2 metastatic lymph nodes in 10 patients ( 19.6 % ) , three in 1 patient ( 2 % ) , 4 in 3 patients ( 5.9 % ) and 5 or more in 25 patients ( 47.1 % ) . metastases were confirmed in all 14 cc and all 10 mediastinal specimens ; thus in levels vi and vii there were no false negative results . out of 59 lateral neck specimens metastases were found in the levels ii and iii in 37 cases ( 72 % ) and in the level iv in 35 cases ( 68 % ) . the occurrence of bilateral metastases did not depend on the type of thyroid cancer , but was often observed in younger patients ( p = 0.07 ) and in patients with primary surgery performed at younger age ( p = 0.049 ) . furthermore , these two groups more frequently required radical neck dissection ( p < 0.01 in both groups ) . in six specimens from the viith level the technical difficulties included disturbed anatomy , scars , infiltration of subclavicular vessels or of the junction between subclavian and internal jugular vein ( 5 patients ) , infiltration of the larynx or esophagus ( 2 patients ) , infiltration of trachea ( 1 patient ) . postoperative vocal cord paresis was observed in 13 ( 25.5 % ) , transient in 5 ( 9.8 % ) , permanent in 8 ( 15.7 % ) patients . in seven patients recurrent laryngeal nerve ( rln ) was injured due to direct tumor invasionin three ( mtc ) nerve was intentionally sectioned and in four ( wdtc ) nerve was detached from the tumor . in six patients rln visualization was impossible due to rigidity of the scar tissue ; paresis occurred postoperatively . six patients suffered from severe dyspnoea : in three laterofixation and in three emergency tracheotomy was indispensable . permanent tracheostomy was maintained in five ( 9.8 % ) patients : in three with vocal cord paresis and in two after laryngectomy . four patients had postoperative chyle leakage : in two cases low output , stopped after conservative treatment after 68 days , in one patient high - output leak was controlled on the 5th day by direct ligation . in the 4th case the patient suffered from chylothorax , required two reoperations , that occurred unsuccessful and the patient expired on the 36th day . massive bleeding occurred intraoperatively in three patients ( in one from common carotid artery , in two from the stump of internal jugular vein ) . one patient died due to massive bleeding from common carotid artery on the 2nd day after surgery . the postoperative pth serum level was within the normal range ( 1565 pg / ml ) in 30 ( 58.8 % ) patients . however , hypocalcaemia requiring vitamin d and/or calcium supplementation appeared in 22 patients ( 43.1 % ) . it is important to note , that 8 out of 14 patients operated on iv , vi , vii levels developed hypoparathyroidism ( 57 % ) . there was no correlation between the rate of complications and patients age , gender , time from primary surgery or cancer histology . furthermore , the number of dissected lymph nodes and the presence of bilateral metastatic changes also did not influence significantly the frequency of complications . secondary nd performed in the left subclavian or in the upper mediastinal area were connected with gross complications : 5/8 cases of rln paresis , 2 chyle leakage and 1 chylothorax and 2 massive bleedings . two of the patients had perioperative complications and expired soon after the procedure ( 2nd and 36th day)both with wdtc . out of the other patients with wdtc : one patient died of disseminated tc 2 years after neck dissection , at age of 74 years ; two patients ( 6 % ) developed new recurrence in the upper mediastinum , not qualifying for the additional nd , 5 patients ( 15.2 % ) developed lung metastases and one bone metastases ( 3 % ) . seven patients ( 21.2 % ) indicated biochemical cure at the first control examination after the reoperative surgery ( tg level < 2 ng / ml evaluated on thyrotropin stimulation ) , in ten patients tg was < 2 ng / ml in the follow - up ( 30.3 % ) , in six patients ( 18.2 % ) tg level decreased after operation . seven patients ( 21 % ) still presented with notably elevated tg levels > 30 ng / ml . none of the patients with mtc achieved calcitonin level lower than 10 pg / ml . nine patients developed distant metastases in 1016 months time ( lung metastases in 7 patients , bone metastases in 3 patients ) . none of the patients with poorly differentiated thyroid cancer achieved tg level < 2 ng / ml ) . one patient developed disseminated lung and bone metastases and is still under control of our clinic ( 4 years after neck dissection ) . distant metastases were observed more frequently in patients with mtc and pdtc ( p = 0.007 ) . the american thyroid association ( ata ) and national comprehensive cancer network ( nccn ) recommends treatment options for recurrent or persistent wdtc : radioiodine therapy for radioiodine sensitive disease and surgery for clinically significant disease . secondary surgery in cc has been approved in the literature [ 4 , 9 , 10 ] , although is associated with high risk of complications and moderate possibilities of tumor eradication . therefore , the question is , how to obtain the balance between the cure and complication rates and whether the patient will benefit from the surgery . in patients with medullary cancer the sites frequently involved in recurrent or persistent disease include retrocarotid nodes , nodes deep to laryngeal recurrent nerve , retroclavicular nodes , retrosternal nodes and nodes running along the superior thyroid vascular pedicle medial to the carotid bifurcation . leaving positive nodes in tracheoesophageal groove may expose the patient to risk of tracheal , esophageal or even laryngeal invasion . . found nodal metastases in central and bilateral central compartment in 86.7 and 42.2 % cases respectively but little attention was devoted to the neighboring iv level . metastasizing pattern in our group revealed pn+ in level iv , vi and vii in 41 , 16 and 10 patients respectively ; in the ivth level over 70 % , in cc 100 % of taken nodes were pn+ ; 17 % of patients had metastases bilaterally . comparing primary and recurrent disease distribution stated , that there was no difference in frequency in levels i , ii , iii and v but level iv was more common in the recurrent cases . salvage neck surgery in our group confirmed the necessity of clearing all lower neck basins . in our group it is important to confront the surgical feasibility and morbidity in patients with recurrences in the lower neck anatomical region . the previous studies demonstrated that there is no additional morbidity in secondary procedures and complication rate is similar to initial surgical procedure , if performed by experienced surgeon [ 2 , 14 ] . numerous authors described the cc salvage surgery as safe and efficient oncological procedure exhibiting minimal morbidity [ 1 , 4 , 5 , 10 , 15 ] . on the other hand , hartl and travagli underlined additional morbidity when central nd is performed as secondary procedure . although the boundaries and the anatomical structures that require dissection are the same , the scar tissue and neovascularization complicates the access to this region ; the sternohyoid and sternothyroid muscles are adherent to the trachea in dense scar tissue . thus , reoperative surgery of the cc is associated with higher risk of vocal folds paresis . according to mirghani cc nd can be performed with minimal morbidity , when recurrent laryngeal nerve is not invaded . in our group in 3/15 medullary cancers the nerve was intentionally sectioned , in 4/33 wdtc detached from the tumor . in eight cases ( 15.7 % ) permanent vocal cord paresis was observed ; in three ( 5.9 % ) tracheotomy had to be maintained . it has been stated , that parathyroid glands are at increased risk for injury and permanent hypoparathyroidism is the most common adverse event in cc secondary surgery . permanent hypoparathyroidism has been reported to range between 2.7 and 9 % . in our study long - term follow - up revealed permanent hypoparathyroidism in 22 patients ( 43.1 % ) . the outcomes of reoperative surgery for wdtc can be categorized as the absence of biochemical disease or clinically detectable disease . in our center the required tg level assessed on tsh stimulation was determined to be lower than 2 ng / ml . according to hughes et al . biochemical complete response obtained in 26 % of patients may be concentrated on locoregional control of disease to prevent complications due to local invasion rather than elimination of biochemically active cancer . in our study stimulated tg < 2 mg / ml was observed in seven patients with wdtc directly after reoperative procedure ( 21.2 % ) and in ten patients in the follow - up ( 30.3 % ) . six patients with wdtc developed distant metastases . in mtc or pdtc the final results of secondary surgery were poor . none of the patients with mtc achieved calcitonin level lower than 10 pg / ml and none of the patients with pdtc achieved biochemical remission . it should be therefore concluded , that mtc and pdtc need more rigorous preoperative work up to ensure that distant metastases are excluded , otherwise the prognosis will be unfavorable despite successful neck dissection . our study reports the large cohort of patients undergoing secondary nd due to thyroid cancer . surgical approach and possibility of complications in this group present a challenge . in mtc and pdtc the long - term results were unsatisfactory ( calcitonin / tg still elevated , frequent distant metastases ) . in wdtc the secondary neck dissection should be performed in cases in which there are strong indications for surgical procedure . metastases localization in levels vi , vii , iv was connected with high complication rate . on the other hand total clearance of these basins proved to be crucial for the satisfactory oncological outcomes .

the purpose of the study was to assess the feasibility of secondary neck dissections ( nd ) in different types of thyroid cancer ( tc ) , to evaluate the influence of nd extent on morbidity and to describe biochemical and clinical outcomes . 51 patients previously operated for tc ( 33-well differentiated tc - wdtc , 15 medullary tc - mtc , 3 poorly differentiated tc - pdtc ) presenting detectable nodal disease . reoperations covered i vii neck levels . radical neck dissection was performed in 22 patients , selective neck dissection in 29 patients . 14 central compartment ( cc ) , 10 mediastinal and 41 level iv excisions were performed . postoperative complications occurred in 13 patients : 4 chyle leaks , 3 massive bleedings , 8 permanent vocal cord pareses , hypoparathyroidism in 22 patients ( 43.1 % ) , 2 patients expired in perioperative period . in wdtc : in seven patients thyroglobulin level normalized directly after nd , in ten patients in the follow - up ; six patients developed distant metastases . none of the patients with mtc achieved calcitonin level < 10 pg / ml ; nine patients developed distant metastases . none of the patients with pdtc achieved tg < 2 mg / ml ; two patients died , the third developed distant metastases . secondary nd in tc present a challenge by means of surgical approach and possibility of complications . in mtc and pdtc the long - term results were unsatisfactory . in wdtc , the secondary nd should be performed due to strong indications . metastases localization in levels iv , vi , vii were connected with high complication rate , but these surgeries were crucial for satisfactory oncological outcomes .

Introduction Materials and methods Results Histopathological findings Complications and morbidity Outcomes of secondary nodal surgery Discussion

PMC5405111

replication stress is a well - characterized tumor characteristic and has recently been considered as a potential new hallmark of cancer ( macheret and halazonetis , 2015 ) . replication stress leads to the activation of the dna damage response ( ddr ) , which is associated with the early stages of cancer development ( bartkova et al . , 2005 , gorgoulis et al . , hypoxia ( low oxygen levels ) is one of the most physiologically relevant driving forces of replication stress and is characterized by an increased number of stalled replication forks and significantly reduced replication rates . importantly , hypoxia - induced replication stress occurs in the absence of dna damage ( olcina et al . , hypoxia - induced replication stress is one of the factors proposed to contribute to the selection pressure to lose key components of the ddr , including p53 ( gorgoulis et al . , 2005 , hammond et al . , 2007 , graeber et al . , 1996 ) the degree of tumor hypoxia is an indicator of poor patient prognosis due to resistance to most current therapies and increased metastatic spread ( begg et al . , 2011 , the importance of ribonucleotide reductase ( rnr ) lies in its unique ability to catalyze the rate - limiting step of the reduction of ribonucleotides ( ndps ) to the corresponding deoxyribonucleotides ( dndps ) , the precursors of dna ( kolberg et al . the large rrm1 dimer contains the catalytic site , whereas the smaller dimer ( rrm2 or rrm2b ) contains an oxygen - requiring di - iron tyrosyl - radical site , which is essential for catalysis ( kolberg et al . , 2004 , rnr is highly regulated , as nucleotide imbalances can be detrimental to cell fate ( aye et al . , 2015 ) . elevated deoxyribonucleotide triphosphate ( dntp ) pools have been correlated with increased mutagenesis , while insufficient dntps cause replication stress and promote genomic instability ( bester et al . oxygen is essential for mammalian rnrs to oxidize the di - iron center found in the smaller subunit ( rrm2 or rrm2b ) ( huang et al . , 2014 ) . this generates the tyrosyl radical , which via a long - range pathway , transfers an electron to the catalytic site of rrm1 , enabling the reduction of ndps to dndps ( lundin et al . , 2015 , kolberg et al . the mechanism by which iron is incorporated into the small subunit , and how oxygen activates the di - iron center for radical initiation , is still poorly understood and under intense investigation ( huang et al . the oxygen dependency of the small rnr subunit has led to the logical assumption that rnr is inactive in severely hypoxic conditions , something that has been verified for the rrm1/rrm2 ( r1/r2 ) version of the rnr enzyme ( brischwein et al . , 1997 , chimploy et al . , 2000 , thelander et al . , 1983 , reichard , 1993 , probst et al . , 1989 , nordlund and reichard , 2006 ) . in contrast , we demonstrate that rnr is able to maintain dndp formation by switching subunits in hypoxic conditions ( < 0.1% o2 ) . this switch is critical to preserve sufficient nucleotide levels for ongoing replication and avoid dna damage accumulation and apoptosis in hypoxia . interestingly , we found that the rrm1/rrm2b ( r1/r2b ) version of the rnr enzyme retains activity in hypoxia due to adaptation of rrm2b to these conditions through two distinct mechanisms . specifically , we determined that most of the tyrosyl radical in rrm2b remained stable when exposed to < 0.1% o2 , a property that was diminished after disruption of the helix b open conformation ( k37/k151 ) and the subsequent loss of the crosstalk between the two monomers . furthermore , computational simulations suggest that y164 plays a role in the fine - tuning of the oxygen - entering frequency of rrm2b , meaning it is likely to be a better oxygen - sequestering agent than rrm2 . collectively , our results reveal a critical role for the rnr ( r1/r2b ) enzyme in hypoxic conditions in maintaining sufficient dntps for ongoing replication and therefore preventing dna damage , genomic instability , and subsequent loss of viability . cells in hypoxic conditions ( < 0.1% o2 ) do not accumulate dna damage , although they are characterized by the presence of replication stress , which leads to the activation of the ddr ( figures s1a and s1b ) ( olcina et al . , 2013 ) . as predicted , hypoxia - induced replication stress leads to the accumulation of single - stranded dna ( ssdna ) , which can be detected through the visualization of replication protein a ( rpa ) foci in s - phase cells ( pires et al . , 2010 ) . however , the percentage of cells in hypoxia with rpa foci decreases after prolonged exposure to these conditions ( > 6 hr ) ( figure 1a ) . therefore , our hypothesis is that although uncoupling of the helicases and polymerases occurs in hypoxia , replication can proceed . using dna fiber analysis , we confirmed that dna replication is significantly slower in hypoxia ( 25% of normal rate ) but is not completely abrogated ( figure 1b ) . dntp incorporation assays were carried out in rko cells exposed to hypoxia and demonstrated that , although the dntp pools were disrupted ( with the purines [ dgtp , datp ] being more affected than the pyrimidines [ dctp , dttp ] ) , there was nucleotide availability in hypoxic conditions ( figures 1c and 1d ) . it has been suggested that pyrimidine levels persist due to compensatory activities of the pyrimidine salvage pathways or as a result of the deoxyuridine present in culture medium , which cells could uptake and phosphorylate , therefore contributing to pyrimidine levels , particularly dttp ( eriksson et al . interestingly , the pyrimidine salvage pathway has been reported to be more efficient than the purine pathway , suggesting an explanation for why purine levels are more sensitive to hydroxyurea ( hu ) and potentially hypoxia ( reichard , 1988 ) . our data demonstrate sufficient nucleotide availability in hypoxia to allow replication to proceed , albeit at a compromised rate ; thus raising the possibility that rnr is able to retain some activity in hypoxia . expression analysis of the rnr complex showed that the rrm2b protein is significantly induced ( 2- to 3.5-fold , depending on cell line ) in response to hypoxia in cell lines , including colorectal , osteosarcoma , glioblastoma , and esophageal cancer cells , whereas the rrm2 small subunit decreased over time and the rrm1 remained unchanged or showed a slight decrease ( figures 1e , 1f , and s1c s1f ) . as rko cells showed the highest level of rrm2b expression , we chose it as a model line for the majority of our experiments ( figure s1 g ) . rrm2b increased 4.6-fold after 24 hr in hypoxic conditions , whereas rrm2 and rrm1 mrna levels decreased 12.3- and 2.5-fold , respectively ( figures 1 g and s1h ) . importantly , in silico tgca ( the cancer genome atlas ) analysis of colorectal adenocarcinoma patient cohorts demonstrated that rrm2b expression correlates significantly with the expression of a verified hypoxia signature ( figure 1h ) , suggesting that the oxygen - dependent overexpression of rrm2b also occurs in vivo ( li et al . , 2014 ) . in contrast , rrm1 and rrm2 expression did not correlate with the same hypoxic signature ( figures s1i and s1j ) . interestingly , overexpression and genetic alterations in rrm2b correlated with worse overall and disease - free survival in colorectal cancer patients ( figures s1k s1n ) . the transcription factor hif-1 ( hypoxia - inducible factor 1 ) is known to mediate significant gene expression changes in response to hypoxia and has roles in dna replication , dna repair , and respiration ( hubbi et al . , 2013 , fukuda et al . , 2007 , crosby et al . , 2009 ) . therefore , we investigated if the induction of rrm2b in hypoxia was dependent on hif-1 by utilizing rko and rko cells exposed to hypoxia ( figures 2a , s2a , and s2b ) . interestingly , both the mrna and the protein levels of rrm2b were induced in hypoxia irrespective of hif-1 status , in contrast to the well - documented hif-1 target glut1 . next , using rko cells exposed to either 2% or < 0.1% o2 , we investigated the oxygen dependency of the induction of rrm2b protein . rrm2b was induced in response to the lower level of hypoxia ( < 0.1% o2 ) , where a robust p53 induction was also observed but did not increase in response to 2% o2 despite hif-1 stabilization ( figure 2b ) . this finding is in agreement with our previous studies demonstrating that the lower level of hypoxia ( < 0.1% o2 ) induces replication stress and that this is the signal that initiates the ddr ( including p53 stabilization ) ( hammond et al . , 2002 , olcina et al . , 2013 ) . rrm2b was first characterized as a p53-regulated rnr subunit ( p53r2 ) ( tanaka et al . , 2000 ) . here , further analysis of the molecular pathways mediating rrm2b induction in hypoxia demonstrated that the hypoxic overexpression of rrm2b occurs in a p53-dependent manner ( figures 2c , 2d , and s2c s2h ) . to rule out the possibility of an indirect mechanism of induction of rrm2b by p53 , chromatin immunoprecipitation ( chip ) assays were carried out and demonstrated that p53 binds directly to the p53-response element at the rrm2b locus leading to transcriptional overexpression ( figures 2e , 2f , and s2i s2k ) . interestingly , although p53 expression was increased in response to the dna damaging agent adriamycin , this did not correlate with increased p53 binding to the p53-response element in rrm2b ( figures 2e and 2f ) . most importantly , analysis of the tcga colorectal adenocarcinoma patient cohorts showed that rrm2b expression significantly correlated with a recently identified group of hypoxia - inducible p53-dependent genes ( leszczynska et al . , 2015 ) , suggesting that hypoxia- and p53-dependent expression of rrm2b occurs in human cancers ( figure 2 g ) . interestingly , in p53 null cell lines ( h1299 , hct116 ) a mild ( 1.3- to 1.7-fold ) increase in rrm2b protein levels was also observed in hypoxia ( figures 2c and 2h ) . these findings suggest that additional post - translational p53-independent mechanisms exist for rrm2b stabilization and therefore the importance of rrm2b in hypoxic conditions . in order to investigate the biological significance of hypoxia - induced rrm2b , we first verified that it forms a complex with the rrm1 subunit to reconstitute the r1/r2b holoenzyme in < 0.1% o2 . immunoprecipitation assays demonstrated that increased levels ( 5.3-fold ) of rrm2b protein were bound to the rrm1 subunit in hypoxia whereas the levels of rrm2 bound to rrm1 decreased by 1.8-fold ( figures 3a , s3a , and s3b ) . next , we asked if the hypoxia - formed r1/r2b enzyme was functional . small interfering rna ( sirna)-mediated loss of rrm2b led to significantly lower intracellular dntp levels in hypoxia ( 50%55% less pyrimidines and 25%30% less purines compared to the control [ sictl ] ) ( figures 3b and s3c ) . in contrast , the loss of rrm2b did not significantly affect the dntp pools in normoxic conditions ( figure s3d ) . in addition , fluorescence - activated cell sorting ( facs ) analysis demonstrated that s - phase u2os cells lacking rrm2b incorporate 37.5% less brdu than the control - treated cells in hypoxia ( figures 3c and s3e ) . these findings demonstrate that depletion of rrm2b in hypoxia leads to further disruption of the dntp pools and indicate that ongoing replication is disrupted . to further investigate the hypoxic role of rrm2b , we used crispr / cas9 technology to construct a rrm2b knockout cell line ( rko ) ( figures s3f s3i ) . rrm2b - depleted rko cells ( both knockout and sirna treated ) showed a persistent formation of rpa foci during long ( 24 hr ) exposures to hypoxia , suggesting an accumulation of ssdna and failure to complete dna replication ( figures 3d and s4a s4c ) . furthermore , cells with reduced levels of rrm2b formed 53bp1 foci indicating the presence of double - strand breaks ( dsbs ) specifically in hypoxia ( figures 3e , 3f , and s4d s4f ) . no evidence of elevated dna damage was observed in control cells or in cells lacking rrm2b in normoxia ; this was attributed to the rrm2-dependent supply of dntps when oxygen is abundant . the increased dsbs observed in hypoxic cells with depleted rrm2b correlated with ( 1 ) a significant loss of viability measured by colony survival assay ( 77% fewer cells survived after 24 hr of hypoxia when rrm2b was silenced in comparison to sictl ) ( figure 3 g ) and ( 2 ) the induction of apoptosis as determined both morphologically and by induction of parp cleavage ( figures 3h and s4 g ) . notably , xenograft tumors of rko and rko cells showed delayed tumor growth and increased radiosensitivity when rrm2b was lacking ( figures 3i and s4h ) . additionally , a significant increase in apoptosis specifically in the hypoxic regions of the rrm2b tumors was observed ( figures 3j3l ) as determined by the presence of cleaved caspase-3 ( apoptosis ) in the pimonidazole ( pimo)-positive ( hypoxic ) regions . increased apoptosis in the hypoxic fraction of the tumors offers a likely explanation for the increased radiosensitivity observed ( figure 3i ) . the data presented so far suggest that the basal levels of dntps provided by r1/r2b ( as opposed to r1/r2 ) are sufficient for ongoing replication in hypoxic cells , preventing the collapse of replication forks that would ultimately lead to dsbs and loss of viability . these data indicate that rrm2b contributes to the increased resistance of the most aggressive fraction of tumors ; therefore , we investigated the hypoxic adaptation of rrm2b by determining the enzymatic properties of both forms of rnr enzyme ( r1/r2 and r1/r2b ) in normoxia and hypoxia . rrm1 , rrm2 , and rrm2b recombinant proteins were overexpressed and purified from e. coli ( figures s5a and s5b ) , without additional iron or reductants , to prevent the variability associated with in vitro assembly of the active cluster of the tyrosyl radical ( cotruvo and stubbe , 2011 ) . we quantified the iron bound to rrm2 and rrm2b using inductively coupled plasma ( icp ) mass spectrometry and found comparable iron incorporation between the two recombinant proteins ( figure s5c ) . the level of iron incorporation was 10% of the level that has been reported in a fully loaded rrm2 , which would in turn predict lower enzymatic activity ( aye et al . the abilities of the two forms of the rnr enzyme ( r1/r2b and r1/r2 ) to reduce cytidine diphosphate ( cdp ) ( substrate ) to deoxycytidine diphosphate ( dcdp ) ( product ) were assayed in normoxic and hypoxic conditions ( < 0.1% o2 ) . 0.1% o2 and normoxia with no statistically significant difference in the total amount of dcdp formed under either condition after 30 min ( figure 4a ) . in fact , the r1/r2b enzyme continued converting cdp to dcdp for over 2 hr in hypoxia ( figures 4b and s5d ) . in contrast , the r1/r2 enzyme did not sustain activity in hypoxia ( figure 4c ) and stopped producing dcdps after 15 min in < 0.1% o2 ( figures 4d and s5d ) . importantly , we verified that the r1/r2 enzyme did not stop dcdp formation in hypoxia due to lack of available cdp ( figure s5e ) . collectively , these data suggest that the compromised r1/r2 activity observed at < 0.1% o2 was a result of limited oxygen availability . it is important here to state that the oxygen does not affect the stability of the tyrosyl radical , and it is only required for tyrosyl radical formation ( stubbe , 2003 ) once assembled , the tyrosyl radical can catalyze multiple turnovers until regeneration is necessary , with mammalian r1/r2 being stable for 1525 min in vitro ( cotruvo and stubbe , 2011 ) . the key difference observed between r1/r2b and r1/r2 in hypoxia offers a mechanistic explanation for why the r1/r2b form of rnr is preferred in hypoxic conditions . it should be noted that , as previously described , the overall activity of the rrm2b - containing rnr enzyme is significantly lower than that of the rrm2-containing rnr ( shao et al . , 2004 ) ( figures 4b and 4d ) . therefore , although our data suggest that r1/r2b is the preferred rnr enzyme in hypoxia , the levels of available nucleotides are still reduced compared to normoxia , thus explaining the continued replication stress in these conditions ( figure 1a ) . given the differences in normoxic and hypoxic activity between rrm2b and rrm2 , we compared the potential oxygen accessibility for the proteins using a modeling approach . using the only available published structures of rrm2b ( pdb : 3hf1 ) and rrm2 ( pdb : 3vpn ) ( smith et al . , 2009 ) , we investigated the theoretical differences and/or similarities in the dynamics of oxygen diffusion between the two proteins using classical molecular dynamics ( md ) simulations . the intention of this study was to understand how molecular oxygen accesses the core of the protein and to probe whether any differences could be related to the activities of rrm2/rrm2b in normoxia versus hypoxia . our analysis revealed three principal oxygen cavity tunnels ( t1t3 ) in both proteins , which could be employed as access points ( figures 4e , 4f , and s5f ) . analysis of t1t3 suggested that rrm2b could act as a better oxygen - sequestering agent than rrm2 . specifically , we found that the predicted oxygen - entering frequencies are greater for rrm2b ( 68% at monomer 1 and 22% at monomer 2 ) than for rrm2 ( 14% at monomer 1 and 0% at monomer 2 ) ( table 1 ) , which indicate differential oxygen - turnover susceptibilities between rrm2b and rrm2 . t2 , and t3 ( figure s5 g ) suggest that the origin of the selectivity between rrm2b and rrm2 for oxygen turnover resided primarily in tunnels t2 and t3 , meaning that the energy barriers for oxygen to cross t2 and t3 are higher for rrm2 than for rrm2b . we then performed electron paramagnetic resonance ( epr ) spectroscopy to monitor the stability of the tyrosyl radical of the two proteins in hypoxic conditions ( figures 4 g and 4h ) . strikingly , we observed that 66% 3.35% of the tyrosyl radical in rrm2b remained stable for 1 hr at 37c in < 0.1% o2 , while in the same conditions , only 43% 4% remained stable in rrm2 ( figure 4i ) . collectively , our results provide compelling evidence that rrm2b is able to retain activity in hypoxia while the activity of rrm2 is compromised and therefore highlight a specific role for rrm2b in the hypoxic stress response . despite the fact that rrm2 and rrm2b share 83% sequence homology , there are distinct functional and structural differences ( shao et al . , 2004 , smith et al . , 2009 , zhou et al . , 2010 , xue et al . , 2006 , wang et al . , 2009b ) . the switch of c202 in rrm2 to y164 in rrm2b results in an open phenylalanine channel specifically in rrm2b ( smith et al . , 2009 ) , which is connected with oxygen t3 ( figures s5f and s5h ) . it has been proposed that f183 in rrm2b versus y221 in rrm2 contributes to the differences in the enzymatic activity between the two proteins ( zhou et al . , 2010 ) . in addition , rrm2b has unique antioxidant capabilities , which are partially due to the y241 and y331 residues ( h279 and y369 in rrm2 ) ( xue et al . , 2006 ) . finally , rrm2b through k37 and k151 ( e76 and e190 in rrm2 ) exhibits greater crosstalk between its secondary structures , which stabilizes helix b in an open conformation ( smith et al . , 2009 ) . based on these key differences between rrm2 and rrm2b ( figure s6a ) and in order to determine the regions of rrm2b responsible for hypoxic adaptation , we constructed , overexpressed , and purified seven recombinant rrm2b mutants : y164c , y164f , f183y , k37e / k151e , y241h , y331f , and q127k ( figure s6b ) . the q127k mutant was generated as a negative control , as it diminishes enzymatic activity due to disruption of the stability of the radical cluster ( zhou et al . , 2010 ) . the seven purified mutants showed the same secondary structure , determined by circular dichroism , as the wild - type rrm2b recombinant protein ( figure s6c ) . the levels of iron incorporation were also similar for the mutant with the exception of q127k , as expected ( figure s6d ) . sustained reduction of cdp to dcdp was observed for 2 hr for most mutants , except y164c and k37e / k151e , which both appeared to stop reducing substrate after 30 min in hypoxia , reminiscent of r1/r2 ( figures s6e s6k ) . for these two mutants , we then compared dcdp formation in normoxia and hypoxia and found that the ability to reduce cdp to dcdp in hypoxia was compromised in both the k37e / k151e and y164c mutants ( figures 5a and 5b ) . these results indicate that these specific residues are critical to the role of rrm2b in hypoxia and that without them , rrm2b activity is not sustained , similar to rrm2 . to further probe the role of k37/k151 and y164 , we observed that while most of the rrm2b mutants behaved as the wt - rrm2b , which retains 60%79% of its tyrosyl radical in hypoxia ( figure s7a ) , the k37e / k151e mutant retained only 28% of its tyrosyl radical in these conditions ( figures 5c and 5d ) . since k37/k151 residues affect helix b of rrm2b monomer 2 , the bending angle of the helices was estimated and showed that helix b in rrm2b is in an open conformation ( 2025 bending angle ) , whereas in rrm2 , it is closed ( 810 ) ( figure s7b ) . interestingly , md simulations for the k37e / k151e variant showed a closure of helix b after 300 ns from 20 to 8 bending angle , therefore resembling the closed conformation of rrm2 ( figures s7b and s7c ) . although these are theoretical studies that provide only indications of the protein conformation , the distortion of helix b in monomer 2 of the k37e / k151e mutant could suggest loss of the essential crosstalk between the two monomers , which may explain the loss of tyrosyl radical stability in hypoxia as demonstrated by our epr analysis . additionally , md simulations showed a 4.8-fold lower oxygen entering frequency for the y164c mutant in comparison to wt - rrm2b , while we did not observe any changes in the oxygen entering frequency for the k37e / k151e mutant ( table 2 ) . this could be explained by disruption of the y164-f197 and y164-f198 contacts in monomer 1 of rrm2b , which could cause side - chain rearrangement of f95 in the y164c mutant . this rearrangement may result in f95 moving closer to f197 , leading to closure of the rrm2b specific phenylalanine channel ( figures 5e and s5h ) , therefore explaining the lower oxygen - entering frequency in y164c mutant . finally , in order to demonstrate that our findings are relevant in cells experiencing hypoxia and that the hypoxic adaptation of rrm2b depends on the identified residues ( y164 and k37/k151 ) , we performed rescue experiments by transfecting our rko cells with the following constructs : prrm2b , prrm2b , prrm2b , and pcdna3.1 ( ctl ) . as expected , when wt - rrm2b was reintroduced , a significant increase in the intracellular nucleotide levels was observed in hypoxia , whereas reintroduction of y164c and k37e / k151e showed significantly lower dntp levels compared to wt - rrm2b ( figures 5f , 5 g , s7d , and s7e ) . additionally , by monitoring apoptosis , we observed that wt - rrm2b rescued the null phenotype from apoptosis , whereas loss of the key residues ( y164 and k37/k151 ) abrogated this rescue ( figures 5h and 5i ) , therefore validating our in vitro experiments and theoretical studies . overall , we demonstrate the critical importance of the hypoxic induction of rrm2b to mitigate replication stress and determine the molecular adaptations of rrm2b to hypoxia to support this function ( figure 5j ) . we identified that in response to the physiological stress of hypoxia , rnr responds by isoform switching favoring rrm2b over rrm2 . in hypoxia the activity of rrm2 is severely compromised due to the lack of available oxygen , leading to replication stress . rrm2b is able to retain its activity in hypoxia and is therefore induced to compensate and facilitate ongoing replication . this property of rrm1/rrm2b , although not sufficient to resolve replication stress , does preserve replication fork integrity and prevent the accumulation of dna damage in hypoxia . we verified that depletion of rrm2b results in lower dntp levels in hypoxic cells and that this has detrimental consequences for cell fate ( failure to complete dna replication , dna damage , and loss of viability ) . importantly , loss of rrm2b in a xenograft model showed delayed tumor growth , increased radiosensitivity , and increased apoptosis specifically in hypoxic areas , further highlighting the biological importance of the hypoxic induction of rrm2b . our data suggest that rrm2b is capable of retaining activity in hypoxia through two mechanisms : ( 1 ) increased oxygen - entering frequency and ( 2 ) enhanced stability of the tyrosyl radical . our md analyses suggested that y164 could increase the oxygen - entering frequency through oxygen tunnel t3 in monomer 1 by keeping f95 an optimum distance from f197 . we also found that through the k37/k151 residues , the rrm2b protein retains 66%70% of its tyrosyl radical in hypoxia . interestingly , monomer 2 of rrm2b showed a higher oxygen - entering frequency than rrm2 ( 22% and 0% , respectively ) , suggesting that the two monomers of the small rnr subunits could react differently in the overall production of the tyrosyl radical . it is possible that monomer 2 contributes more to the tyrosyl radical in rrm2b than in rrm2 , possibly due to its helix b open conformation , and that this could be more relevant in hypoxic conditions . in support of this hypothesis , epr spectra of the double mutant , k37e / k151e , showed a marked reduction in the tyrosyl radical content in hypoxia , but not in normoxia . we propose that the tyrosyl radical of monomer 2 is better protected in rrm2b , therefore contributing to the prolonged activity of the enzyme in hypoxic environments . our proposed model , where both monomers contribute to tyrosyl radical formation , is in agreement with the established finding that the tyrosyl radical is equally distributed between each small subunit , suggesting the possibility of generation of two tyrosyl radicals per dimer ( cotruvo and stubbe , 2011 ) . it is important to note that our md simulations were carried out with the only currently available crystal structure of human rrm2b protein ( pdb : 3hf1 ) , which has limitations in its active site configuration and specifically in the iron center . however , we considered it unlikely that as a result of this , there would be large conformational changes in the global structure of the protein compared to its native state , at least to a degree that would influence our md analysis . indeed , this is supported by the results of our recombinant mutant analysis and , most importantly , our rescue experiments using our rrm2b knockout cell line . collectively , this work highlights the importance of y164 , k37 , and k151 for rrm2b activity in hypoxia . it is important to note that the recombinant proteins used in our biochemical assays were assembled without additional iron . the biochemistry of rnr , especially the reconstitution of the tyrosyl radical , is extremely complex and not fully understood . additional studies that also consider the relevant factors for accurate assembly are required and will likely further illuminate the role of rrm2b in both normoxia and hypoxia . to date , mammalian rrm2b has been primarily associated with dna repair and mitochondrial dna synthesis ( wang et al . however , it is probable that rrm2b has been evolutionarily maintained in order to be used as the hypoxic - specific rnr small subunit , especially as variants of rrm2b protein have been found to be responsible for continued cell division in anoxia tolerant vertebrates ( sandvik et al . , 2012 ) . here , we propose that one of the principal functions of rrm2b is to act as the hypoxic - specific rnr subunit in order to be able to react promptly when this physiologically relevant stress occurs . it is tempting to speculate that through antioxidant and catalase - like properties ( xue et al . , 2006 ) , rrm2b might even play a role in increasing immediate oxygen availability . interestingly , a number of previous reports have demonstrated that rrm2b is frequently affected by copy - number changes , typically showing gains , in a broad range of cancers ( chae et al . this has led to the suggestion that rrm2b is a tumor promoter ( aye et al . , 2015 ) . our study demonstrates that increased rrm2b expression in hypoxia maintains replication and prevents dna damage , therefore providing a plausible explanation for why rrm2b is so often amplified in cancers . our data suggest that targeting r1/r2b enzyme specifically in hypoxic tumor cells might be an effective therapeutic strategy . rnr is a well - established therapeutic target , and a number of rnr inhibitors ( such as gemcitabine , clofarabine , hydroxyurea , and triapine ) are being used clinically ( manegold et al . , 2000 , aye and , 1998 , hehlmann et al . , 1993 , nutting et al . , 2009 ) . interestingly , delivery of sirna against rrm2 by phosphorothionate oligodeoxynucleotides ( gti-2040 ) ( lee et al . , 2003 ) is in clinical trials for various solid tumors ( juhasz et al . , 2006 , leighl et al . , 2009 , oh and park , 2009 ) . it is tempting to speculate that if this approach was modified to target rrm2b and was employed alongside patient stratification to identify those with significant tumor hypoxia , it may be an effective way to target the most aggressive and treatment - resistant fraction of tumors . reagent or resourcesourceidentifierantibodiesgoat polyclonal anti - rrm1 ( t-16)santa cruzcat # sc-11733goat polyclonal anti - rrm2 ( n-18)santa cruzcat # sc-10844goat polyclonal anti - p53r2 ( n-16 ) ( rrm2b)santa cruzcat # sc-10840mouse monoclonal anti - chk1 ( g-4)santa cruzcat # sc-8408mouse monoclonal anti - p53 ( do-1)santa cruzcat # sc-126rabbit polyclonal anti - p53 ( fl-393)santa cruzcat # sc-6243mouse monoclonal anti--actin antibody ( ac-15)santa cruzcat # sc-69879mouse anti - human hif-1 clone 54bd transduction laboratoriescat # 610958mouse anti - brdu clone b44bd transduction laboratoriescat # 347580rat monoclonal anti brdu clone bu1/75 ( icr1)bio - radcat # obt0030cxmouse monoclonal anti - rrm2 clone 1e1bio - radcat # mca3434zrabbit polyclonal anti - kap-1bethyl / universal biologicalscat # a300 - 274rabbit polyclonal anti - phospho kap-1 ( s824)bethyl / universal biologicalscat # a300 - 767rabbit polyclonal anti - phospho kap-1 ( s473)biolegendcat # 644602 rrid : ab_2241094rabbit polyclonal anti-53bp1novus biologicalscat # nb100 - 904rabbit polyclonal anti - phospho - p53 ( ser15)cell signalingcat # 9284rat monoclonal anti - rpa32/rpa2 ( 4e4)cell signalingcat # 2208rabbit polyclonal anti - phospho - chk1 ( ser296)cell signalingcat # 2349rabbit polyclonal anti - phospho - chk1 ( ser317)cell signalingcat # 2344rabbit polyclonal anti - phospho - chk1 ( ser345)cell signalingcat # 2341rabbit polyclonal anti - parpcell signalingcat # 9542rabbit polyclonal anti - cleaved caspase-3 ( asp175)cell signalingcat # 9661anti - mouse igg , hrp - linkedcell signalingcat # 7076anti - rabbit igg , hrp - linkedcell signalingcat # 7074rabbit polyclonal anti - beta tubulin antibodyabcamcat # ab6046mouse monoclonal anti - pimonidazole clone 4.3.11.3)chemicon internationalcat # hp1 - 100alexa fluor 680 goat anti - mouse igg ( h+l)invitrogencat # a21057alexa fluor 680 goat anti - rabbit igg ( h+l)invitrogencat # a21076alexa fluor 680 donkey anti - goat igg ( h+l)invitrogencat # a21084irdye 800cw donkey anti - rabbit iggli - corcat # 926 - 32213irdye 800cw donkey anti - mouse iggli - corcat # 926 - 32212alexa fluor 488-conjugated goat anti - rabbit igginvitrogencat # a11070alexa fluor 488-conjugated donkey anti - rabbit igginvitrogencat # a21206alexa fluor 488-conjugated goat anti - mouse igginvitrogencat # a11017alexa fluor 488-conjugated donkey anti - goat igginvitrogencat # a11055alexa fluor 594-conjugated goat anti - rabbit igginvitrogencat # a11072alexa fluor 594-conjugated goat anti - rat igginvitrogencat # a11007alexa fluor 594-conjugated goat anti - mouse igginvitrogencat # a11020alexa fluor 594-conjugated donkey anti - goat igginvitrogencat # a11058alexa fluor 555-conjugated goat anti - rat igginvitrogencat # a21434alexa fluor 488-conjugated goat anti - mouse f(ab)2 fragmentinvitrogencat # a11017pierce recombinant protein a / gthermo fisher scientificcat # 21186bacterial and virus strainsbl21-gold(de3 ) competent cellsagilent technologiescat # 230132one shot top10 chemically competent e. colithermo fisher scientificcat # c404010chemicals , peptides , and recombinant proteinshydroxyurea ( hu)sigmacat # h8627doxorubicin hydrochloride ( adriamycin)sigmacat # d15155-bromo-2-deoxyuridine ( brdu)sigmacat # b5002propidium iodide ( pi)sigmacat # s7109protease inhibitor cocktailrochecat # 04693159001cldu ( 5-chloro-2-deoxyuridine)sigmacat # c6891idu ( 5-iodo-2-deoxyuridine)sigmacat # i7125cytidine 5-diphosphocholine sodium salt dehydrate ( cdp)sigmacat # c9755protein g sepharose , fast flowsigmacat # p3296isopropyl -d-1-thiogalactopyranoside ( iptg)sigmacat # i6758deoxyadenosine 5-triphosphate , [ 8 - 3h(n ) ] ( datp - h3)perkin elmercat # net268250ucdeoxythymidine 5-triphosphate ( ( dttp ) tetrasodium salt ) ( dttp - h3)perkin elmercat # net520a250uctrizol reagentthermo fisher scientificcat # 15596018dharmafect 1dharmaconcat # t-2001lipofectamine ltxthermo fisher scientificcat # 15338100prolong goldthermo fisher scientificcat # p36931critical commercial assayshypoxyprobe-1 kit for the detection of tissue hypoxiachemicon internationalcat # hp1 - 100real - time and dynamic monitoring of cell proliferation and viability for adherent cellsacea biosciences inc/ cambridge biosccat # 00380601050sybr green pcr master mixapplied biosystemscat # 4309155verso cdna synthesis kitthermo fisher scientificcat # ab1453bhistrap hp columnsge healthcarecat # 17 - 5248 - 02qiaprep spin miniprep kitqiagencat # 27106quickchange ii xl site - directed mutagenesis kitagilent technologiescat # , 1998rkorrm2b/this papern / aexperimental models : organisms / strainsmice : female balb / c nude were used for xenograft experimentscharles river , ukoligonucleotidessirna - rrm2b ( sequence : ugaguuuguagcugacagauu)sigmapiao et al . , 2009sirna#2 - rrm2b ( sequence : ggaacaagcuuaaagcaga)ambion / life technologiess224156sirna - p53 ( sequence : guaaucuacugggacggaa)ambion / life technologiesleszczynska et al . , 2015allstars negative control sirnaqiagensi03650318primers for rrm2b forward - chip ( sequence : cttgctgggaaatcttgacc)sigmatanaka et al . 2007primers used for site - directed mutagenesis table s1n / an / aprimers used for dntp incorporation assay table s1n / an / aprimers used for quantitative pcr ( qpcr ) table s1n / an / arecombinant dnaplasmids used in this work are listed in table s2n / an / asoftware and algorithms7500 fast real - time pcr thermocycler was used with v2.0.5 softwareapplied biosystemshttp://www6.appliedbiosystems.com / support / software/7500/the software caver 3.0 was used for the analysis of the evolution of lateral fenestrations during the md simulationsn / an / athe particle mesh ewald ( pme ) algorithm was used for evaluation of electrostatics interactionsdarden et al . , 1993n / athe multi time step algorithm verlet - i / r - respa was used to integrate the equations of motiontuckerman et al . , 1992 , verlet , 1967n / athe pops ( parameter optimsed surfaces ) algorithm was used for calculation of the solvent - accessible surface area ( sasa ) of both proteins rrm2b and rrm2cavallo et al . , 2003 , fraternali and van gunsteren , 1996n / athe settle algorithm was used for constrained the lengths of covalent bonds involving hydrogen atomsmiyamoto and kollman , 1992n / aotherfor the analysis of rrm2b expression and genetic alterations in colorectal cancer datasets the cbioportal and prognoscanhttp://www.cbioportal.org/ ; http://www.abren.net/prognoscan/gao et al . , 2013 , , 2009gene expression omnibus ; smith et al . , 2010https://www.ncbi.nlm.nih.gov / geoaccession number gse17536icp - ms was performed with icp - ms trace element analysishttps://www.earth.ox.ac.uk / research / services / geochemical - analysis / icpms / n / a further information and requests for reagents may be directed to , and will be fulfilled by , the lead contact , dr . ester m. hammond ( [email protected] ) . source of cell lines used in the study is reported in the reagent and resource table . source of cell lines used in the study is reported in the reagent and resource table . cells were grown in dmem with 10% fbs , in a standard humidified incubator at 37c and 5% co2 . rko was constructed in this work using crispr - cas9 technology as previously described ( ran et al . , 2013 ) . rrm2b gene has three isoforms and in order to construct a full knock - out cell line two 20-bp target sgrna sequences were used targeting exon 1 : ttcggcggagtctgcgcgat ( isoforms 1 and 3 ) and aaatgttattcgccgcggtc ( isoform 2 ) . lipofectamine ltx ( invitrogen ) was used for plasmid transfections according to manufacturer s recommendations ( the plasmids used are listed in table s2 ) . dharma - fect 1 reagent ( thermo fisher scientific ) was used for sirna knockdown according to manufacturer s instructions . rko or u2os cells were transfected with the sirna sequences reported in the reagent and resource table in a final concentration of 50 nm . hydroxyurea ( hu ) was used at a concentration of 2 mm for 6 hr . hypoxia treatments were carried out in a bactron ii anaerobic chamber ( shell labs ) or an in vivo2 400 ( baker ruskinn ) ( for oxygen concentrations at 2% ) . for experiments at oxygen concentrations were periodically verified using an oxylite probe ( oxford optronix , uk ) . for immunoblots , cells were lysed in utb ( 9 m urea , 75 mm tris - hcl ph 7.5 and 0.15 m -mercaptoethanol ) and sonicated briefly . the odyssey infrared imaging technology was used ( li - cor biosciences ) and the odyssey analysis system was used for quantification of the immunoblots . in each case , experiments were carried out in triplicate and a representative blot is shown unless otherwise stated . the antibodies used in this study are listed in the reagent and resource table . for immunofluorescence , cells were seeded on autoclaved cover glasses ( menzel - glaser ) . at the end of each experiment cells were fixed with 4% fixation buffer ( 4% ( w / v ) paraformaldehyde in pbs ) , lysed with 1% pbs - triton x-100 and then incubated with blocking buffer ( 2% ( w / v ) bsa in 0.1% pbs - triton x-100 ) . incubation with the appropriate primary and then secondary antibody followed ( diluted in 2% ( w / v ) bsa - 0.1% pbs - triton x-100 ) ( antibodies used are listed in the reagent and resource table ) . at least 100 cells were counted per condition . due to the presence of 53bp1 foci in the nuclei of unstressed cells , induction of dna damage rna was prepared using trizol ( invitrogen / life technologies ) . for qpcr expression analysis cdna was reverse transcribed from total rna using verso kit ( thermo scientific ) . qpcr was performed using sybr green pcr master mix kit ( applied biosystems ) in a 7500 fast real - time pcr thermocycler with v2.0.5 software ( applied biosystems ) . mrna fold change was calculated using a 2 method in relation to the 18s reference gene . facs analysis was performed as previously described ( olcina et al . , 2013 ) . u2os cells were treated with either rrm2b sirna or negative control sirna and were placed in normoxia or < 0.1% o2 ( 3 hr ) . samples were pulsed with brdu ( 20 m ) for 30 min before collection . sub confluent cells ( 50%60% confluency ) were sequentially pulse labeled with 25 mm cldu and 250 mm idu for 20 min each and left at 21% o2 in a 37c incubator ( normoxic samples ) . cells were washed once with fresh warm media before the addition of the second ( idu ) label . for hypoxic treated samples , cells were first placed for 2 hr in the hypoxic chamber ( < 0.1% o2 ) and then treated with 25 mm cldu for the indicated times , followed by the addition of 250 mm idu for as long as needed for all samples to be in hypoxia for 6 hr in total . at the end of the treatment , labeled cells were lysed and spread with spreading buffer ( 200 mm tris - hcl , ph 7.4 , 50 mm edta , 0.5% sds ) in a tilting the slide . dna fibers were then fixed with methanol / acetic acid ( 3:1 ratio ) and stained with rat anti - brdu monoclonal antibody ( for detection of cldu labeled tracts ) and mouse anti - brdu monoclonal antibody ( for detection of idu labeled tracts ) . fibers were imaged using a bio - rad radiance or lsm780 ( carl zeiss microscopy ltd ) confocal microscope and analyzed using imagej software ( nih ) . the length of the fiber tracts that had incorporated both labels ( cldu and idu ) was measured and replication rates were calculated with the following formula ( vcldu ( kb / min ) = [ ( x 0.132 m ) 2.59 kb / m ] / t ( min ) , where x = length of cldu ) . dntp pool determination in whole - cell extracts was carried out as previously described ( dangiolella et al . , 2012 ) . in brief , cells were seeded in glass dishes and treated either in < 0.1% o2 ( for the indicated times ) or with 2 mm hu ( 6 hr ) . the normoxic samples were processed immediately after the beginning of the hypoxic and/or hu treatment . samples were then boiled , centrifuged and the supernatant was dried by centrifugal evaporation and finally dissolved in h2o . for the preparation of each primer mix a different set of primers was used ( listed in table s1 ) supplemented with either [ 3h]datp or [ 3h]dttp ( perkinelmer ) . for dgtp and dctp determination 1 u of thermo sequenase dna polymerase ( ge healthcare ) was used ; for dttp determination 1.25 u klenow fragment ( thermo fisher scientific ) and for datp determination 0.625 u klenow fragment ( thermo fisher scientific ) was used . reactions were incubated for 1 hr either at 48c ( for dgtp and dctp mixtures ) or at room temperature ( for dttp and datp mixtures ) . following incubation , the reaction mixture were spotted onto whatman de81 paper ( ge healthcare ) and let dry . the papers were then washed with 5% na2hpo4 , followed by rinsing with distilled h2o and 100% etoh . after being dried , the radioactivity on the papers was measured in a ls 6500 multi - purpose scintillation counter ( beckman coultertm ) using 3.5 ml optiphase hisafe 3 ( perkinelmer ) counting fluid . data present percentage of dntp incorporation compared to the positive control of each experiment . in experiments where both sirna and hypoxia treatment were used , a normalization to each normoxic control was preceded the final analysis . briefly , rko cells were lysed in lysis buffer ( 100 mm nacl , 20 mm tris - hcl ph 7.4 , 5 mm mgcl2 , 0.5% np-40 ) with freshly added phosphatase / protease cocktail inhibitors ( roche ) . the lysates were incubated with protein g sepharose beads ( sigma ) and the antibody of interest rrm1 ( santa cruz biotechnology : sc-11733 ) or a control igg antibody ( invitrogen : a10535 ) with agitation , at 4c overnight . antibodies used were : rrm1 ( santa cruz biotechnology : sc-11733 ) , rrm2b ( santa cruz biotechnology : sc-10840 ) , rrm2 ( abd serotec , mca3434z ) and for the detection of rrm2 the pierce recombinant protein a / g ( thermo fisher scientific , 21186 ) secondary antibody was used . chip in rko cells treated as indicated in figure legend was carried out as previously described ( leszczynska et al . , 2015 ) . for each sample 2 g of combined mouse p53 ( do-1 ; sc-126 ) and rabbit p53 ( fl-393 ; sc-6243 ) ( santa cruz ) antibodies were used for immunoprecipitation . combined non - specific mouse ( 7076 ) and rabbit ( 7074 ) ( cell signaling ) were used as control iggs . a no antibody control sample was also included in each experiment . bound fraction and input were analyzed by qpcr using specific primer set for the rrm2b locus : cttgctgggaaatcttgacc ( tanaka et al . , 2007 ) . fold enrichment is expressed as a % of input and is normalized to total p53 in each sample . rko cells were treated either with rrm2b sirna or negative control sirna and exposed to normoxia or hypoxia ( < 0.1% o2 for 24 hr ) . subsequently all cells were placed in normoxic incubator and left for 9 days to form colonies , which were visualized by crystal violet staining . apoptosis assay were performed as previously described ( leszczynska et al . , 2015 ) . in brief , both adherent and floating cells were collected and fixed with 4% paraformaldehyde . samples were then washed and the nuclei were stained with prolong gold mounting medium with dapi ( thermo fisher scientific ) . apoptosis was plotted as the percentage of cells with fragmented dna per field of view , with at least ten fields of view quantified per experiment . the xcelligence real - time cell analyzer ( acea biosciences ) dp instrument equipped with an e - plate was used for proliferation assays . the attachment , spreading and proliferation of the cells were monitored every 15 min for 60 hr . for quantification all animal procedures were performed in accordance with current uk legislation and were approved by the university of oxford biomedical services ethical review committee , oxford , uk . for the tumor growth curves ( xenografts ) , female balb / c nude mice or female athymic nude mice ( charles river , uk ) were randomized and injected subcutaneously with 3 10 rko or rko cells in 25% ( v / v ) matrigel and serum - free dmem . tumor growth was monitored until they reached approximately 300 - 400 mm3 ( volume = height x depth x width x / 6 ) . for the irradiation experiment , the tumors in half of the animals were irradiated with 10 gy when tumor volumes reached 100 mm . tumors were measured regularly , and tumor growth was plotted as a mean of tumor volumes sem . mice were injected with 50 mg / kg of pimonidazole ( pimo ) 2 hr before the end of the experiment . tumors were split in half and either preserved in formalin for wax embedding or were snap frozen and embedded in optimal cutting temperature compound ( oct ) . , tumors embedded in oct were sectioned ( 5 m ) and froze down at 20c . sections were immediately fixed with 4% paraformaldehyde and then quenched with 50 mm nh4cl . blocking was performed with 1% bsa in tbs , followed by incubation with mouse on mouse ( m.o.m . ) blocking reagent ( vector laboratories ) . the sections were stained with pimo and cleaved caspase-3 primary antibodies , followed by secondary antibodies ( listed in the reagent and resource table ) and mounted with prolong gold mounting medium with dapi ( invitrogen / life technologies ) . raw rna - seq data for 382 colorectal adenocarcinoma tumors were downloaded from the tcga project ( accessed through cbioportal : http://www.cbioportal.org/ ) . to examine tumor - associated hif - activity ( referred to as hypoxia signature ) , raw data for each sequenced gene were rescaled to set the median equal to 1 , and hif - activity was quantified by averaging the normalized expression of 44 target genes , associated with hif activity ( encoding adm , gfbp3 , edn2 , pfkfb4 , flt1 , tfr2 , bnip3l , tgfa , bnip3 , pgk1 , egln1 , ldha , egln3 , cp , tgfb3 , pfkfb3 , hk1 , tfrc , edn1 , cdkn1a , ca9 , hmox1 , serpine1 , lox , ndrg1 , ca12 , pdk1 , vegfa , ero1l , rora , p4ha1 , mxi1 , slc2a1(glut1 ) , stc2 , mif , ddit4 , eno1 , cxcr4 , plod1 , p4ha2 , gapdh , pgam1 , tmem45a and pim1 ) ( li et al . , 2014 ) . log10 conversion of the hypoxia signature was plotted against log10 conversion of raw data for rrm1 , rrm2 and rrm2b . two - tailed p value shown on each graph for each pearson and spearman r ( correlation coefficient ) . to examine tumor - associated p53-activity ( referred to as p53 group ) , raw data for each sequenced gene were rescaled to set the median equal to 1 , and p53-activity was quantified by averaging the normalized expression of 6 p53 target genes , associated with hypoxia - induced p53 activity ( encoding btg2 , cyfip2 , inpp5d , kank3 , phlda3 and sulf2 ) ( leszczynska et al . , 2015 , log10 conversion of the p53 signature was plotted against log10 conversion of raw data for rrm2b ( also rescaled to set the median equal to 1 ) . two - tailed p value shown on each graph for each pearson and spearman r ( correlation coefficient ) . for the analysis of rrm2b expression and genetic alterations in colorectal cancer datasets the cbioportal ( http://www.cbioportal.org/ ) and prognoscan ( http://www.abren.net/prognoscan/ ) tools were used on the 5th dec 2016 ( gao et al . , 2013 , cerami et al . , 2012 , mizuno et al . , 2009 ) . using cbioportal the tcga ( provisional ) dataset was analyzed ( rna seq v2 rsem ) for alterations , including mutations , putative copy - number alterations , mrna expression , mutations and survival probability ( 629 cancer patients ) . for the prognoscan analysis , rrm2b expression was checked in all available colorectal cancer datasets and kaplan - meier graphs were extracted only for the statistically significant ( p < 0.05 ) dependence between rrm2b expression ( probe 223342_at ) and survival probability . this analysis used the publicly available dataset at gene expression omnibus ( https://www.ncbi.nlm.nih.gov/geo ) with the accession number gse17536 ( smith et al . , 2010 ) . the 6 his - tagged hrrm1 ( pet28a - rrm1 ) was kindly provided by prof joanne stubbe ( massachusetts institute of technology , cambridge , usa ) , expressed in e.coli bl21-gold ( de3 ) competent cells ( agilent technologies ) and as previously described ( ando et al . , the 6 his - tagged hrrm2 and hrrm2b ( pet28a - rrm2 , pet28a - rrm2b ) were kindly provided by dr . yun yen ( beckman research institute at city of hope , duarte , usa ) . the proteins were expressed in e.coli bl21-gold ( de3 ) competent cells ( agilent technologies ) and purified as previously described ( shao et al . , 2004 ) with minor modifications . specifically , an overnight culture of the transformed bacteria was diluted 80-fold in 600 ml of 2 yt medium containing 50 g / ml kanamycin . each culture was grown at 37c until od600 nm = 0.7 - 0.9 and then induced by 1 mm isopropyl-1-thio--d - galactopyranoside ( iptg ) and incubated overnight at 18c . cells were harvested by centrifugation and the pellets were lysed with appropriate amount ( 5 ml / gr of pellet ) lysis buffer ( 50 mm nah2po4 ph 7.0 ; 0.1% triton x-100 ; 10 mm -mercaptoethanol ; freshly added edta - free protease inhibitors and dnase i ) at 4c with vigorous agitation until the lysate was homogeneous . the lysate was then sonicated ( 60% amplitude 30 s on / 30 s off ) and clarified by centrifugation at 48,000 g for 20 min at 4c . soluble lysate was passed through a his - trap hp purification column ( ge healthcare ) , washed with at least 30-fold bed volume of wash buffer ( 50 mm nah2po4 ph 7.0 ; 800 mm nacl ; 50 mm imidazole ; 0.1% triton x-100 ; 10 mm -mercaptoethanol ) and finally eluted with elution buffer ( 50 mm nah2po4 ph 7.0 ; 300 mm nacl ; 125 mm imidazole ) . the protein then underwent buffer exchange into 25 mm tris - hcl , ph 7.5 before being concentrated and stored at 80c . the apo - forms of rrm2 and rrm2b recombinant proteins used in icp - ms experiments were overexpressed and purified as previously described ( wang et al . , 2009a ) . site - specific mutations in rrm2b ( y164c ; y164f ; f183y ; k31e / k151e ; y241h ; y331f and q127k ) were generated by pcr , using the pet28a - rrm2b as a template and the quickchange ii xl site - directed mutagenesis kit ( agilent technologies ) . expression and purification of the hrrm2b mutated proteins were performed in the same way as for hrrm2b . the enzyme concentration ( r1/r2b or r1/r2 ) was the same in all cases and the limited factor was the presence or not of oxygen ( normoxia versus hypoxia ) . specifically , a final concentration of 1.25 m of purified rrm1 was incubated with 2.5 m of either rrm2 or rrm2b protein . the reaction mixture contained 0.1 mm cdp , 50 mm hepes ( ph 7.5 ) , 6 mm dtt , 8 mm magnesium acetate , 2 mm atp and 1 mm nadph in a final volume of 400 l . the rnr proteins purified were active without reassembling the iron center ( shao et al . ( < 0.1% o2 ) the reaction mixture was assembled and the assay was carried out within an anaerobic chamber ( bactron , shel labs ) . reactions were initiated by the addition of cdp , incubated at 37c and aliquots of 50 l were quenched by the addition of 10 l of 3% tricarboxylic acid over a time course of the reaction . graphs present either percentage of dcdp formation when normoxia and hypoxia were compared ( data were normalized against the normoxic samples at 37c at 30 min where the dcdp formation was considered 100% ) or in m when the accumulation of dcdp formed in < 0.1% o2 at a 0 - 120 min time course was examined . cdp and dcdp levels following rnr activity assays were determined by ion - pairing hplc with uv detection ( 270 nm ) . the column was an ace c 18 , 3 m , 125 3 mm ( hichrom ) maintained at 35c . a gradient separation was achieved using 10% methanol , 10 mm kh2po4 , 10 mm tbaoh , ph 6.9 ( a ) and 30% methanol , 50 mm kh2po4 , 6 mm tbaoh , ph 7.0 ( b ) , with a linear gradient of 25 47% b over 8 min , 47 80% b over 1 min , then returning to the started conditions 3 min . the flow rate was 0.6 ml / min and the running time was 16.5 min . circular dichroism ( cd ) spectra ( chirascan cd / fluorimeter spectrometer ( applied photophysics , uk ) of hrrm2b and the mutants ( 0.2 mg / ml ) were recorded in the wavelength range 260 to 185 nm in 100 mmol / l potassium phosphate buffer ( ph 7.5 ) . all measurements were carried out at 37c using a 0.1-cm path length quartz curette . the data pitch was 0.1 nm with a 1-nm bandwidth at a scan speed of 1.0 nm / s . all cd data were expressed as the mean residue ellipticity , [ ] , degcmdmol . the thermo finnigan element 2 icp - ms was used for quantitation of iron content in the recombinant purified proteins ( 40 g of protein was used per 50 l of reaction ) . calibrations were obtained using external standards ( a series of standards of known fe concentrations were prepared and analyzed to gain a calibration linear , prior to the measurement of the samples ) . an external standard was diluted and measured from a dilution of high purity standards 10 ppm icp - ms-68 a standard . all blanks , standards and samples were also spiked with 1 ng / g rh , so that any general instrument drift could be normalized . dilutions were made using a 2% hno3 solution , prepared using in - house distilled nitric acid and 18.2 mohm di water . all data results were first reported as elemental concentrations and then calculated in nm of fe per m of protein . cw - epr spectra were collected in the center for advanced esr ( caesr ) in the inorganic chemistry laboratory at the university of oxford . x - band measurements utilized a bruker - biospin micro emx spectrometer equipped with a premiumx microwave bridge , a cylindrical te011-mode resonator ( shqe - w ) , an esr-900 liquid helium cryostat , and an oxford instruments itc-503 s temperature controller . measurements were performed within the limit of resolution by temperature , 40k , and under non - saturating conditions . the protein concentration for all epr samples was 200 m except for rrm2 protein , which was 70 m . the protein samples ( rrm2b , rrm2 or rrm2b mutants ) were diluted in 50 mm hepes , ph 7.5 and were incubated either at 37c for 5 min with oxygen present ( normoxia ) or at 37c for 60 min in a bactron ii anaerobic chamber ( < 0.1% o2 ) . at the end of each treatment 20% ( v / v ) glycerol was added for vitrification during the low - temperature recordings . samples were then transferred to epr tubes and quick - frozen by immersion in liquid nitrogen . spin quantitation was performed using the bruker spectrometer hardware spin calibration , given the input dimensions of the precision wilmad pq-706 tubes . the results were verified with a 1 mm cu edta in 50 mm hepes ph 7.5 , 20% ( v / v ) glycerol that was measured under non - saturating conditions at 100 k with 50 w microwave power . system setup : md simulations of o2 transport on hrrm2b ( pdb : 3hf1 ; resolution 2.60 ) and hrrm2 ( pdb : 3vpn ; resolution 2.25 ) were performed ( smith et al . , 2009 ) . the 3vpn x - ray structure is that of the e106d variant of native rrm2 , and comprises residues m65 to m350 . four simulations were performed in which 200 randomly chosen water molecules outside the solvated protein were replaced by 200 o2 molecules ( 0.5 m ) termed the 100% oxygen level simulation . an additional two sets of simulations were performed to control for concentration effects ; the first set up has a tenfold reduction in oxygen concentration , termed 10% oxygen level simulation with effective concentration of 50 mm , for rrm2b and rrm2 . the second set does not include any oxygen molecules , termed 0% oxygen level simulation . each system was solvated in a box of dimensions ( 80 , 80 , 92 ) using the solvate plug - in of vmd ( humphrey et al . , 1996 ) , with the total system size comprising 61,000 atoms . in accordance with the uniprot sequence alignment two mutants of native rrm2b were prepared with the mutate plugin in vmd ( humphrey et al . , 1996 ) , namely y164c and k37e / k151e . calculations were done with namd 2.9 ( phillips et al . , 2005 ) , using the charmm 27 protein force - field ( brooks et al . , 2009 ) with the cmap correction ( buck et al . , 2006 ) , together with the tip3p water model ( jorgensen et al . , 1983 ) . oxygen lennard - jones parameters were taken from charmm ( cohen et al . , 2005 , the iron centers were modeled using charmm 27 force - field , with iron lennard - jones parameters o = 0.00 kcal / mol and rmin/2 = 0.65 . oxygen ( o2 ) was modeled using charmm 27 lennard - jones parameters from heme oxygen , o = 0.12 kcal / mol and rmin/2 = 1.7 , as well as bond spring constants and bond spring lengths for o2 as 1.23 and 600 kcal / mol / , respectively . rrm2b contains the same diiron / dityrosyl cofactor that rrm2 does , and both rrm2b and rrm2 are biologically active as homodimers . in rrm2b , the active site iron coordination environment in monomer-1 is a mono - iron site , and monomer-2 has a di - iron site . in rrm2b , the active site iron coordination environment after 1,000 steps of conjugate - gradient minimization , four classical molecular dynamics ( md ) simulations of the o2-containing solvated protein were carried out for 300 ns in the npt ensemble . the high oxygen concentration was necessary to ensure sufficient sampling of protein cavities by o2 on the accessible timescale of current simulations . the particle mesh ewald ( pme ) algorithm was used for evaluation of electrostatics interactions beyond 12 , with a pme grid spacing of 1 , and namd defaults for spline and values ( darden et al . , 1993 ) . a cut - off at 12 was applied to non - bonded forces , both electrostatics and van der waals forces were smoothly switched off between the cut - off distance of 12 , and the switching distance of 10 , using the default namd switching function . a verlet neighbor list with pairlist distance of 14 was used to only evaluate non - bonded neighboring forces within the pairlist distance ( verlet , 1967 ) . the lengths of covalent bonds involving hydrogen atoms were constrained by the settle algorithm ( miyamoto and kollman , 1992 ) in order to be able to use a 2-fs time - step . the multi time step algorithm verlet - i / r - respa ( tuckerman et al . , 1992 , verlet , 1967 ) was used to integrate the equations of motion . non - bonded short - range forces were computed for each time step , while long - range electrostatic forces were updated every 2 time steps . the pressure was kept at 1 atm by the nos - hoover langevin piston ( langevin , 1908 , nos , 1984a , nos , 1984b ) , with a damping time constant of 50 fs and a period of 100 fs . the temperature was maintained at 300 k by coupling the system to a langevin thermostat , with a damping coefficient of 1 ps . the standard mpi version 2.9 of the md simulation code namd was employed ( phillips et al . , 2005 ) . a free license can be obtained at http://www.ks.uiuc.edu / research / namd/. version 1.9 of visual molecular dynamics ( vmd ) was employed for analysis , with license obtained at http://www.ks.uiuc.edu / research / vmd/. the solvent - accessible surface area ( sasa ) of both proteins rrm2b and rrm2 was calculated using the pops ( parameter optimsed surfaces ) algorithm ( cavallo et al . , 2003 , fraternali and van gunsteren , 1996 ) . the bending angle of helices was estimated using bendix in vmd ( dahl et al . , 2012 ) . the diffusion - reaction model ( wang et al . , 2013 ) presented in equation ( 1 ) was adopted to describe the diffusion of o2 into the rnr active site:(1)enz+gaskoutkin[o2]enzgas . this is a kinetic two - step model consisting of an initial diffusion step of the gas molecule , followed by a metal - gas molecule chemical reaction step ( wang et al . , 2011 , wang and blumberger , 2012 ) . here , a classical force - field ( ff ) was employed , and only the diffusion and binding of o2 gas molecules to the rnr iron center were modeled:(2)rnr(fe)+o2k1k+1rnr(fe)o2 . the simulation of o2 diffusing in the solvated rnr enzymes yields diffusion statistics that enable the construction of a probability density map of the o2-distribution within rnr ( rrm2b or rrm2 ) . this distribution is then divided into a set of m discrete protein cluster states that correspond to the probability maxima . first , a distance criterion is established to determine when an oxygen molecule has entered into the active site of hrrm2b and hrrm2 , defined as the difference between the center - of - mass ( com ) of the iron ion and the o2 molecule . second , cut - off criteria of 8 , 9 and 10 were employed to generate statistics of the o2 entering . an o2 molecule is counted as having entered the cluster if dcom ( o2-fe ) < c where c is 8 or 9 . as previously described , o2 entry into wt rrm2b and rrm2 was identified to occur primarily through tunnels t1 , t2 and t3 . based on this assignment , equilibrium adaptive - biasing force ( abf ) md simulations ( chipot and pohorille , 2007 , darve and pohorille , 2001 , darve et al . , 2008 , van gunsteren , 1989 ) were performed on oxygen entry into tunnels t1 , t2 and t3 in monomer-1 to determine relative differences in the thermodynamics of oxygen entry into rrm2b and rrm2 . in total , six abf simulations per rnr were performed , totalling 0.5 s of md simulation dynamics . each abf simulation was performed with a force bias on a single oxygen molecule meaning these simulations constitute single - molecule biased md simulations , using the collective variable center - of - mass ( com ) distance between fe(iii ) and o2 , (rn)=d(o2fe(iii ) ) , with ranging from 3 to 15 , in bins of width 0.25 . each abf simulation obtains ergodic sampling by applying every 500 timesteps a biasing force fabf ( 3 ) onto the newtonian equations of motion , whose value is updated on - the - fly ( den otter , 2000 , den otter and briels , 1998 ) from the result , free - energy gradient along , the potential of mean force ( pmf ) along is obtained from numerical integration of the gradient.(3)fabf=ra=f()r(4)(dg()d)=u(rn)1in|j|=f(). tunnel t1 in monomer-1 extends for 30 and connects the two faces of the protein . it was simulated as two separate tunnels of length 15 in monomer 1 , denoted tunnel t1 and t1 , and was compared to a single simulation of tunnel t1 in monomer 2 . the software caver 3.0 was used for the analysis of the evolution of lateral fenestrations during the md simulations , using 100 evenly chosen snapshots from the md simulations , which is every 2 ns . the outer surface of the protein is calculated by rolling a large spherical probe around the surface of the protein and then internal cavities are identified using a smaller spherical probe ( a radius of 0.8 was used here , and 12 probe spheres used ) . all identified tunnels are grouped into clusters based on relative proximities and with an 8 cut off for each cluster node , meaning that tunnels differing from the node by more than 8 are excluded . the values for shell radius and depth ( 15 ) influence the definition of the protein molecular surface . the statistical significance of differences between datasets was determined assessed by using the following test . student s t test ( two - tailed paired , except for dntp incorporation assays , where a two - tailed unpaired test was applied ) was used in all experiments where two means were compared . one - way analysis of variance ( anova ) was used to compare several means and two - way anova was used for determination in a response that is affected by two factors ( one- or two - way anova analysis is indicated at the figure legends ) . statistical significance was assumed if p < 0.05 or lower and is noted in the figures . error bars represent mean standard error of the mean ( s.e.m . ) . i.p.f . conceived , designed , performed , and interpreted the majority of the experiments and wrote the manuscript . k.b.l . performed the xenografts , immunohistochemistry ( ihc ) , some experiments , and tcga analysis . e.m.h . conceived the project ; conceived , designed , and interpreted the majority of the experiments ; and wrote the manuscript .

summarycells exposed to hypoxia experience replication stress but do not accumulate dna damage , suggesting sustained dna replication . ribonucleotide reductase ( rnr ) is the only enzyme capable of de novo synthesis of deoxyribonucleotide triphosphates ( dntps ) . however , oxygen is an essential cofactor for mammalian rnr ( rrm1/rrm2 and rrm1/rrm2b ) , leading us to question the source of dntps in hypoxia . here , we show that the rrm1/rrm2b enzyme is capable of retaining activity in hypoxia and therefore is favored over rrm1/rrm2 in order to preserve ongoing replication and avoid the accumulation of dna damage . we found two distinct mechanisms by which rrm2b maintains hypoxic activity and identified responsible residues in rrm2b . the importance of rrm2b in the response to tumor hypoxia is further illustrated by correlation of its expression with a hypoxic signature in patient samples and its roles in tumor growth and radioresistance . our data provide mechanistic insight into rnr biology , highlighting rrm2b as a hypoxic - specific , anti - cancer therapeutic target .

Introduction Results Discussion STARMethods Author Contributions

PMC4247472

over the last 15 years , laparoscopy has been inexorably integrated into urologic practice . however , during this transition period , urologic training programs are only now beginning to provide systematic and fundamental minimally invasive surgery ( mis ) training to residents . additionally , there remains a significant proportion of practicing urologists who have limited laparoscopic experience , but who wish to add this to their therapeutic armamentarium . the road to laparoscopic expertise involves a steep and prolonged learning curve , which may discourage physicians from undertaking this challenge [ 1 , 2 ] . with the advent of modern technology , surgical robotics have come of age and now represent a viable solution to technically complex mis [ 35 ] . the benefits of robotic assistance over conventional laparoscopy including increased degrees of freedom , elimination of tremor , 3d visualization , and motion scaling have proved so advantageous as to introduce the possibility of mis without laparoscopic training [ 6 , 7 ] . in a similar vein , the impact of advanced surgical technologies on surgeons of varying mis skills has not been evaluated previously . just as laparoscopy has been integrated into training programs , as surgical robotics becomes more common , the question of how these advanced platforms influence surgical education for future trainees needs to be addressed . with the proliferation of robotic technology , should trainees expect to learn robotic skills rather than conventional laparoscopy ? in the personal computer world , we have come to expect technological leaps at every quarter , as advances in miniaturization and engineering efficiency continue apace . computational speed does not equal power , however , as architecture and design have been found to be significant and limiting factors , and more evidence - based metrics were developed to suit these purposes . analogous to this phenomenon , surgical robotics is a burgeoning field with limitless potential for the future . standard , validated , objective evaluations have not yet been widely adopted to evaluate their performance , and this remains a limiting factor in their comparisons . as well , task - specific robotics further complicates comparisons , as one robot may not be suited to all tasks . in an effort to evaluate the surgical performance of different advanced surgical platforms in standardized tasks we compared standard laparoscopy , zeus ( computermotion , sunnyvale , ca ) surgical robot and davinci ( intuitive surgical , sunnyvale , ca ) surgical performance across varying operator skill levels . between april 2004 and june 2005 , 12 participants were recruited to the study : four staff surgeons with laparoscopic expertise ( laparoscopic aneurysm repair , pyeloplasty , cholecystectomy ) , four senior level postgraduate trainees ( pgy ) with moderate levels of laparoscopic training , and four laparoscopic nave medical student interns ( msi ) . thirty - minute acclimation sessions on each platform were allowed prior each study trial for all study groups . each participant was required to complete four trials each of two standardized drylab modules : suturing and knot tying with 5 - 0 prolene sutures ( fig . 1 ) on each of the three test platforms ( lap , zeus , davinci ) . the suture task consisted of driving the needle through dots spaced 3 mm apart on a latex sheet , then running the suture six passes through consecutive dot series . the knot - tying task consisted of an identical setup ; however , instead of running the suture , the participants were required to throw three half hitches to knot the prolene suture . introduction to the tasks were allowed , as were informal practice task runups to the trial . following completion of the task on each platform , participants filled out surveys designed to subjectively score system performance in eight domains : visualization , fluidity , efficacy , precision , dexterity , tremor , tactile feedback and coordination . this survey was developed in - house through review of the current literature as well as interviews with experts in the field . cstar is currently working towards validating this tool for future minimally invasive clinical trials . participants were required to pass a 5 - 0 prolene suture immediately into and out of the adjacent marked dots spaced 3 mm apart on a latex sheet , and run six passes through consecutive dot series . setup is identical for knot - tying tasks ; however , participants are required to drive one pass of a 5 - 0 prolene suture through adjacent marked dots , followed by three throws of a half - hitch knot drylab module setup for suturing skills . participants were required to pass a 5 - 0 prolene suture immediately into and out of the adjacent marked dots spaced 3 mm apart on a latex sheet , and run six passes through consecutive dot series . setup is identical for knot - tying tasks ; however , participants are required to drive one pass of a 5 - 0 prolene suture through adjacent marked dots , followed by three throws of a half - hitch knot a standard black box laparoscopic simulator was used for these experiments consisting of a plexiglass enclosure with three ports on the angled upper surface for one laparoscope and two working ports . for conventional laparoscopy zeus and davinci trials were conducted in a similar fashion , placing the robotic laparoscope and working elements through their respective ports on the simulator . a hollow aluminum block with one face cut off an ic chip was recruited for the template to generate the target rows of ink dots on the latex sheet . twelve - centimeter lengths of 5 - 0 prolene suture were used for all tasks in this study . endowristed continuous variables were analyzed using single - sided unpaired student 's t - tests , and categorical variables from likert scales were analyzed using kruskal all participants completed all tasks for the study . overall platform performance times and errors are illustrated in fig . 2a , b. zeus robot - assisted tasks demonstrated significantly inferior performance compared to conventional laparoscopy and davinci platforms in both task times ( suturing : 485.7 s vs 302.0 vs 156.7 s ; knot tying : 456 s vs 183.7 s vs 93.5 s , respectively ) and task errors ( suturing : 12 vs 5 vs 2 ; knot tying : 12 vs 4 vs 1 , respectively ; p < 0.05 ) . fig . 2 a overall platform performance times for suturing and knot - tying modules . zeus assisted task times were significantly longer compared to conventional laparoscopy and davinci platforms for both suturing and knot - tying tasks . a trend towards significance was demonstrated in task performance times between davinci and laparoscopy ( * p < 0.05 , * * p < 0.05 ) . zeus - assisted task errors were significantly more numerous compared to conventional laparoscopy and davinci platforms for both suturing and knot - tying tasks . a trend towards significance was demonstrated in task performance errors between davinci and laparoscopy ( * p < 0.05 , * * p < 0.05 ) a overall platform performance times for suturing and knot - tying modules . zeus assisted task times were significantly longer compared to conventional laparoscopy and davinci platforms for both suturing and knot - tying tasks . a trend towards significance was demonstrated in task performance times between davinci and laparoscopy ( * p < 0.05 , * * p < 0.05 ) . zeus - assisted task errors were significantly more numerous compared to conventional laparoscopy and davinci platforms for both suturing and knot - tying tasks . a trend towards significance was demonstrated in task performance errors between davinci and laparoscopy ( * p < 0.05 , * * p < 0.05 ) when stratified by skill level , suturing task performance paralleled overall platform performance in that zeus robotic - assisted procedures were lengthy and introduced more task errors than either the laparoscopic or davinci platforms . these findings were most significant at the msi skill level , and incremental loss of significance between platforms was observed with increasing skill level ( fig . the zeus robotic platform demonstrated significantly inferior times compared with laparoscopic and davinci platforms across skill levels . however , with increasing surgeon skill , there is an incremental loss of significant performance time benefit seen with laparoscopic and davinci platforms ( * p < 0.05 , * * p < 0.05 ) . the zeus robotic platform demonstrated significantly higher error rates compared with laparoscopic and davinci platforms across skill levels . in a similar fashion as to performance times , surgical experience appears to blunt the significance of platform dependence ( * p < 0.05 , * * p < 0.05 ) a suturing task performance times stratified by skill level of participant . the zeus robotic platform demonstrated significantly inferior times compared with laparoscopic and davinci platforms across skill levels . however , with increasing surgeon skill , there is an incremental loss of significant performance time benefit seen with laparoscopic and davinci platforms ( * p < 0.05 , * * p < 0.05 ) . b suturing task performance errors stratified by skill level of participant . the zeus robotic platform demonstrated significantly higher error rates compared with laparoscopic and davinci platforms across skill levels . in a similar fashion as to performance times , surgical experience appears to blunt the significance of platform dependence ( * p < 0.05 , * * p < 0.05 ) in a similar analysis , knot - tying task performance stratified by skill level again demonstrated the inferiority of the zeus platform compared to conventional laparoscopy and davinci . with knot - tying task times , significant differences were observed across skill levels ; however , knot - tying errors demonstrated significance with msi and pgy trainees only ( fig . davinci robotic - assisted knot - tying times were significantly shorter compared to laparoscopic and zeus - assisted platforms ( * p < 0.05 , * * p < 0.05 ) . b knot - tying errors stratified by skill level of participant . this trend was maintained across msi and pgy skill levels , but lost when staff surgeons performed this task a knot - tying task times stratified by skill level of participant . davinci robotic - assisted knot - tying times were significantly shorter compared to laparoscopic and zeus - assisted platforms ( * p < 0.05 , * * p < 0.05 ) . b knot - tying errors stratified by skill level of participant . this trend was maintained across msi and pgy skill levels , but lost when staff surgeons performed this task overall subjective assessment by platform demonstrated a significant benefit to the davinci platform versus both zeus and conventional laparoscopy . analysis of subjective data by individual domains grouped by skill level is shown in fig . the degree of conformity of the data across platforms increased except for the domain tactile feedback , which increases in disparity . fig . 5 a c subjective assessment of surgical platform across eight domains stratified by platform type for msi , pgy , and staff study participants . note that likert scores ranged from 0 to 10 where 0 was the worst possible score , and 10 was the best ( * p < 0.05 ) a c subjective assessment of surgical platform across eight domains stratified by platform type for msi , pgy , and staff study participants . note that likert scores ranged from 0 to 10 where 0 was the worst possible score , and 10 was the best ( * p < 0.05 ) as technology continues to be refined in the setting of advanced mis platforms , the previously daunting task of introducing laparoscopic approaches into established practices is becoming increasingly facile . the question of whether increasing surgical experience can facilitate this process while foregoing formal laparoscopic training has so far been unanswered . additionally , although a single surgical robotic platform dominates the market at this point , head - to - head comparisons of existing technologies across surgical skill levels has not been examined [ 1012 ] . we show in this study that not all robotic platforms are created equal in their impact on surgical performance and surgeon preference . the davinci robotic assistant outperformed both zeus and conventional laparoscopy in all task times and error rates as well as proving subjective superiority . the increased degrees of freedom , enhanced visualization provided by binocular laparoscopic vision , smooth motion scaling and translation , as well as ergonomically superior controls combined to provide a stable , consistently efficacious platform for task performance regardless of skill level . the zeus robotic platform scored subjectively and objectively worse in all areas than the davinci platform or laparoscopy . with increasing surgical experience , however , the negative effects associated with zeus assistance were reduced . conventional laparoscopy fell somewhere in between these results with most participants yielding better performance than with the zeus platform and inferior performance than with the davinci platform in all tasks . the finding that msi participants performed worse in zeus trials versus conventional laparoscopy is interpreted as a result of removing two degrees of freedom , a lack of tactile feedback , and 2d visualization with inexperienced surgeons . additionally , the zeus operating platform provides restrictions on arm movement in terms of radii that are traversable at each joint , a generally unrefined human - machine interface , and poor visualization . the combination of these factors seems to impact the operator more if the subject lacks surgical experience , as it represents additional complexities to be overcome in completing the task at hand . operator compensation can occur for deficits of platform functionality as seen in correlating pgy and staff trials , but this appears to be a learned behavior . machine interface is a crucial component of any new surgical technology , as the inability to realize the full potential of the technology at hand will result in frustration and poor adoption . all subjective scoring in the msi group demonstrated davinci superiority across domains except in tremor reduction and coordination , although a trend towards significance was found . the lack of surgical experience may have led to the finding of non - significance in the domain of tactile feedback in the msi group who may not yet appreciate the subtleties of surgery . the pgy group demonstrated significant differences across all subjective domains in favor of the davinci platform , except in tactile feedback in which laparoscopy was superior . this was the predicted result of senior pgy trainees who were operating on a daily , continuous basis with some sensitivity to the tools of the trade . finally , experienced staff surgeons were indifferent as to platform preference except in demonstrating a preference for laparoscopy in the domain of tactile feedback . again , this is the expected result as experienced surgeons likely had the technical knowledge to surpass deficiencies of platform dependence noting only the most obvious of inequalities . historically , surgeons have relied on the sense of touch to provide invaluable direction during surgery ; however , current robotic systems do not yet have integrated haptics to provide feedback to the surgeon at the console . while this deficiency is observed in the results of the subjective scoring in this study , it is not borne out in the objective results of task times or errors . this is likely because the in vitro model is too simplistic in nature to mimic the tissue planes , tensile fragilities , and their complex interactions found in vivo . even without the ability to provide force - feedback , the davinci robot excelled at suturing and knot tying without increased error rates secondary to tearing of the latex membrane . the effect of surgical experience was also examined in this study and demonstrated globally improved performance with increasing experience . if the gold standard is considered the staff experience with each platform , then we can see that the graduation of surgical experience from msi to pgy to staff decreases the learning curve of the skill , whereas the platform type dictates the overall difficulty level of the task . analysis of performance trends across repeated trials again demonstrated quicker skills acquisition and earlier plateau with increasing surgical experience . the most inexperienced participants produced more random results with respect to skills adaptation , an indicator of the steep learning curve , confirmed by the correspondingly prolonged task times . our study suggests that increasing surgical experience can negate the significant effects of platform advantage , which explains why highly proficient surgeons whether in open , laparoscopic or robotic arenas may report equivalent results of similar surgeries with different approaches . our study also implies that experienced laparoscopic surgeons can recruit the davinci platform with a short learning curve and utilize it to facilitate more complex procedures not otherwise feasible through pure laparoscopy . even with procedures currently performed through pure laparoscopic means , we support the possibility that in experienced hands , davinci robotic assistance can facilitate or improve on these operations as well . in terms of surgical education , this study demonstrates the significant benefits of davinci robotic assistance in task performance across trainee skill levels . it also highlights the fact that while performance is enhanced , there is a narrow range in which the most technologically advanced platform is superior to conventional laparoscopy . while some might argue that with increasing task complexity , one would see a concomitant increase in the difference between these two systems , in all likelihood there remains a baseline performance function for each platform , for which surgical experience alone may make up the difference . a search of the current literature regarding surgical robotics elicits numerous studies comparing laparoscopy to one or another robotic platform , but few which compare all three directly in terms of task performance [ 1315 ] . as well , results from these studies are at odds with each other with similar platform comparisons . this is likely a function of the fact that the zeus surgical system has undergone several revisions since its introduction , and as a result , its performance has likely been improved . however , as mentioned previously , this model may be overly simplistic to account for the multifactorial environment of operating in vivo . as well , subjective scoring was carried out using an in - house developed survey , which was constructed in a standard manner , however , not validated on a separate dataset . our group in the canadian surgical technology and advanced robotics ( cstar ) facility at our center is currently constructing a validated subjective tool to assess platform performance . additionally , the results of this study only imply that robots may benefit the experienced surgeon in practice who is laparoscopically nave . as senior residents were used as the comparator to trained mis surgeons , this remains a limitation of this study . in summary , we confirm the results of previous literature documenting the inferior performance of zeus robotic surgical system versus laparoscopy as well as demonstrate that the davinci surgical platform is superior to conventional laparoscopy for both task performance and usability . in fact , in finding the zeus platform inferior to conventional laparoscopy , this reinforces the notion that as new technologies are introduced into the surgical armamentarium , each must be carefully and comprehensively evaluated in the provision of benefit over established means .

laparoscopy has found a role in standard urologic practice , and with training programs continuing to increase emphasis on its use , the division between skill sets of established non - laparoscopic urologic practitioners and urology trainees continues to widen . at the other end of the spectrum , as technology progresses apace , advanced laparoscopists continue to question the role of surgical robotics in urologic practice , citing a lack of significant advantage to this modality over conventional laparoscopy . we seek to compare two robotic systems ( zeus and davinci ) versus conventional laparoscopy in surgical training modules in the drylab environment in the context of varying levels of surgical expertise . a total of 12 volunteers were recruited to the study : four staff , four postgraduate trainees , and four medical student interns . each volunteer performed repeated time trials of standardized tasks consisting of suturing and knot tying using each of the three platforms : davinci , zeus and conventional laparoscopy . task times and numbers of errors were recorded for each task . following each platform trial , a standardized subjective ten - point likert score questionnaire was distributed to the volunteer regarding various operating parameters experienced including : visualization , fluidity , efficacy , precision , dexterity , tremor , tactile feedback , and coordination . task translation from laparoscopy to zeus robotics appeared to be difficult as both suture times and knot - tying times increased in pairwise comparisons across skill levels .

Introduction Methods Results Discussion

PMC4058214

ulcers are an open sore of the skin or mucus membrane characterized by sloughing of inflamed dead tissue . ulcers are lesions on the surface of the skin or a mucous membrane characterized by a superficial loss of tissue . ulcers are most common on the skin of the lower extremities and in the gastrointestinal tract , although they may be encountered at almost any site . there are many types of ulcer such as mouth ulcer , esophagus ulcer , peptic ulcer , and genital ulcer . of these peptic ulcer . the two most common types of peptic ulcer are called gastric ulcer and duodenal ulcer . gastric ulcers are located in the stomach , characterized by pain ; ulcers are common in older age group . although patients with gastric ulcers have normal or diminished acid production , yet ulcers may occur even in complete absence of acid . duodenal ulcers are found at the beginning of small intestine and are characterized by severe pain with burning sensation in upper abdomen that awakens patients from sleep . a duodenal ulcer is more common in younger individuals and predominantly affects males . in the duodenum , peptic ulcer can be life threatening with symptoms like bloody stool , severe abdominal pain , and cramps along with vomiting blood . the pathophysiology of peptic ulcer disease involves an imbalance between offensive ( acid , pepsin , and helicobacter pylori ) and defensive factors ( mucin , prostaglandin , bicarbonate , nitric oxide , and growth factors ) . peptic ulcers are once believed to be caused by spicy food and stress ; these have been found merely to be aggravating factors and the real causes have been found by research to include bacterial infection ( helicobacter pylori ) or reaction to various medications , particularly nsaids ( nonsteroidal anti - inflammatory drugs ) . helicobacter pylori , nsaids drugs , emotional stress , alcohol abuse , and smoking are the principal etiological factors associated with peptic ulcer . the gram - negative bacterium helicobacter pylori remains present between the mucous layer and the gastric epithelium and is strategically designed to live within the aggressive environment of the stomach . initially , helicobacter pylori resides in the antrum but over time migrates toward the more proximal segments of the stomach . peptic ulcer is one of the world 's major gastrointestinal disorders and affecting 10% of the world population . annual incidence estimates of peptic ulcer hemorrhage and perforation were 19.457 and 3.814 per 100,000 individuals , respectively . the average 7-day recurrence of hemorrhage was 13.9% and the average long - term recurrence of perforation was 12.2% . in the indian pharmaceutical industry , antacids and antiulcer drugs share 6.2 billion rupees and occupy 4.3% of the market share . in this modern era also 7580% of the world populations still use herbal medicine mainly in developing countries , for primary health care because of better cultural acceptability , better compatibility with the human body , and lesser side effects . preliminary photochemical screening of this medicinal plant identified the presence of important secondary metabolites like flavonoids and tannins which are the active principles of antiulcer activity . present study was conducted to review medicinal plants considered as gastroprotective and healing agents on ulcers in ayurvedic resources and beside that to gather evidence for their effectiveness and biological mechanisms in modern investigation . in order to achieve this aim , indian ayurvedic book meteria medica and electronic databases including science direct , pubmed , scopus , and google scholar were explored for each of the medicinal plants for peptic ulcers and all retrieved articles were evaluated to achieve any in vitro , in vivo , or clinical evidence for their efficacy and possible mechanisms . the retrieved studies either demonstrate obviously effectiveness of these herbs or indirectly their efficacy on the involved mechanisms in the treatment of peptic ulcers . meteria medica provides lots of information about ethno medicinal herbs , which are valuable as antiulcer agents and their use experimentally was evaluated and proved by many researchers for its antiulcer activity . following compiled data acacia arabica ( family mimosaceae ) , is common all over india in dry and sandy localities . it is commonly known as babul tree and locally called as karuvelam . chemical constituents reported in this plant are gum containing arabic acid combined with calcium , magnesium , and potassium and also small quantity of malic acid , sugar , moisture 14% , and ash 3 - 4% . bark contains a large quantity of tannin ; pods contain about 22.44% tannin . in ayurvedic . bruised tender leaves formed into a poultice and applied to ulcers act as stimulant and astringent . in recent studies . aqueous extract of a. arabica gum showed protection against meloxicam - induced intestinal damage and attenuated intestinal enzymes activity . adansonia digitata belonging to the family malvaceae is commonly known as boabab or monkey - bread tree of africa . it is one of the largest and long - lived trees in the world , met with chiefly in bombay , gujarat , and coromandal coast and ceylon . chemical constituents in this plant are pulp that contains phobaphenes , mucilage and gum , glucose , tartrate and acetate of potash , and other salts . bark contains wax , soluble and insoluble tannin , acid gum , albuminous carbonate and chloride of sodium and potassium , and a glucoside adansonin . in ayurvedic . fresh juice of the leaves mixed with powdered ginger together with the expressed juice of the fresh root of salvadora indica is applied with considerable benefit to indolent syphilitic ulcer . aegle marmelos which is commonly known as a bael tree belonging to the family rutaceae is the plant that chiefly grows on throughout india . chemical constituents in this plant are flavonoids , tannins , and saponins . in folk medicine . the fruit of a. marmelos is traditionally used for the treatment of ulcer among the kani tribes in kanyakumari district , tamil nadu , india . in recent studies . ulcers are induced by aspirin plus pylorus ligated gastric ulceration in rats and aqueous extract of leaves is to be administered orally for 21 days , daily dose of 1 gm / kg . the result indicated a significant reduction in the ulcer lesion count compared to control . allium sativum belonging to the family liliaceae is commonly known as garlic and locally called as vellapundu . chemical constituents in this plant arean acrid volatile oil which is the active principle , starch , mucilage , albumen , and sugar . the juice , more particularly its oil constituents , is rich in organically bound sulphur , iodine , and salicylic acid combinations , apart from important nutrient and complementary substances containing vitamins . in ayurvedic . mustard or coconut oil in which garlic has been fried is an excellent application for maggots infesting ulcers , ulcerated surfaces , and wounds . garlic juice mixed with 3 or 4 parts of ordinary or distilled water has been used as a lotion for washing wounds and foul ulcers . in recent studies . the extract of a. sativum bulb juice was administered at the doses of 250 and 500 mg / kg orally in rats , against cysteamine induced gastric ulcer . the extract significantly increases healing of gastric ulcer and prevents the development of experimentally induced gastric and duodenal ulcers in rats . active constituents . aloe vera belonging to the family liliaceae is commonly known as aloe gel . chemical constituents in this plant are aloin , isobarbaloin , and emodin . in ayurvedic . first the pain diminishes and after a few weeks the ulcers heal . in recent studies . aloe vera powder was mixed with gum acacia ; the solution was administered orally in rats at dose of 200 it is cultivated in gardens all over india which is locally called as sitapalam . chemical constituents in this plant are alkaloids , flavonoids , saponins , and tannins . seeds yield oil and resin ; seeds , leaves , and immature fruit contain an acrid principle . in ayurvedic . leaves made into a paste without adding water are applied to unhealthy ulcers . in recent studies . the aqueous leaf extract protected against pylorus ligation and ethanol induced gastric ulcer in rats . active constituents . azadirachta indica ( family meliaceae ) is indigenous to and cultivated nearly all over india and in bengal . chemical constituents reported in this plant are nimbidin , phenolic compounds , saponin , and flavonoids . the volatile fatty acids probably consist of a mixture of stearic and oleic acids with a small amount of lauric acid . in ayurvedic . a poultice of leaves mixed with sesamum seeds is very useful in unhealthy ulcerations . in recent studies . azadirachta indica leaf extract protected against pylorus ligation and cold restraint stress induced gastric ulcer in rats . active constituents . stearic and palmitic acid isolated from the nimbidin fraction of neem seeds oil is considered . it is grown on the sind , rajputana , eastern bengal , berars , assam , khandesh , and mysore which is locally called gukkulu . chemical constituents in this plant are volatile oil , gum - resin , and bitter principles . in ayurvedic . guggul gum is mixed with lime juice or coconut oil ; it is applied as a plaster or in the form of a lotion in indolent ulcers . gum obtained from other species , b. pubescens found in sind , karachi , and baluchistan , is used as ointment in bad ulcers such as delhi sores , combined with sulphur , catechu , and borax . bauhinia variegate ( family caesalpiniaceae ) is indigenous to and grow on the sub - himalayan tract and the forests of india and burma . it is commonly known as orchid tree and locally called shemmandarai . chemical constituents reported in this plant are quercetin , rutin , apigenin , and apigenin 7 - 0-glucoside . bark contains tannin ( tannic acid ) , glucose , and a brownish gum . in ayurvedic . a preparation known as kanchanara guggula made of the following ingredients is useful in ulcers : take the bark of bauhinia variegate ( 10 parts ) , 3 myrobalans , ginger , black - pepper , long - pepper , bark of crataeva nurvala , cardamoms , cinnamon , and tejpatra leaves , each one part . powder them all and add guggula ( 15 parts ) to make a pill mass . this is given in doses of half a tola every morning with a decoction of sphaeranthus mollis or of triphala or of catechu . the ethanolic and aqueous extract of root of b. variegate was administered at the doses of 200 and 400 mg / kg orally , in rats against pylorus ligation , ethanol , and aspirin induced gastric ulcer . the extract significantly inhibited gastric mucosal damage and reduced the basal gastric acid secretion . berberis aristata ( family berberidaceae ) is grown on the nilgiris and all over the temperate himalayas , from bhutan to kunawer . chemical constituents reported in this plant are roots and wood which are rich in a yellow alkaloid berberine bitter substance , which dissolves in acids and forms salts of the alkaloid ; root contains two more alkaloids . in ayurvedic . crude extracts known as rasaut ( in hindi ) are prepared from the root ; bark mixed with honey is useful application to ulcerations of the skin . it is native of the sea - coasts of tree mediterranean , now extensively cultivated in europe and america , and is known as sugar - beet . it is also cultivated in gardens in many parts of india for the sake of its flesh roots and leaves . chemical constituents in this plant are an active principle betin . in ayurvedic . a decoction of the root with a little vinegar added is excellent for all kinds of ulcers and running sores . leaves made into a pulp and used as poultice 3 to 4 times a day rapidly heal obstinate ulcers . in recent studies . the ethanol stem bark extract of c. arborea was administered at the doses of 300 and 600 mg / kg orally in rats for 5 days against ethanol , cold restraint stress , and pylorus ligation induced ulcer models . the extract significantly increases healing of gastric ulcer as compared to control . chemical constituents in this plant are papain , chymopapain , pectin , carposide , carpaine , carotenoids , and antheraxanthin . in folk medicine the unripe fruit can be cooked as parts of salads , jellies , and stews while the ripe fruits are usually eaten raw without the skin or seed . intake of the unripe fruit of the plant has been linked with an antiulcer effect . in recent studies . the aqueous seed extract of c. papaya was administered at the doses of 50 and 100 mg / kg orally , in rats against ethanol induced gastric ulcer . the extract protected the gastric mucosa against ethanol effect . c. papaya extract significantly reduced the gastric juice volume and gastric acidity . active constituents . chymopapain and papain are widely known as being useful for digestive disorders and disturbances of the gastrointestinal tract . chemical constituents in this plant are euphorbon , resin , gum , caoutchouc , malate of calcium , and so forth . in ayurvedic . plant juice is largely used with clarified or fresh butter as an application to unhealthy ulcers and scabies . this sacred peepul is a large tree round wild and cultivated all over india by the hindus . chemical constituents in this plant are bark containing tannin , caoutchouc ( cochtone ) , and wax . in ayurvedic . bark is useful in ulcers in infusion or decoction ( simple kashayam ) with a little honey . in recent studies . the hydro alcoholic extract leaves of f. religiosa were studied at two dose levels ( 250 and 500 mg / kg , oral ) in rats against absolute ethanol , aspirin , and pylorus ligation induced gastric ulcer . chemical constituents in this plant are yields gum , a trace of albumen and colouring matter , ash containing a trace of manganese , brown resin and chlorophyll and a principle allied to quercetin or querritrin , and glucoside rutin . in ayurvedic . it is native to china and grown widely as an ornamental plant through india . chemical constituents in this plant are flavonoids , anthocyanins , quercetin , cyanidin , kaempferol , and hydrocitric acid . in folk medicine . the root of h. rosa sinensis is traditionally used for the treatment of ulcer among the kani tribes in kanyakumari district , tamil nadu , india . in recent studies . the aqueous and alcohol extracts of h. rosa sinensis roots possessed significant antiulcer activity in pylorus ligated rats at the doses of 250 and 500 mg / kg . thus , it has been scientifically proven that these extracts possess enough potential as an antiulcerogenic agent . this small weed is common all over india , growing plentifully in moist localities . chemical constituents in this plant are an oleaginous white crystalline substance vellarin which is the active principle of the leaves , resins and some fatty aromatic body , gum , sugar , tannin , albuminous matter , and salts , mostly alkaline sulphates . in ayurvedic . for ulcerations , the powder , in 3 to 5 grain doses , may be given thrice daily ; at the same time some of the powder may be sprinkled on the ulcers or preferably poultices of the fresh leaves may be applied . this small erect shrub is cultivated extensively in northern india , especially in bengal , bihar , orissa , sind , oudh , southern india , madras , and bombay . chemical constituents in this plant are indican ( a glucoside ) , the oxidized form of luc - indigo , or indigo - white , what is produced from the fermentation of the fresh green plant . in ayurvedic . leaves crushed are used as stimulant poultice or plaster in various skin affections and to cleanse and to heal wounds and ulcers . it is common all over india , cultivated chiefly as a hedge and garden plant . chemical constituents in this plant are leaves that yield a colouring matter ( henna dye ) 12 to 15% hanno , tannic acid , a kind of tannin , and an olive green resin soluble in ether and alcohol . chemical constituents in this plant are alkaloids , sterols , saponins , tannins , and flavonoids . in ayurvedic . leaf extracts were dissolved in rice bran oil and given orally for ulcer . traditionally the plant is reported to have antiulcer activity . in recent studies . the flower decoction was administered in the doses of 250 , 500 , and 1000 mg / kg orally , in rats with gastric lesions in dose - dependent manner . chemical constituents in this plant are flavonoids , quercitin , naringin , saponins , tannins , gums , and mucilage . in ayurvedic . decoction of the fresh leaves and seeds are consumed for intestinal ulcer . in recent studies . ethanolic extract of the leaves of mimosa pudica have been reported to possess antiulcer activity in a dose - dependent manner and these leaf extracts may be useful as a natural antioxidant in treatment of ulcer . active constituents . chemical constituents in this plant are bitter glucoside soluble in water and insoluble in ether , a yellow acid , resin , and ash 6% . fresh vegetable contains 88.75% moisture , albuminoids 1.62% , soluble carbohydrates 85.41% , woody fiber 1.51% , and ash 8.53% . in ayurvedic . whole plant powdered is used for dusting over leprous and other intractable ulcers and in healing wounds ; when mixed with cinnamon , long pepper , rice , and chaulmugra oil it forms a good ointment in malignant ulcers . in recent studies . alcoholic and aqueous extract of m. charantia fruit at the doses of 200 and 400 mg / kg separately are used against pylorus ligation , aspirin , and stress induced ulcer in rats . moringa oleifera ( moringaceae ) is commonly known as drum - stick , horse radish tree . it is native to the western and sub - himalayan region , india , pakistan , asia minor , africa , and arabia . chemical constituents in this plant are alkaloids , flavonoids , saponin , tannins , zeatin , quercetin , kaempferom , and terpenoids . in folk medicine . the medicinal value of the different parts of the plant has long been recognized in folklore medicine . the leaf tea treats gastric ulcers by kani tribals of pechiparai hills , tamil nadu , india . flower buds of m. oleifera are widely consumed in pakistan and have been reported to possess antiulcer activity . in recent studies . the alcoholic leaves extract of m. oleifera was administered in the doses of 125 , 250 , and 500 mg / kg orally , in rats against pylorus ligation , ethanol , cold restraint stress , and aspirin induced gastric ulcer . myrica nagi ( myricaceae ) is commonly known as box myrtle ; bay - berry . it is an evergreen plant of the subtropical himalayas , simla district , syihet , and southwards to singapore and found also in the khasia mountains and the hills of burma . chemical constituents in this plant are bark that contains tannin , saccharine matter , and salts . the ground bark yields a colouring principle named myricotin . in ayurvedic . a poultice made by bruising the bark and simmering it in water and stirring in indian meal till it obtains the proper consistence cures scrofulous ulcers ( tukina ) . fruits when boiled yield a kind of wax called myrtle wax which is used as a healing application to ulcers . chemical constituents in this plant are ripe berries that contain an essential volatile oil ( oil of myrtle ) , resin , tannin , citric acid , malic acid , and sugar . . in ayurvedic the fruit , myrtle berry , is carminative and given in the form of infusion for internal ulcerations . in recent studies . a topical formulation of m. communis in low doses demonstrated wound healing activity in rat excision wounds . m. communis fruits protected against gastric ulcer caused by ethanol , indomethacin , and pylorus ligation in rats via suppressing gastric secretion and acidity and enhancing its mucosal barrier . active constituents . chemical constituents in this plant are alkaloids , tannins , saponins , flavonoids , and sterols . in ayurvedic . a tea prepared with the leaves of tulsi is commonly used for intestinal disorders . in recent studies . the fixed oil of o. sanctum was administered in the doses of 1 , 2 , and 3 ml / kg intraperitoneally , in the rats in which ulcer is induced by aspirin , indomethacin , alcohol , and stress - induced ulceration . it is cultivated generally in hotter parts of india.chemical constituents in this plant are barks that contain tannin and ash that contains considerable quantity of potassium carbonate . in ayurvedic . this is a principal food crop of india , ceylon , burma , china , japan , and siam and is spread over the tropical and subtropical regions of both hemispheres . chemical constituents in this plant are rice that contains more starch than any other starchy grains , but no appreciable fat , a very small quantity of proteins , and a trace of mineral matter . in ayurvedic . where there is an irritable or inflammatory state of the stomach , rice gruel or conjee water , as it is commonly called , ( decoction 1 in 40 ) or thicker liquid made by boiling the rice powder in water , with a pinch of salt and a squeeze of lemon , makes a good drink and without the lime - juice and salt in gastric ulcer . schnabel in american journal of medical science reports good results from the use of rice - water mixture in the treatment of gastric and duodenal ulcers . in recent studies . the extract of o. sativa bran ( rice bran oil ) was administered at the dose of 1 ml / day for 4 days against swimming stress induced and pylorus ligation induced ulcer in rats . chemical constituents in this plant are fruits that contain an essential oil of a light yellow colour . in ayurvedic . infusion ( 1 in 10 ) of fruit is used in doses of 12 to 1 ounce like that of fennel seeds as carminative , gastric and intestinal disorders , and so forth , . phyllanthus niruri ( euphorbiaceae ) is commonly known as stonebreaker or seed - under - leaf . chemical constituents in this plant are alkaloids , saponins , tannins , flavonoids , carbohydrates , and glycosides . in ayurvedic . whole plant pounded with its root and combined with rice water is used as poultice for ulcers . in recent studies . the metanolic aerial part extract of p. niruri was administered at the dose of 400 mg / kg orally in rats and significantly inhibited the development of ulcer induced by indomethacin . active constituents . alkaloids-4-methoxy - securinine , ellagic acid , beta sitosterol , gallic acid , and hypophyllanthin are considered . pinus longifolia ( coniferae ) is commonly known as long - leaved pine . it is common on the slopes of the himalayas , north western frontier province from afghanistan to kashmir , the punjab , u.p . to bhutan , assam , and upper and lower burma . chemical constituents : its sapwood yields on incision an oleoresin from which turpentine is obtained which contains 20% volatile oil of turpentine called pinene with a small quantity of limonene and about 80% of residue which is very largely used under the name of calophony or resin . in ayurvedic . wood is useful to cool the burning sensation of the body and as an application in ulcerations . it is the source of the resin usually employed as a stimulating application for ulcers . this persian herb is found also in north - west india , the punjab , and sind and cultivated to a small extent in bengal , mysore , and coromandel coast . the genus plantago comprises about 50 species , of which ten are natives of india . chemical constituents in this plant are mucilage , fixed fatty oil , and albuminous matter , in large quantities . in ayurvedic . the decoction in doses of 2 to 3 drachms , plain or mixed with sugar , is very beneficial in gastritis , gastric , and duodenal ulcers . chemical constituents in this plant are bark that contains tannin 27.4% , resin , and crystals of calcium oxalate . leaves contain resin , fat , cellulose , tannin , volatile oil , chlorophyll , and mineral salts . in ayurvedic . locally , decoction of the leaves is employed in unhealthy ulcers and is an efficacious gargle for swollen gums and ulceration of the mouth . in recent studies . the methanol leaf extract of p. guyava was administered at the doses of 500 and 1000 mg / kg orally , in rats for 10 days against ethanol induced gastric ulcer . chemical constituents in this plant are ellagic acid , gallic acid , isoquercitrin , myricitrin , and tannic acid . in ayurvedic . it is generally used in the form of powder or extract ; dose of the powder is 20 to 30 grains . locally the paste mixed with charcoal powder is applied to unhealthy ulcers . in recent studies . the hydro alcoholic extract of r. coriaria was administered at the doses of 145 and 248 mg / kg orally in rats against ethanol induced gastric ulcer . it is an ornamental plant and is found in the plains of western himalayas to sri lanka . chemical constituents in this plant are saponins , tannins , and triterpenes . in folk medicine . sesbania grandiflora leaves prepared in the form of soup and taken orally by the valaiyan tribe of alagarkoil hills , madurai district , tamil nadu , india , are used as vermifuge and against peptic ulcer . 50 ml of leaf decoction is taken orally on an empty stomach as vermifuge and against stomach ailments by tribal and rural people of sirumalai hills , dindigul district , tamil nadu , india . leaves are boiled in cow milk and orally taken in kikuku village , muleba district , tanzania , for treatment of peptic ulcers . the boiled leaves are taken orally for ulcer by paliyar tribals in dindigul district of tamil nadu , india . in recent studies . the ethanol leaf extract of s. grandiflora was administered at the dose of 400 mg / kg orally , in rats against aspirin , ethanol , and indomethacin induced gastric ulcer . the extract significantly inhibited gastric mucosal damage and reduced the basal gastric acid secretion . active constituents . tannins and saponins it is common in the sub - himalayan regions and the forests of western bengal . chemical constituents in this plant are ursolic acid , tri and tetrehydroxy ursenoic acid , asiatic acid alpha and beta amyrin , and mangiferonic acid uvaol . in ayurvedic . take s. robusta : 5 , cinnabar : 2 , mastiche : 3 , calamus draco : 3 , and ghee ( 10 parts ) . mix and make an ointment ; it is used for foetid ulcers . in recent studies . the extract of s. robusta was administered at the doses of 150 and 300 mg / kg orally in rats against ethanol and pylorus ligation induced gastric ulcer . the extract significantly increases the gastroprotective activity as compared to control . active constituents . chemical constituents in this plant are alkaloids , saponins , flavonoids , and phytosterols . in folk medicine . the fresh leaves are consumed for intestinal ulcer by paliyar tribals in dindugal district , tamil nadu , india . in recent studies . aqueous leaf extract of solanum nigrum protected against pylorus ligation induced gastric ulcers in rats . active constituents . this evergreen tree which is indigenous to south india is cultivated throughout india and burma . chemical constituents in this plant are pulp that contains tartaric acid 5% , citric acid 4% , malic and acetic acids , tartaric of potassium 8% , invert sugar 2540% , gum , and pectin . seeds contain albuminoids , fat , carbohydrates 63.22% , fibre , and ash containing phosphorus and nitrogen . decoction of the leaves is used as a wash for indolent ulcers and promotes healthy action . in recent studies . the methanolic extract of the seed coat of t. indica at doses of 100 and 200 mg / kg significantly reduces the total volume of gastric juice and free and total acidity of gastric secretion in pylorus ligation induced ulcer model as compared to control . active constituents . terminalia chebula ( combretaceae ) is commonly known as myrobalan ; ink - nut ; gullnut . this tree is wild in the forests of northern india , central provinces , and bengal and common in madras , mysore , and in the southern parts of the bombay presidency . chemical constituents in this plant are tannin ( tannic acid ) 45% and a large amount of gallic acid , lucilage , a brownish yellow colouring matter , and chebulinic acid which when heated in water splits up into tannic and gallic acids . in ayurvedic . ashes of triphala mixed with sindhu salt ( saindhava , that is , potassium nitras or nitricum ) are dusted over syphilitic ulcers for washing away the exudation from the ulcers . equal parts of dried myrobalans in combination with emblic and beleric myrobalans and catechu , both finely powdered and rubbed into a thick paste with sufficient ghee or some bland oil , make an excellent ointment as an application to aphthae for chronic ulcerations and ulcerated wounds . in recent studies . methanolic extract of t. chebula was administered in the doses of 250 and 500 mg / kg orally . the extract showed significant reduction in gastric volume , free acidity , and ulcer index as compared to control . some of the herbal drugs have been chemically characterized and the entities involved in the activity have been isolated . these are summarized in table 1 . from this study we can conclude that studies with plant sources can result in novel and effective pattern of treatment . current stalemates of modern medicine in the management of various ailments incline research tendencies to traditional medicine . in this respect , all of the remedies presented here had adequate evidence from traditional or scientific source for their efficacy in management of ulcers . according to the old hypothesis , acid secretion was thought to be the sole cause of ulcer formation and reduction in acid secretion was thought to be the major approach towards therapy . now treatment of ulcer mainly targets the potentiation of the defensive system along with lowering of acid secretion . chemical substances derived from plants have been used to treat human diseases since the dawn of medicine . roughly 50% of new chemical entities introduced during the past two decades are from natural products . , efforts should be directed towards isolation and characterization of the active principles and elucidation of the relationship between structure and activity . there are various medicinal plants and their extracts ( containing active chemical constituents , e.g. , tannins and flavonoids ) that have significant antiulcer activity in in vivo experiments on animal models . furthermore , detailed analysis of the active constituents of natural drugs should be directed towards clinical relevance . although the clinical efficacy of these preparations is reported by traditional practices , they have not been scientifically validated . ayurveda , the oldest medicinal system in the world , provides leads to find therapeutically useful compounds from plants . therefore , ayurvedic knowledge supported by modern science is necessary to isolate , characterize , and standardize the active constituents from herbal sources for antiulcer activity . the combination of traditional and modern knowledge can produce better drugs for the treatment of peptic ulcer with fewer side effects . it is apparent that experimental evaluation of herbal drugs for the treatment of gastric ulcer is rather impressive but very few have reached clinical trials and still few have been marketed . this shows that the benefits of research are not reaching the people to whom medical research is directed and hence the time , manpower , and resources are not efficiently utilized . hence , pharmacologists need to take more active interest in evaluation of herbal drugs for potential antiulcer activity and standardization of such herbal drugs to be clinically effective and globally competitive .

ulcer is a common gastrointestinal disorder which is seen among many people . it is basically an inflamed break in the skin or the mucus membrane lining the alimentary tract . ulceration occurs when there is a disturbance of the normal equilibrium caused by either enhanced aggression or diminished mucosal resistance . it may be due to the regular usage of drugs , irregular food habits , stress , and so forth . peptic ulcers are a broad term that includes ulcers of digestive tract in the stomach or the duodenum . the formation of peptic ulcers depends on the presence of acid and peptic activity in gastric juice plus a breakdown in mucosal defenses . a number of synthetic drugs are available to treat ulcers . but these drugs are expensive and are likely to produce more side effects when compared to herbal medicines . the literature revealed that many medicinal plants and polyherbal formulations are used for the treatment of ulcer by various ayurvedic doctors and traditional medicinal practitioners . the ideal aims of treatment of peptic ulcer disease are to relieve pain , heal the ulcer , and delay ulcer recurrence . in this review attempts have been made to know about some medicinal plants which may be used in ayurvedic as well as modern science for the treatment or prevention of peptic ulcer .

1. Introduction 2. Findings and Discussion 3. Conclusion

PMC2665887

avian influenza virus type a , subtype h5n1 , is becoming the world s largest pandemic threat due to its high virulence and lethality in birds , quickly expanding host reservoir , and high rate of mutations.(1 ) antigenic drift has given rise to new strains that are resistant to existing drugs , and antigenic shift is resulting in new virulent subtypes of the flu virus , underscoring the need to design novel therapeutics . influenza s two major membrane glycoproteins , hemagglutinin ( ha ) and neuramindase ( na ) , together play important roles in the interactions with host cell surface receptors . na facilitates viral shedding by cleaving terminal sialic residues on host cell surface proteoglycans , which are bound by ha.(2 ) the neuraminidase enzymes are phylogenetically categorized into two groups : group-1 , which includes n1 , n4 , n5 , and n8 , and group-2 , which includes n2 , n3 , n6 , n7 , and n9.(3 ) although active - site residues are largely conserved across both groups , different na subtypes exhibit varied drug susceptibility(4 ) and resistance profiles . since the first na crystal structure was published in 1983,(7 ) a number of structure - based computational studies against the group-2 subtypes have added significant insight to our understanding of substrate recognition and inhibitor design . currently available na inhibitors , including oseltamivir(8 ) and zanamivir,(9 ) have been designed against crystal structures of group-2 enzymes ( ref ( 2 ) and references therein ) . oseltamivir , which has been stockpiled by many nations in efforts to avert a possible pandemic , is the only orally available drug effective against h5n1 ; yet , oseltamivir - resistant strains have already been isolated . recent studies of the avian - type n1 enzyme have enriched our understanding of the binding process . the first crystal structures of a group-1 na in apo form and in complex with currently available drugs(12 ) revealed that , although the binding pose of oseltamivir was similar to that seen in previous crystallographic complexes , the 150-loop adopted a distinct conformation , opening a new cavity adjacent to the active site . under certain crystallization conditions , however , the 150-loop adopted the same closed conformation as previously seen in group-2 na structures , suggesting a slow conformational change may occur upon inhibitor binding.(12 ) it was conjectured that the new structural observation of the n1 strain s open 150-loop could be exploited in structure - based drug discovery efforts . despite this detailed structural information , the interpretation of the loop dynamics based on crystal structures alone is a difficult task . to complement the crystallographic structures , all - atom explicit solvent molecular dynamics ( md ) simulations of the apo and oseltamivir - bound systems were carried out.(14 ) the extensive simulations suggested that the 150-loop and adjacent binding site loops may be even more flexible than observed in the crystal structures.(14 ) in the apo simulations , the 150-loop was seen to open more widely than observed in the crystal structures , and its motion was often coupled to an outward movement of the adjacent 430-loop . these coupled motions significantly expanded the active - site cavity , increasing its solvent - accessible surface area compared with both the open and closed crystal structures . subsequent computational solvent mapping ( cs - map ) experiments assessed the ability of small , solvent - sized molecules to bind within close proximity to the sialic acid binding region and the newly discovered 150- and 430-cavities.(15 ) the consensus binding sites ( i.e. , hot spots ) of these probes , as determined by the cs - map algorithm , have been shown to relate directly to experimental druggability and are thus able to identify important binding site features . mapping analyses of the nonredundant md conformations revealed the presence of additional hot spot regions in the 150- and 430-loop regions , confirming the druggability of these newly revealed sites . furthermore , small - molecule docking to the new 150- and 430-cavities indicated favorable binding of several compounds to these areas , in addition to the sialic acid binding site.(19 ) a complete understanding of the comparative human- and avian - type neuraminidase structural dynamics is lacking , despite the relevance of this knowledge to antiviral development . our current understanding of the active - site dynamics for these medically relevant enzymes is predominantly based on x - ray crystallography experiments(12 ) and the aforementioned explicitly solvated md simulations of the open 150-loop n1 enzyme . in this work , in order to assess the dynamical differences in the 150-loop , 430-loop , and sialic acid binding regions between the avian and human subtypes , we present a series of generalized born molecular dynamics ( gb md)(20 ) simulations of the n1 and n9 na enzymes . gb md , which employs a continuum representation of the solvent water molecules and salt ions , offers computational efficiency over the more rigorous poissonboltzmann solvers or explicit solvent simulations.(21 ) the implicit solvent approach essentially reduces solvent friction and has been shown to enhance conformational sampling for a wide variety of peptide , protein , and nucleic acid systems . to date , however , the application of gb md to larger biomolecular systems , such as the neuraminidase enzyme , has been much fewer in number than its application to smaller proteins and peptides . this work therefore presents an important methodological example that highlights the utility of employing generalized born implicit solvent models for elucidating the dynamics and energetics of large protein systems . although crystallographic experiments provide critical time / ensemble - averaged structure and ligand binding information , atomic - level simulations yield additional insights and relevant biophysical information that is otherwise inaccessible with standard experimental techniques . as oseltamivir does not rely on water - mediated interactions with the protein active site , unlike other sialic acid analogue na inhibitors,(26 ) the n1 and n9 oseltamivir - bound systems are particularly well suited for treatment with implicit solvent . the comparative dynamics that we present here allow us to gain insights into the flexibility of the 150- and 430-loops , which are important due to their proximity to the sialic acid binding site . a more complete understanding of the dynamical differences between the subtypes , especially in the 150- and 430-loop regions , may assist in the design of antiviral compounds that are able to take advantage of the newly revealed ligand binding areas . here we show that the extensive conformational sampling gained through the gb md simulations allows us to attain a significantly enhanced conformational sampling speedup , as compared to explicit solvent md . the dynamics of residues that have been shown by computational solvent mapping to be potentially important in the binding of ligands to this expanded area are investigated and discussed , and a comparison of the resulting structures to the explicitly solvated md simulations is provided . the closed - loop oseltamivir - bound n1 simulation started from one of the monomers from the 2hu4 ( pdb code ) structure . the open - loop oseltamivir - bound n1 simulation started from one of the monomers from the 2hty structure . the closed - loop n9 oseltamivir - bound simulation was initiated from one of the monomers from the 2qwk structure . for each of the monomer systems , several residues on the n - terminus were removed ( first six residues in n1 , v83n88 ; first nine residues in n9 , r82g90 ) . coordinates of the three corresponding apo systems were obtained by removing the inhibitor oseltamivir , respectively . to study the impact of the adjacent subunits on loop structure and dynamics , a tetramer system comprised of the closed - loop n1 neuraminidase in complex with oseltamivir ( 2hu4 ) protonation states for histidines and other titratable groups were determined at ph 6.5 by the pdb2pqr web server and manually verified.(27 ) hydrogen atoms were added by using the leap module in amber . the ff99sb force field , which has modifications to backbone torsional terms , was used for the protein.(28 ) parameters for oseltamivir were obtained from antechamber module using the generalized amber force field ( gaff)(29 ) with resp hf 6 - 31 g * charges as described previously.(14 ) all md simulations were carried out with amber 8.(30 ) temperature was maintained by using langevin dynamics with a collision frequency of 5 ps . the time step was 2 fs , and all bonds involving hydrogen were constrained with the shake algorithm with a tolerance of 10 . seven simulations of the n1 ( group-1 , avian ) and n9 ( group-2 , human ) neuraminidases are summarized with their system name , initial pdb structure identifier , brief description , total simulation time , and number of atoms . the gb model has been demonstrated to provide improved conformational sampling as compared to simulations with explicit representation of solvent molecules . the improved sampling arises from an acceleration of transitions due to the lack of solvent viscosity , as well as through reduced cost of calculating forces of explicit solvent . a modified gb model ( gb , igb = 5 ) was employed for more accurate solvation of large proteins.(23 ) the born radii were adopted from bondi with modification ( mbondi2 ) and an offset of 0.09 . the salt concentration was set to 0.2 m. a reaction field cutoff ( rgbmax = 25 ) was used to speed - up the calculation of effective born radii . no cutoff was used for the long - range interactions for the monomer systems , while a cutoff of 50 was used for the tetramer system . the starting structure was minimized for 2000 steps to remove close contacts and then gradually heated to 300 k at 50 k intervals during six simulations of 200 ps . positional restraints on backbone atoms were gradually decreased from 3 to 0 kcal/(mol ) in several stages . each production run was 16 ns for the monomer systems and 5 ns for the tetramer system . system performance benchmarks are as follows : for the tetramer system , 0.8 ns per day using 1024 processors on the ibm bluegene / l platform at san diego supercomputer center ; for the monomer system , 9 ns per day on the ncsa abe platform using 256 processors ( 32 nodes ) , and 2 ns per day on the ibm bluegene / l machine using 256 processors . in order to generate reduced , representative structural ensembles for the simulations and for the cs - map calculations , root - mean - square deviation ( rmsd ) conformational clustering was performed , based on a previously reported clustering algorithm.(31 ) to be able to compare to previous clustering results , the gromos method within gromacs ( g_cluster ) was employed.(33 ) structures were extracted in 10 ps intervals over each of the simulations ; 1.6 10 trajectory structures for each simulation were superimposed using all c atoms to remove overall rotation and translation . the rmsd - clustering was performed on a subset of 62 residues that line the entire binding - site area , which we define here as the binding - site residues : 117119 , 133138 , 146152 , 156 , 179 , 180 , 196200 , 223228 , 243247 , 277 , 278 , 293 , 295 , 344347 , 368 , 401 , 402 , and 426441 . these residues were clustered into batches of similar configurations using the atom - positional rmsd of all atoms ( including side chains and hydrogen atoms ) as the similarity criterion . a cutoff of 1.3 was chosen on the basis of previous work using explicit solvent.(15 ) using equidistant snapshots extracted from the gb md trajectories , the total binding free energy was computed using the mm - gbsa scheme . for computational efficiency , a single trajectory approach was used and the calculations were performed on 800 snapshots with a sampling interval of 20 ps . where emm represents the sum of electrostatic , van der waals , and internal energies ( we note that the internal energies cancel out in eq 2 due to the single trajectory approach ) . gsolvation is the desolvation free energy penalty , estimated from gb and solvent - accessible surface area ( sasa ) calculations which yield gpolar and gnonpolar . a surface tension coefficient ( ) of 0.0072 kcal/(mol ) is used to calculate the nonpolar solvation free energy contribution . the modified gb model ( gb , igb = 2),(23 ) the modified born radii ( mbondi2 ) , and 0.2 m salt concentration were adopted , to be consistent with the gb md simulations . the best results ( i.e. , most closely reproduced experimental binding constants ) were obtained with igb2 . ts is the product of temperature ( at 300 k ) and solute entropy , derived from normal - mode analyses of the solute coordinates after energy minimization of solute structures to within a root - mean - square of the energy gradient of 1.0 kcal/(mol ) . the normal - mode analysis was carried out on eight snapshots at 2 ns sampling intervals due to its prohibitive computational cost on large proteinligand systems . the minimum ts results were selected to compute the total binding free energy in eq 1 . the seven simulations with various starting conditions allow us to investigate the comparative dynamics of the avian- and human - type neuraminidase enzymes in several states , including the apo and oseltamivir - bound n1 enzyme starting with the open 150-loop crystal structure ( n1ao , n1ho , respectively ) , the apo and oseltamivir - bound n1 enzyme starting with the closed 150-loop crystal structure ( n1ac , n1hc ) , the apo and oseltamivir - bound n9 crystal structures ( n9a , n9h ) , and the tetramer closed 150-loop oseltamivir - bound n1 ( table 1 ) . as the human - type n9 enzyme has never been crystallized with the 150-loop in the open conformation , the starting conditions are with a closed 150-loop . overall , the backbone rmsd establishes that all of the systems are stable during the trajectories ( supporting information , figure s1 ) . an rmsd - based clustering analysis of 62 residues lining the binding site allows us to compare the relative structural populations of n1 and n9 in the different trajectories . the clustering procedure was performed in a manner identical to that described by landon et al.(15 ) ( using a 1.3 cutoff ) and thus provides a basis of comparison for the explicit solvent and gb md simulation cluster structures ( table 2 ) . the central member structures of the most dominant clusters for each of the simulations illustrate the range of structural fluctuation in this loop region ( figure 1 ) . the clustering results indicate that the gb md simulations enhance the conformational sampling of the n1 enzyme , as compared to the explicit solvent simulations , while maintaining the relative population trends established in the latter . loop conformations are shown in cartoon representation for the most dominant configurations of ( a ) the n1-apo - closed ( orange ) and the n1-oseltamivir - bound - closed ( red ) , ( b ) the n1-apo - open ( orange ) and the n1-oseltamivir - bound - open ( red ) , and ( c ) the n9-apo - closed ( orange ) and the n9-oseltamivir - bound - closed ( red ) trajectories . the n1 open ( blue ) and n1 closed ( green ) crystal structures are shown for comparison . first , regardless of the open vs closed 150-loop configuration , the apo systems are more flexible in the binding - site region compared to the oseltamivir - bound systems . these results are consistent with the explicitly solvated simulations.(14 ) second , the relative cluster populations of the open 150-loop n1 systems indicate that the gb simulations are able to sample a larger conformational space than the explicitly solvent simulations , despite the fact that the gb simulations were 1 order of magnitude shorter in time scale . this is due to the reduced solvent friction in the gb md simulations and highlights the utility of the gb method for exploring receptor conformational space . in terms of the comparative dynamics between the n1 and n9 systems , both the n1 and n9 apo systems are represented by 55 clusters , indicating that the dynamics are similar between the subtypes in the absence of substrate . in contrast , the n1hc system has more clusters than the n9h system , suggesting that the n9 subtype is more stable than the n1 subtype when oseltamivir is bound . the dynamics of residues lining the sialic acid , 150- , and 430-cavities are of interest for drug design investigations as well as to better understand the fundamental basis of molecular recognition in the neuraminidase enzymes . in order to characterize the dynamics of residues within the n1 and n9 systems , a root - mean - square fluctuation ( rmsf ) analysis the dynamics of hot - spot residues ( table 3 ; supporting information , figure s3 ) are of particular interest , as they have been suggested to provide additional opportunities for drug discovery that could potentially mitigate the effects of drug resistance.(15 ) the first column lists the residue type , the second column lists the residue number , and the third through eighth columns list the rmsf per residue for the n1-apo - open , n1-apo - closed , n1-holo - open , n1-holo - closed , n9-apo , and n9-holo systems , respectively . most notably , with the exception of y406 , all the hot - spot residues lining the sialic acid cavity in n9 exhibit much lower rmsf values than the same residues in the n1 strain ( table 3 ) . this is also illustrated in the equilibrium binding poses of oseltamivir , which , in the n9 subtype , undergoes the least amount of movement from the original crystal structure pose versus the n1 subtypes ( figure 2 ) . the increased positional fluctuations of the sialic acid binding residues in the n1 subtype may contribute to the reduced efficacy of several known inhibitors against n1 . in addition , the n9h system shows significant stabilization of the 150-loop upon binding of oseltamivir ( table 3 ) . the increased rigidity in this area substantiates available crystallographic data that , to date , have not provided evidence of 150-loop mobility in the n9 subtype . overall orientations ( left panel ) and active - site interactions ( right panel ) of oseltamivir bound to ( a ) the n1-closed , ( b ) the n1-open , and ( c ) the n9-closed system . the original conformations of the 150- and 430-loops are shown in blue and red , respectively , to highlight the loop motion observed in the course of the dynamics . the presence of oseltamivir in the closed 150-loop n1 system actually increases the rmsf of the sialic acid and 150-loop hot - spot residues ( table 3 ) . this is in contrast to both the open 150-loop n1 and n9 systems , which exhibit reduced fluctuations in the 150-loop in the presence of oseltamivir . the stabilization of the 150-loop residues in the n1ho system is in agreement with the explicitly solvated md simulations.(14 ) taken together , these findings suggest relative structural instability of the n1 system in this region due to the presence of inhibitor as compared to n9 , lending support to the potential importance of targeting the 150- and 430-cavities for developing inhibitors that bind more strongly to the n1 subtype . at approximately 6 ns into the n1hc trajectory , a strained salt bridge forms between r152 and the carboxylate group of oseltamivir . is not seen in any of the other gb or explicit solvent simulations , it is likely that this salt bridge is an artifact of the continuum solvent approach . the formation of overstabilized salt bridges , especially those involving arginine residues , is a known weakness of the gb model.(38 ) in all other trajectories , r152 , which is within a stable region of the 150-loop , forms varying salt bridge interactions with neighboring aspartate residues in the protein . hot - spot residue rmsf values for n1hc simulation time segments before ( 05.2 ns ) and after ( 10.015.2 ns ) formation of the strained salt bridge indicate that the positional fluctuations of residues in the vicinity of r152 are slightly dampened after the salt bridge is formed ( data not shown ) . the trends in rmsf values for both time segments , however , are the same as the mean values reported in table 3 . the crystal structures of the n1 enzyme published by russell et al . first revealed the flexibility of the 150-loop.(12 ) adjacent to the sialic acid binding site , the outward movement of the 150-loop expanded the n1 active site and was suggested to present a new opportunity for antiviral drug design . explicitly solvated md simulations presented by amaro et al . sampled a single openclose loop transition in chain d of the oseltamivir bound system , as demonstrated by the instantaneous rmsd of the 150-loop from the closed ( 2hu4 ) and open ( 2hty ) 150-loop crystal structures during 40 ns of tetramer simulations ( figure 1 in ref ( 14 ) ) . in addition , the explicit md sampled a so - called wide - open 150-loop structure that significantly altered the size and topology of the n1 binding pocket . in this work , a similar analysis of the six monomer systems provides dynamical and structural insights into this region . for the open 150-loop n1 system , the apo and holo systems each demonstrate two openclose loop transition events ( figure 3 ) . in comparison to the explicit solvent md , which sampled only one transition event in the n1ho system over 160 ns of simulation , the four loop transition events sampled here point to the advantage of the gb method for sampling conformational transitions on a much faster time scale compared to explicit solvent md . similar to the explicit solvent md , the most dominant central member cluster structure of the n1 apo system is significantly more open than the open 150-loop crystal structure ( figure 1 ) . the general agreement between the resulting structures determined by both computational methods substantiates the idea that the n1 subtype generally exists in a more open conformation in the absence of ligand . by contrast , the closed 150-loop n1 systems remain relatively stable across the trajectories , and no loop transition events are sampled ( figure 3 ) . root - mean - square deviations of the 150-loop ( comprising residues n146r152 ) from the md with respect to the open ( gray ) and closed ( black ) crystal structures are shown for the n1-apo - closed ( a ) , the n1-oseltamivir - bound - closed ( b ) , the n1-apo - open ( c ) , the n1-oseltamivir - bound - open ( d ) , the n9-apo - closed ( e ) , and the n9-oseltamivir - bound - closed ( f ) structures . in the ( closed - loop ) apo n9 system , the openclose loop transition analysis indicates that the enzyme adopts an intermediate conformation ( figure 3 ) . this is also illustrated in the most dominant cluster representative structure , which indicates that the 150-loop in the apo n9 system takes on characteristics of both the closed and open loops but is definitively more open . our analysis indicates that the most populated structure in the n9a system has an open 150-cavity . this structural finding suggests that , in the absence of inhibitor , both subtypes take on a more relaxed and open conformation in the 150-loop region . in contrast , the holo n9 system remains in a more closed conformation throughout ( figures 1 and 3 ) . it is also worth noting that , in each of the most dominant structures extracted from all six monomer simulations , the 430-loop adopts a more open conformation ( figure 1 ) . these results reveal motion in the 430-loop region for the n9 enzyme and , more broadly , indicate that motion in the 430-loop region is independent of bound ligand . importantly , this suggests that compounds designed to target the more open 430-cavity have the potential to be viable against both n1 and n9 subtypes . in order to quantify the individual contributions to oseltamivir binding in the n9 and n1 systems , the theory of end - point energy , also known as mm - pb(gb)sa , calculations relies on a set of assumptions , elegantly reviewed in ref ( 39 ) . a simple thermodynamic cycle and single - trajectory postprocessing allows the efficient computation of the various contributions . here , the application of the mm - gbsa approach allows us to decompose the terms that contribute to ligand binding while providing relative free energies of binding that correlate well with experimental data for both n9 and n1 subtypes ( table 4 ) . the absolute values of the binding free energies , however , are off by approximately 57 kcal / mol in all three cases , which is not surprising given the assumptions present in the underlying theory and also considering that we neglected to include the internal energy change ( i.e. , strain energy ) for oseltamivir binding . . showed that the range of ligand strain energy may be in the range of 5 to 5 kcal / mol for known inhibitors of na , although they recognized the inherent difficulty in determining accurate absolute quantities due to insufficient sampling.(26 ) the average contributions to the oseltamivir binding free energies to the n9 , n1-open , and n1-closed systems over the 16 ns trajectories are given in kcal / mol ; standard errors of the mean are given in parentheses . eelec and evdw are the molecular mechanics electrostatic and van der waals contributions , respectively . gsolv , polar is the polar component of the solvation free energy , whereas gsolv , nonpolar is the nonpolar component . ts is the contribution from the rotational ( rot ) , translational ( trans ) , and vibrational ( vib ) entropies . experimental binding free energies ( gbinding , exp ) are provided for comparison .. in theory , if the configurational space sampling is sufficient , the n1 open- and closed - loop systems should converge to the same binding free energy , since they are the same enzyme ( merely different starting configurations of the 150-loop ) . although our results are not identical between the two systems , it is promising that they are within the standard error of the mean for each . a plot of the convergence of the binding free energies as a function of trajectory length indicates that the differences between running 1 and 16 ns in terms of the enthalpic contributions are minor ( supporting information , figure s4 ) . it also indicates that the salt bridge artifact between r152 and oseltamivir in the n1hc system ( which was not present before 6 ns ) has a negligible effect on the binding free energy . the absolute values of the electrostatics terms are similar to those recently published by chachra and rizzo for the n1 system , using explicit solvent simulations.(41 ) although they and others have shown that within a single na enzyme , the electrostatics term trends well with experimental binding free energies , our results suggest this is not the case when comparing across human and avian subtypes . notably , it is only with the inclusion of the entropic component that the free energies of binding agree with what is known experimentally ( table 4 ) . our work indicates that the n1 systems ( whether initiated from a closed- or open-150 loop ) pay a larger entropic penalty upon binding oseltamivir . these results are in good correspondence with the loop transition sampling events ( figure 3 ) and the per - residue rmsf values ( table 3 ) , which indicate increased flexibility in the n1 subtype . thus , we would expect the n1 systems to pay a higher entropic penalty upon complexation with oseltamivir , as compared to n9 , which appears to be intrinsically less flexible . future antiviral development efforts against the n1 subtype may benefit from considering these entropic contributions . as a control for our monomer simulations , we also present data from a 5 ns gb simulation of the closed 150-loop n1 tetramer system . the tetramer system , with 21 732 atoms ( table 1 ) , exhibited excellent parallel performance scaling : we were able to generate 0.8 ns per day when scaled to 1024 processors on the ibm bluegene machine at the san diego supercomputer center . the comparison of the tetramer n1hc system to the monomer n1hc system reveals largely similar behavior , especially in the sialic acid , 150- , and 430-loop regions . the openclose loop transition behavior was also similar for each of the monomers within the tetramer ( figure 3 ; supporting information , figure s5 ) . the exception is chain b of the tetramer , in which the 150- and 430-loops ( data not shown ) shift to a more open conformation after 3 ns . notably , the interaction of r152 with the carboxylate group of oseltamivir occurs in two of the four chains of the tetramer , indicating the intermittent occurrence of this interaction . we anticipate that this extended arginine interaction may occur in all four chains if the tetramer simulations were run for longer than 5 ns . again we note that this salt bridge is likely overstabilized and could be an artifact of the gb electrostatics . a comparison of the rmsf difference per residue of the tetramer to the monomer systems does indicate slight structural variation between the simulations in a region far from the active site . in particular , in the monomer simulations , a helix on the opposite side of the 430-loop , comprised of residues d103f115 ( hereafter referred to as the 110-helix ) , exhibited larger rmsf values ( supporting information , figure s6 ) and in several cases appeared to completely unravel . in the physiologically relevant tetrameric form of the enzyme , the 110-helix makes contact to a neighboring subunit . correspondingly , in the tetramer simulation , the contacts formed among the subunits appear to stabilize this region . the percentage of secondary structure formed ( helix ) within the core of this 110-helix ( residues s105g109 ) as a function of simulation time was investigated ( table 5 and figure s6 ) and indicates that the tetrameric contacts play a role in stabilizing this region of the enzyme . although the tetramer simulation was performed only for the n1 enzyme , we expect similar results for the n9 subtype . recent explicit solvent simulations of the monomer form of the n9 subtype also indicate that restrained dynamics were necessary to maintain the proper backbone fold,(41 ) whereas explicit solvent simulations of the tetramer enzyme were stable.(14 ) our work here supports these observations , although the increased structural fluctuations are localized to the 110-helix and the termini strand , not generalizable to the entire protein backbone . this study points to the promise and utility of generalized born implicit solvent models for elucidating the dynamics and energetics of large - scale atomistic protein systems . the reduced solvent friction in the simulations enhances the receptor conformational space sampling for the n1 and n9 neuraminidase enzymes , as compared to explicitly solvated md simulations . the 16 ns gb md trajectories sample twice as many openclose loop transition events as compared to 160 ns of explicit solvent simulations , suggesting a conformational sampling speed - up of at least 10 times that of equivalent explicit solvent simulations . our work also shows that end - point free energy calculations performed with the mm - gbsa protocol are able to recapture experimentally known oseltamivir binding profiles , thus providing an important computational framework to understand the various contributions to substrate recognition in the neuraminidase enzymes . moreover , our estimates of the entropic contributions to the binding free energies suggest that these contributions can not be neglected when comparing binding affinities across the subtypes . this work also provides several provocative insights into the structural dynamics of the avian- and human - type na enzymes , including that the apo n9 enzyme may also present 150- and 430-cavities to antiviral compounds . especially promising is the persistence of the open 430-cavity in both the oseltamivir - bound and apo systems . consequently , we anticipate that novel antiviral compounds targeting the recently discovered 150- and 430-cavities in the n1 subtype may also be effective against n9 , and we suggest the targeting of these new pockets as a general anti - influenza drug development strategy . this could have important implications for developing new therapeutics that maintain efficacy over currently known resistant strains , for both seasonal and potentially pandemic influenza subtypes .

the comparative dynamics and inhibitor binding free energies of group-1 and group-2 pathogenic influenza a subtype neuraminidase ( na ) enzymes are of fundamental biological interest and relevant to structure - based drug design studies for antiviral compounds . in this work , we present seven generalized born molecular dynamics simulations of avian ( n1)- and human ( n9)-type nas in order to probe the comparative flexibility of the two subtypes , both with and without the inhibitor oseltamivir bound . the enhanced sampling obtained through the implicit solvent treatment suggests several provocative insights into the dynamics of the two subtypes , including that the group-2 enzymes may exhibit similar motion in the 430-binding site regions but different 150-loop motion . end - point free energy calculations elucidate the contributions to inhibitor binding free energies and suggest that entropic considerations can not be neglected when comparing across the subtypes . we anticipate the findings presented here will have broad implications for the development of novel antiviral compounds against both seasonal and pandemic influenza strains .

Introduction Methods Results and Discussion Conclusions

PMC3636808

marathon running became a mass fitness phenomenon in the 1980s.1 recent studies reported that master runners increased participation and improved performance in flat city marathon running , such as in the new york city marathon.2,3 master runners were defined as athletes typically older than 35 years of age and systematically training for and competing in organized forms of sport specifically designed for older adults.4 an age - related decrease in the number of finishers in master sports has been reported.5 however , the community of master runners is still growing quickly in marathons;3 conversely , the number of younger participants increased at a lower rate.2 older master athletes are able to achieve exceptional performances . for example , the canadian marathoner ed whitlock achieved , at the age of 80 years , a marathon race time of 3 hours , 15 minutes , and 54 seconds at the toronto marathon held in 2011.6 in 2009 , an 86-year - old man became a double champion in the european championship for road running . he won the 10 km road run with a time of 58 minutes and 1 second , setting a new european record for men aged 85 years and older . two days later , he became a european champion in the same age group for the half - marathon , with a time of 2 hours and 17 minutes.7 several studies investigated participation and performance trends in master ultramarathoners where participation increased and performance improved.8,9 in marathon running , a few studies focused on the number of finishers and the performance in master marathoners competing in flat city marathons.2,3,10 the number of finishers in master runners increased at a higher rate than the number of younger marathoners,3 and their performances improved over time.2,3 lepers and cattagni3 showed an increase in performance for men older than 64 years and for women older than 44 years , competing between 1980 and 2009 in the new york city marathon . participation and performance trends for master runners in a city marathon , such as the new york city marathon , have been investigated,2,3 but are not known for master runners competing in a mountain marathon . apart from city marathon running,11 mountain marathon running is increasing in popularity.12 we intended to investigate whether the number and the performance of master runners would be similar in a mountain marathon when compared to a flat city marathon . because of the selection bias ( ie , starting places not being freely available for everyone in some of the major marathons , such as the new york city marathon ) , we intended to compare a mountain marathon ( jungfrau marathon ) with a flat city marathon ( lausanne marathon ) with no limitation on the number of starters , and which were held in the same country ( switzerland ) between 2000 and 2011 , regarding changes in participation and performance trends in master runners . we hypothesized for both races that : ( 1 ) the number of master runners would increase ; and ( 2 ) the performance of master runners would improve over the years . race times and ages of all finishers in the jungfrau marathon and the lausanne marathon between 2000 and 2011 were analyzed . the data were obtained from the race websites of the jungfrau marathon and the lausanne marathon.13,14 the jungfrau marathon was selected as the mountain marathon since this race was held in 2007 and 2012 as the official world championship in mountain running . the study was approved by the institutional review board of st gallen , switzerland , with a waiver of the requirement for informed consent , given that the study involved the analysis of publicly available data . the jungfrau marathon in switzerland was established in 1993 and is one of the most popular mountain marathons in the world.13 the race is held annually with the start in interlaken ( 565 meters ( m ) above sea level ) and the finish at the kleine scheidegg ( 2095 m above sea level ) . it covers an overall ascent of 1830 m and an overall descent of 305 m. the first quarter of the race is mostly flat and in the first half of the race the athletes had no more ascent than 300 m in the half - marathon distance ( figure 1 ) . the lausanne marathon was established in 1992 and is the second most important swiss city marathon following the zrich marathon.14 the race is held annually in autumn in lausanne on the border of lake lman with 36 m of altitude change ( figure 1 ) . data from 43,636 runners ( including 7567 [ 17.3% ] women and 36,069 [ 82.7% ] men ) who finished the jungfrau marathon between 2000 and 2011 , and 20,464 runners ( including 3104 [ 15.2% ] women and 17,360 [ 84.8% ] men ) who finished the lausanne marathon between 2000 and 2011 were available . to analyze the performance in age groups , all athletes were divided in 10-year age groups : 1524 years , 2534 years , 3544 years , 4554 years , 5564 years , 6574 years , and 7584 years . to calculate the performance ratio and sex differences from each age group , the annual top ten women and men were determined . only those age groups with at least ten finishers in both sexes in at least eleven out of the twelve analyzed years were included . due to the low number of finishers per age group , only athletes in the 2534-year and 5564-year age groups the performance ratio for both sexes and the sex differences in performance were calculated per age group and year , and these were subsequently analyzed regarding their change over time . the performance ratio was calculated using the equation : the performance ratio expresses the performance of the top ten athletes of an age group as a percentage of the performance of the overall top ten athletes and is a good indicator of the age - related decline in performance.3 the sex difference in performance was calculated using the equation : where the sex difference was calculated for every pairing of equally placed athletes ( eg , between female and male first place , between female and male second place , and so on ) before calculating the mean value and standard deviation of all the pairings . to facilitate reading , all sex differences were transformed into absolute values before analyzing . to increase the reliability of the data analyses , each set of data was tested for normal distribution as well as for the homogeneity of variances in advance of the statistical analyses . normal distribution was tested using the dagostino pearson omnibus normality test , and the homogeneity of variances was tested using levene s test . to find significant changes in the development of a variable across years , linear regression was used . to find significant differences between two groups in case of normal distributed data , a student s t - test was used with welch s correction in case of significant different variances between the two compared groups . in case of statistical analyses were performed using the ibm statistical package for the social sciences version 19 ( spss ; ibm corporation , armonk , ny , usa ) and graphpad prism version 5 ( graphpad software inc , la jolla , ca , usa ) . significance was accepted at p < 0.05 ( two - sided for t - tests ) . the jungfrau marathon in switzerland was established in 1993 and is one of the most popular mountain marathons in the world.13 the race is held annually with the start in interlaken ( 565 meters ( m ) above sea level ) and the finish at the kleine scheidegg ( 2095 m above sea level ) . it covers an overall ascent of 1830 m and an overall descent of 305 m. the first quarter of the race is mostly flat and in the first half of the race the athletes had no more ascent than 300 m in the half - marathon distance ( figure 1 ) . the lausanne marathon was established in 1992 and is the second most important swiss city marathon following the zrich marathon.14 the race is held annually in autumn in lausanne on the border of lake lman with 36 m of altitude change ( figure 1 ) . data from 43,636 runners ( including 7567 [ 17.3% ] women and 36,069 [ 82.7% ] men ) who finished the jungfrau marathon between 2000 and 2011 , and 20,464 runners ( including 3104 [ 15.2% ] women and 17,360 [ 84.8% ] men ) who finished the lausanne marathon between 2000 and 2011 were available . to analyze the performance in age groups , all athletes were divided in 10-year age groups : 1524 years , 2534 years , 3544 years , 4554 years , 5564 years , 6574 years , and 7584 years . to calculate the performance ratio and sex differences from each age group , the annual top ten women and men were determined . only those age groups with at least ten finishers in both sexes in at least eleven out of the twelve analyzed years were included . due to the low number of finishers per age group , only athletes in the 2534-year and 5564-year age groups could be included in the data analysis . the performance ratio for both sexes and the sex differences in performance were calculated per age group and year , and these were subsequently analyzed regarding their change over time . the performance ratio was calculated using the equation : the performance ratio expresses the performance of the top ten athletes of an age group as a percentage of the performance of the overall top ten athletes and is a good indicator of the age - related decline in performance.3 the sex difference in performance was calculated using the equation : where the sex difference was calculated for every pairing of equally placed athletes ( eg , between female and male first place , between female and male second place , and so on ) before calculating the mean value and standard deviation of all the pairings . to facilitate reading , to increase the reliability of the data analyses , each set of data was tested for normal distribution as well as for the homogeneity of variances in advance of the statistical analyses . normal distribution was tested using the dagostino pearson omnibus normality test , and the homogeneity of variances was tested using levene s test . to find significant changes in the development of a variable across years , linear regression was used . to find significant differences between two groups in case of normal distributed data , a student s t - test was used with welch s correction in case of significant different variances between the two compared groups . in case of not normal distributed data , the mann statistical analyses were performed using the ibm statistical package for the social sciences version 19 ( spss ; ibm corporation , armonk , ny , usa ) and graphpad prism version 5 ( graphpad software inc , la jolla , ca , usa ) . significance was accepted at p < 0.05 ( two - sided for t - tests ) . the number of finishers in the mountain marathon remained unchanged over the 12-year period ( figure 2a ) , whereas the number of finishers in the city marathon decreased ( figure 2b ) . in the mountain marathon , the number of women accounted for 17.3% of the total field , while they accounted for 15.2% in the city marathon . there was a significant increase in the number of female finishers in the jungfrau marathon ( figure 2a ) , but there was a decrease in the lausanne marathon ( figure 2b ) . the number of male finishers showed no change in the jungfrau marathon compared to a decrease in the lausanne marathon . in both events , athletes in the 3544 age group showed the highest number of finishers for both women and men ( figure 3 ) . in the mountain marathon , the number of female master runners aged > 34 years increased in contrast to female finishers aged < 35 years ( figure 4a ) , while the number of male finishers was constant in all age groups ( figure 4b ) . in the city marathon , the number of female finishers was constant while the number of male finishers in the age groups of 2534 and 3544 years decreased . for the top ten women and the top ten men in the jungfrau marathon ( the mountain marathon ) , the overall running speed remained stable . in the lausanne marathon ( the city marathon ) , the overall top ten women maintained their running speed . however , the overall top ten men became significantly slower as their running time increased by 8% , from 139.8 minutes 2.4 minutes ( 2000 ) to 151.1 minutes 6.4 minutes ( 2011 ) . figure 5 presents the changes in performance ratio for age group runners . in female marathoners , performance improved in athletes aged 3544 years and 5564 years in the city marathon ( lausanne marathon ) . male marathoners race times improved in the 4554-year age group in both the city marathon ( lausanne marathon ) and in the mountain marathon ( jungfrau marathon ) . the analysis of the interaction between the type of competition and age group showed a significant interaction for women ( f = 5.7 ; p < 0.0001 ) , where the type of competition accounted for 35.3% ( f = 1232.5 , p < 0.0001 ) and age group accounted for 58.4% ( f = 224.9 , p < 0.0001 ) of the total variance . for men , the type of competition and age group showed a significant interaction ( f = 9.3 ; p < 0.0001 ) , where the type of competition accounted for 52.4% ( f = 2028.7 , p < 0.0001 ) and age group for 41.2% ( f = 178.4 , p < 0.0001 ) of the total variance . female runners reduced sex differences in performance in the 4554-year age group in both competitions and in the 3544-year age group in the mountain marathon ( jungfrau marathon ) . in the mountain marathon ( jungfrau marathon ) , the sex difference in the performance of the 4554-year age group decreased from 23.4% 1.9% ( 2000 ) to 15.9% 6.1% ( 2011 ) in the mountain marathon and from 34.7% 4.6% to 11.8% 6.2% in the city marathon ( lausanne marathon ) . in the mountain marathon , the sex difference of the 3544-year age group decreased from 19.1% 4.7% ( 2000 ) to 16.6% 1.9% ( 2011 ) . the interaction analysis between the type of competition and age group on running speed showed significant interaction in terms of sex differences ( f = 8.64 ; p < 0.0001 ) , where the type of competition and age group accounted for 71.8% of the total variance , and age group alone accounted for 71.7% ( f = 72.90 ; p < 0.0001 ) of the total variance . the type of competition accounted for less than 0.01% of the total variance ( f = 0.02 ; p = 0.90 ) and was insignificant . the number of finishers in the mountain marathon remained unchanged over the 12-year period ( figure 2a ) , whereas the number of finishers in the city marathon decreased ( figure 2b ) . in the mountain marathon , the number of women accounted for 17.3% of the total field , while they accounted for 15.2% in the city marathon . there was a significant increase in the number of female finishers in the jungfrau marathon ( figure 2a ) , but there was a decrease in the lausanne marathon ( figure 2b ) . the number of male finishers showed no change in the jungfrau marathon compared to a decrease in the lausanne marathon . in both events , athletes in the 3544 age group showed the highest number of finishers for both women and men ( figure 3 ) . in the mountain marathon , the number of female master runners aged > 34 years increased in contrast to female finishers aged < 35 years ( figure 4a ) , while the number of male finishers was constant in all age groups ( figure 4b ) . in the city marathon , the number of female finishers was constant while the number of male finishers in the age groups of 2534 and 3544 years decreased . for the top ten women and the top ten men in the jungfrau marathon ( the mountain marathon ) , the overall running speed remained stable . in the lausanne marathon ( the city marathon ) , the overall top ten women maintained their running speed . however , the overall top ten men became significantly slower as their running time increased by 8% , from 139.8 minutes 2.4 minutes ( 2000 ) to 151.1 minutes 6.4 minutes ( 2011 ) . figure 5 presents the changes in performance ratio for age group runners . in female marathoners , performance improved in athletes aged 3544 years and 5564 years in the city marathon ( lausanne marathon ) . male marathoners race times improved in the 4554-year age group in both the city marathon ( lausanne marathon ) and in the mountain marathon ( jungfrau marathon ) . the analysis of the interaction between the type of competition and age group showed a significant interaction for women ( f = 5.7 ; p < 0.0001 ) , where the type of competition accounted for 35.3% ( f = 1232.5 , p < 0.0001 ) and age group accounted for 58.4% ( f = 224.9 , p < 0.0001 ) of the total variance . for men , the type of competition and age group showed a significant interaction ( f = 9.3 ; p < 0.0001 ) , where the type of competition accounted for 52.4% ( f = 2028.7 , p < 0.0001 ) and age group for 41.2% ( f = 178.4 , p < 0.0001 ) of the total variance . female runners reduced sex differences in performance in the 4554-year age group in both competitions and in the 3544-year age group in the mountain marathon ( jungfrau marathon ) . in the mountain marathon ( jungfrau marathon ) , the sex difference in the performance of the 4554-year age group decreased from 23.4% 1.9% ( 2000 ) to 15.9% 6.1% ( 2011 ) in the mountain marathon and from 34.7% 4.6% to 11.8% 6.2% in the city marathon ( lausanne marathon ) . in the mountain marathon , the sex difference of the 3544-year age group decreased from 19.1% 4.7% ( 2000 ) to 16.6% 1.9% ( 2011 ) . the interaction analysis between the type of competition and age group on running speed showed significant interaction in terms of sex differences ( f = 8.64 ; p < 0.0001 ) , where the type of competition and age group accounted for 71.8% of the total variance , and age group alone accounted for 71.7% ( f = 72.90 ; p < 0.0001 ) of the total variance . the type of competition accounted for less than 0.01% of the total variance ( f = 0.02 ; p = 0.90 ) and was insignificant . this study intended to compare participation and performance trends in runners of various age groups competing in a mountain marathon and a flat city marathon in the same country where the race director set no limit in the number of starters ( in contrast to other marathons where the field is limited ) . we hypothesized for both races that : ( 1 ) the number of master runners would increase ; and ( 2 ) the performance of master runners would improve over the years . in the mountain marathon , the number of female finishers increased in every age group of master runners ; the number of male finishers , however , remained constant . eichenberger et al15 reported similar findings for mountain ultramarathoners , showing that the number of female finishers increased from 2000 to 2011 in the swiss alpine marathon , whereas the number of male finishers remained unchanged . only the number of master runners increased while the number of younger finishers ( ie , < 35 years old ) remained constant . however , in the city marathon , the number of younger finishers ( ie , < 45 years old ) decreased while the number of older male finishers was constant . a possible explanation might be that since the beginning of the lausanne marathon in 1992 , an abundance of newer and larger city marathons has been offered in switzerland , including the zrich marathon and others that have taken place abroad ; therefore , runners now have a wider spectrum of competitions to choose from.11 considering the performance of athletes based on age group , an important finding was that there was an increase in the performance ratio for female master runners aged 3544 years and 5564 years in the city marathon and for male master runners aged 4554 years in both the city and the mountain marathons . marathoners in younger age groups , however , showed no improvements in performance in either marathon . therefore , it seemed that female athletes in the mountain marathon reached their peak running speed and their performance may not improve anymore in the near future . conversely , in the city marathon , women improved in the 3544 and 5564 age groups . jokl et al2 reported an improvement in running speed for master runners at a faster rate than younger athletes in the new york city marathon from 19831999 , which is consistent with our findings in the city marathon for men . a possible explanation for the improvement of male master runners in the city marathon might be that the top ten men became significantly slower over the years . therefore , even maintenance of running speed in master runners might have resulted in a relative improvement among the top ten runners in their respective marathon years . the finding that female master runners exhibited improved performances in city marathon running was consistent with the findings of lepers and cattagni,3 who reported a significant increase in running speed in women older than 45 years . while men between 45 and 54 years of age could improve their running speed in both a mountain and a city marathon , age - matched women could not . women were able to improve race times in the city marathon in age groups 3544 years and 5564 years . women seemed to have reached their peak running speed in mountain marathon running , but this was increasingly true for the younger ( ie , < 44 years old ) runners than for older runners . in the mountain marathon , the sex difference in the of 2529-year age group was 15.4% 1.7% , while the difference in the 6064-year age group was 20.4% 2.1% , which was consistent with the findings in the study by hunter and stevens,16 where the authors noted that the sex differences of running speed increased with advancing age in the new york city marathon . no changes in sex difference for running times were found between the top ten women and the top ten men in both races . nevertheless , significant changes in the sex differences in running times in male athletes in the 4554-year age group were found for both races . additionally , the sex difference decreased in the 3544-year age group in runners from the mountain marathon . these findings suggest that female master runners older than 35 years reduced the sex difference in performance observed in both mountain and city marathon running . studies concerning the sex differences in running exist for elite athletes and for recreational runners.15,1720 the sex difference in endurance performance ( ie , running speed ) was a frequently discussed topic,21,22 and it was found to be existent in different sports disciplines.23 twenty years ago , whipp and ward22 hypothesized that women would achieve the performance of men depending upon the distance . existing findings show that women are unlikely to be able to outrun men in the near future , as the average difference in running speed between men and women decreased for a long time but leveled in recent years.24 cheuvront et al24 reported a sex difference of 8%14% for running distances from 1500 m to 42 km , whereas the sex difference in running speed was on average about 11% in other competitions.18,21 lepers and cattagni3 showed relatively stable sex differences in marathon running times across the different age groups for the last decade in the new york city marathon . considering the sex difference in performance , the interaction between the type of competition and running time was insignificant , and the sex difference in running speed was therefore independent from the type of competition . the time difference between sexes was constant over the years , while the sex difference was smaller between the mountain marathon runners compared to the city marathon runners . the fastest female runner was about 16% slower in the mountain marathon and approximately 18% slower in the city marathon compared to the top ten women in the mountain marathon , with approximately 19% difference in running speed and approximately 22% in the city marathon . cheuvront et al24 similarly found that the sex difference in performance was constant but smaller at around 8%14% in competitions ranging from 1500 m to the marathon distance.24 in longer distances , coast et al25 reported a mean sex difference of 12.4% over distances ranging from 100 m to 200 km . the difference in running speed between winners and the top ten runners can be partly explained by the lower number of female finishers and , therefore , the faster decline in performance in women . we found a highly significant interaction between the type of competition and the age of the top ten runners . to the best of our knowledge , there was never a direct comparison between a mountain and a city marathon in terms of running times and age . nevertheless , other studies suggested that the type of competition and the age of the top ten runners were directly related . eichenberger et al15 found that there was a mean age of approximately 35 years for the top ten runners over the time span of 14 years at the swiss alpine marathon held in davos , switzerland.15 conversely , in city marathon running , the average age of runners was reported to be at around 30 years for both women and men.18,26 since these two types of competitions were not directly compared , our results can not be definitively supported by these findings . this study is limited since variables such as physiological parameters,27 anthropometric characteristics,20,28,29 training characteristics,20,28,30,31 previous experience,32 nutrition,33 and weather were not considered.34 it would be of interest to compare the same women and men competing in both the city and the mountain marathon . female marathoners with lower body mass and lower body mass index compared to male marathoners might hold an advantage and achieve relatively faster race times.30,31 this study is limited since variables such as physiological parameters,27 anthropometric characteristics,20,28,29 training characteristics,20,28,30,31 previous experience,32 nutrition,33 and weather were not considered.34 it would be of interest to compare the same women and men competing in both the city and the mountain marathon . female marathoners with lower body mass and lower body mass index compared to male marathoners might hold an advantage and achieve relatively faster race times.30,31 to summarize , the number of female master runners increased in the mountain marathon . the sex difference in performance decreased in the 3544-year age group in the mountain marathon . in runners aged 4554 years , the sex difference decreased in the mountain marathon and in the city marathon . these findings suggest that female master runners reduced the sex difference in performance in both mountain and city marathon running .

backgroundthe performance in master marathoners has been investigated in flat city marathons but not in mountain marathons . this study examined changes in the sex differences in performance across time in female and male master runners competing in a mountain marathon compared to a flat city marathon.methodsthe association between age and performance of finishers in the jungfrau marathon , switzerland , with 1830 meter changes in altitude and a flat city marathon ( lausanne marathon ) , switzerland , were analyzed from 2000 to 2011.resultsin both events , athletes in the 3544 years age group showed the highest number of finishers . in the mountain marathon , the number of female master runners aged > 35 years increased in contrast to female finishers aged < 35 years , while the number of male finishers was unchanged in all age groups . in the city marathon , the number of female finishers was unchanged while the number of male finishers in the age groups for 2534-year - olds and 3544-year - olds decreased . in female marathoners , performance improved in athletes aged 3544 and 5564 years in the city marathon . male marathoners improved race time in age group 4554 years in both the city marathon and the mountain marathon . female master runners reduced the sex difference in performance in the 4554-year age group in both competitions and in the 3544-year age group in the mountain marathon . the sex difference in performance decreased in the 3544-year age group from 19.1% 4.7% to 16.6% 1.9% in the mountain marathon ( r2 = 0.39 , p = 0.03 ) . in age groups 4554 years , the sex difference decreased from 23.4% 1.9% to 15.9% 6.1% in the mountain marathon ( r2 = 0.39 , p < 0.01 ) and from 34.7% 4.6% to 11.8% 6.2% in the city marathon ( r2 = 0.39 , p < 0.01).conclusionthese findings suggest that female master runners aged 3554 years reduced sex differences in their performance in both mountain and city marathon running .

Introduction Methods Races Data analyses Statistical analysis Results Participation trends Performance trends Discussion Limitations and implications for future research Conclusion

PMC459234

ivermectin ( mectizan ) is a semi - synthetic lactone drug that exhibits broad anti - helminthic specificity . mectizan has become the drug of choice for mass chemotherapy campaigns to eliminate onchocerciasis as a public health problem . in general , a notable exception occurs in some areas where the agent of onchocerciasis , onchocerca volvulus , is sympatric with loa loa , the causative agent of another filarial infection of humans . over the past several years , severe adverse reactions to mectizan treatment have been reported in individuals residing in onchocerciasis endemic areas that are also endemic for l. loa [ 3 - 5 ] . these adverse reactions are characterized by severe neurological complications , including incontinence , coma , and in some cases , death of the patient . interestingly , a large majority of the individuals suffering from such severe adverse reactions have resided in southern cameroon [ 6 - 8 ] . the development of severe reactions to mectizan in l. loa infected individuals has had a negative impact on the design and implementation of mectizan distribution campaigns to control onchocerciasis in areas where o. volvulus and l. loa are co - endemic . parasitological examinations of patients who developed post - mectizan encephalopathy showed a correlation between the risk of developing such reactions and the l. loa microfilarial load . however , the finding that the vast majority of such severe reactions occurred in a localized geographic area also suggested that the parasites endemic to this area might represent a genetically distinct population that is particularly capable of inducing adverse reactions in the host upon being exposed to mectizan. if the parasites from southern cameroon represent a genetically distinct population , it would follow that genetic exchange with other parasite populations would be expected to be limited or non - existent . such reproductively isolated populations often suffer from a genetic founder effect , resulting in a population in which the level of genetic heterogeneity is severely limited , and in which the distribution of alleles in the isolated population is often strikingly different from that seen in non - reproductively isolated populations . furthermore , prolonged reproductive isolation of a population will result in the accumulation of population specific genetic polymorphisms . if this occurs , phylogenic analyses based upon data collected from isolates from within and outside of the isolated population should result in a phylogeny in which the isolates from within the isolated population are grouped together . very little is known concerning the degree of genetic diversity among different populations of l. loa and the role that such genetic polymorphisms may play in the adverse responses to mectizan treatment . simian and human strains of l. loa , which differ in the length of the microfilariae and their periodicity in the host have long been recognized . despite these differences , human and simian l. loa are believed to be the same species , as experimental studies have shown that parasites from these two primate hosts can hybridize . because parasites from the two hosts differ in periodicity and are transmitted by different species of the chrysops genus , it has been proposed that the human and simian parasites " are at an early stage of radiative evolution " . whether hybridization of the simian and human strains is a co - factor in the development of post - mectizan encephalopathy is unknown . some biochemical variation has also been reported among different populations of human l. loa , although these differences have not been explored at the genetic level . perhaps the most well documented case of the importance of genetic factors in the pathogenesis of a human filarial infection is in onchocerciasis . human onchocerciasis exhibits two distinct clinical and epidemiologic disease patterns in the rain forest and savanna bioclimes of west africa . studies have revealed a strong correlation between the classification based upon the epidemiological disease pattern and the pattern of hybridization to strain specific dna sequences derived from a repeated sequence encoded in the nuclear genome of o. volvulus . subsequent studies have demonstrated that variation within this repeat population could be used as a tool for biogeographic studies . in ascaris suum , ostertagia osteragi and haemonchus contortus mitochondrially encoded sequences have proven to contain useful polymorphisms for population based studies . similarly , the internal transcribed spacer ( its ) of the ribosomal rna gene cluster has been used to develop markers capable of distinguishing different populations of haemonchus contortus . to test the hypothesis that l. loa endemic to southern cameroon represents a distinct population , these parasites consisted of infective larvae obtained from four individual wild caught l. loa infected chrysops dimidiata vector flies collected in the village of ngat ( 325'n , 1133'e ) , located approximately 50 km south of the capital of yaound . individual isolates were also obtained from blood collected from l. loa infected expatriate former residents of nigeria , gabon and the democratic republic of the congo at the laboratory of parasitic diseases at the national institutes of health in bethesda , md , usa . dna was extracted from the parasite preparations , following previously described methods . these dna samples were then used as templates in pcr amplification reactions targeting three gene sequences : the mitochondrial 16s rrna gene , the its2 domain of the nuclear rrna gene cluster , and the 15r3 polyprotein gene in l. loa . the 15r3 gene has previously been shown to be polymorphic and useful in the development of a dna based diagnostic assay for this parasite . these gene sequences were chosen for study because , as discussed above , homologues ( or related sequences ) have previously been shown to be informative population based markers in studies of other parasitic nematodes . pcr amplicons resulting from these reactions were subjected to direct dna sequence analysis to identify sequence polymorphisms . all of the dna templates were successfully amplified with the primer set derived from the mitochondrial 16s rrna gene . however , no polymorphisms were noted in the amplification products analyzed ( data not shown ) . amplification products were also obtained from all of the samples targeting the 15r3 polyprotein gene . interestingly , sequences derived from dna extracted from the blood of l. loa infected individuals from gabon , nigeria and the democratic republic of the congo were identical ( figure 1 , panel a ) . one of the isolates from southern cameroon also contained a 15r3 sequence which was identical to that found in the parasite isolates from the other countries . however , the remaining three isolates from southern cameroon contained between 3 and 7 polymorphic sites when compared to the canonical sequence found in the other parasites ( figure 1 , panel a ) . these data suggest that the 15r3 gene sequences from parasites obtained from southern cameroon were at least as polymorphic as those from other l. loa foci in sub - saharan africa . none of the polymorphisms were consistently found among the isolates from southern cameroon , suggesting that these polymorphisms probably did not arise from evolution in a genetically homogeneous , reproductively isolated population ( data not shown ) . analysis of polymorphisms in the 15r3 and its2 gene sequences of l. loa : panel a : pairwise differences in 15r3 amplicon sequences among l. loa isolates . the 15r3 gene fragment was amplified from l. loa genomic dna ( 2.5 l per reaction ) using primers with the sequences 5 ' ggcacaaaacactgcagcagtcct 3 ' and 5 ' cagctgtctcaaatcgaagattct 3 ' . a total of 2.5 units of taq polymerase ( roche applied biochemicals , indianapolis , usa ) was used in each 50 l amplification reaction , together with the reaction buffer supplied by the manufacturer . amplification conditions consisted of an initial denaturation of 5 minutes at 94c , followed by 40 cycles consisting of 1 minute at 94c , 1 minute at 49c , and 2 minutes at 72c . the its2 gene fragment was amplified from l. loa genomic dna ( 2.5 l per reaction ) using primers with the sequences 5 ' taacaatgaagataaagcga 3 ' and 5 ' ttagtttcttttcctccgct 3 ' . a total of 2.5 units of taq polymerase ( roche applied biochemicals , indianapolis , usa ) was used in each 50 l amplification reaction , together with the reaction buffer supplied by the manufacturer . amplification conditions consisted of an initial denaturation of 5 minutes at 94c , followed by 40 cycles consisting of 1 minute at 94c , 1 minute at 50c , and 2 minutes at 72c . the phylogeny was developed using parsimony methods , performing an exhaustive search of the data with the parsimony routines in the paup program package ( v4.0 , release 10 ) . the robustness of the phylogeny was tested by running 1000 synthetic datasets with the bootstrap method in the paup program package . as indicated on the figure , the division of the four sequences into the two clades shown was supported 70% of the time in the bootstrap analysis . amplification of the its2 locus was successful from l. loa dna samples from nigeria , gabon and two of four samples from southern cameroon . again , several polymorphisms were noted among the four samples examined ( figure 1 , panel b ) . however , none of these polymorphisms were specific to and conserved in the two isolates from southern cameroon . thus , an analysis of the sequence data using parsimony methods did not support a phylogeny that grouped the two isolates from southern cameroon ( figure 1 , panel c ) . the data presented above are preliminary , and involve the analysis of a limited number of sequences from a small number of parasite isolates . first , it appears that genetic variation in parasites from the focus in southern cameroon is at least as frequent as in parasites from other locations . second , no polymorphisms were noted which were unique to the parasites from southern cameroon and also shared among the isolates examined from this focus . together , these observations argue against the hypothesis that the parasites found in southern cameroon represent a distinct , reproductively isolated population . this study revealed a considerable amount of sequence variation in the two nuclearly encoded genes examined . if this level of sequence heterogeneity is representative of the rest of the nuclear genome , this suggests that the l. loa population from southern cameroon is fairly genetically heterogeneous . it is therefore possible that a proportion of the parasites from southern cameroon contain allelic variants of genes that result in enhanced virulence and/or pathogenicity in the face of mectizan treatment . this might explain the fact that severe adverse reactions in response to mectizan treatment are confined to a small proportion of the treated population . to test this hypothesis , it will be necessary to obtain parasites from individuals who exhibit the preliminary symptoms of a severe adverse reaction shortly following mectizan distribution , when l. loa microfilariae are still likely to be present in the circulation . tbh was responsible for carrying out the laboratory experiments described in the manuscript , including dna isolation , pcr amplification and dna sequence analysis . ak and mb were involved in the experimental planning and conducted the field work involved in obtaining the samples . he was responsible for obtaining grant support for the project , for assisting in the experimental design , data analysis and for preparation of the manuscript . m. demanou , who assisted with collection of chrysops from which loa loa infective larvae were isolated , and dr . this investigation received financial support from the undp / world bank / who special programme for research and training in tropical diseases ( tdr ) , project # 991105 .

ivermectin ( or mectizan ) is widely used by onchocerciasis and lymphatic filariasis control programs worldwide . generally , mectizan is both safe and well tolerated . an exception to this general pattern is in some areas co - endemic for onchocerca volvulus and loa loa , where a number of severe adverse reactions to mectizan have been noted in l. loa infected individuals . the vast majority of these severe adverse events have occurred in southern cameroon . this suggested the hypothesis that the parasites endemic to southern cameroon might form a distinct population that exhibited a phenotype of eliciting severe adverse reactions in loa - infected individuals upon mectizan exposure . to test this hypothesis , the dna sequences of three potentially polymorphic loci were compared among l. loa parasites from southern cameroon and other endemic foci in sub - saharan africa . analysis of these data suggested that parasites from southern cameroon were at least as genetically diverse as those from other foci . furthermore , no polymorphisms were noted that were unique to and shared among the parasite isolates from southern cameroon . although a limited number of parasite isolates were tested , these results do not appear to support the hypothesis that l. loa parasites from southern cameroon represent a unique , genetically isolated population .

Findings Competing Interests Author's Contributions Acknowledgements

PMC3394405

recent studies have shown that food production and consumption are responsible for 1030% of an individual s total environmental impact . a considerable amount of the total food intake by mass ( 30% ) is represented by fruits and vegetables , which constitute the largest food group consumed worldwide . the effects of their production are revealed in different categories of environmental impacts , like climate change , impacts of land and water use , human- and eco - toxicological effects , eutrophication , acidification , soil fertility degradation , and landscape changes . policy makers and private companies in various countries have recognized the need to quantify these environmental impacts and , on this basis , to identify measures for impact reduction . for instance , a new law in france and a recommendation of the swiss federal office for the environment encourage the labeling of food products with their carbon / environmental footprints . private companies , such as tesco and walmart , calculate the carbon footprint of some of their products and communicate these to their customers , while others use such environmental information for internal decision making regarding products and supply chain management . finally , water footprint studies have gained high interest in the area of food production , revealing the amounts of water consumption and the related impacts . the international organization for standardization ( iso ) is therefore currently considering a standard on water footprint to allow consistent analysis and reporting for product labeling . despite these initiatives for instance , while several life cycle assessment ( lca ) studies on a variety of fruits and vegetables have been published , the comparability of these studies is compromised by differences in system boundaries and background data . in contrast to process - based lca studies , input - output lca studies provide data on total food consumption without having cut - offs in the supply chain , leading to a large gap in the overall impacts . such studies help to identify relevant food groups , but the data are given on an industrial - sector resolution and hence do not allow for identifying improvement potentials within sectors . moreover , international trade is not well captured due to inconsistencies in the underlying statistical data . thus , in addition to these studies , detailed , process - based lca data are needed to support decisions regarding adequate sourcing of food products , means of transportation , agricultural management , and , finally , choices between different food commodities . the goals of the present study were ( a ) to elaborate a consistent and up - to - date life cycle inventory ( lci ) of a large range of fruits and vegetables from different origins , ( b ) to show selected life cycle impact assessment ( lcia ) results and derive general decision guidelines for producers , retailers , policy makers and consumers on how to improve the environmental impacts of fruit and vegetable consumption , and ( c ) to illustrate and discuss the implementation of these guidelines for a specific case of purchasing decision and environmental supply chain management of a main swiss retailer . the functional unit ( fu ) was defined as 1 kg of product at the point of sale . the lca study includes the following fruits and vegetables : apple , avocado , banana , broccoli , cabbage for conserves , carrots , cauliflower , celery root , citrus fruits , cucumbers , eggplant , fennel , grape , green asparagus , bell pepper , iceberg lettuce , kiwi , lettuce , melon , onion , vine tomatoes , papaya , pear , pineapple , potatoes ( lci adapted from ecoinvent ) , radish , red cabbage , round carrots , spinach , strawberries , tomatoes , white asparagus , white cabbage , and zucchini . these products cover more than 80% of the fruits and vegetables sold by one of the two major retailers in switzerland in 2007 , for which the study was originally undertaken . the products were either produced locally or transported to switzerland from 29 different countries . the lci were compiled by extrapolating from a basic set of data for one product to the same product from other origins by varying parameters , such as transport means and distances , irrigation , heating energy for greenhouse production , and cooling energy for storage . inputs and outputs from packaging and the operation of the store were excluded from the analysis as these were shown to be relatively low compared to the overall impact ( supporting information ( si ) , section 1 ) and equal for all fruits and vegetables . vegetables , apples , pears and strawberries were modeled using the swiss agricultural standard production scheme called integrated production as described elsewhere . the other fruits were produced according to the so - called conventional production . tables with agricultural production means for cost calculations were used to set up the inventory of vegetables , apples and pears , whereas for tropical fruit production additional data were obtained from literature and leaflets of agricultural extension services ( si , section 2 ) . good agricultural practice ( gap ) was assumed for all agricultural activities , irrespective of the production site , assuming common global standards throughout the supply chain . this assumption was in accordance with the commissioner of the study , but may need to be revised in cases in which retailers do not make sure that gap is applied . next , a short outline of every parameter considered in the lci is given ; detailed information can be found in the si , section 2 . it was assumed that the land occupied is arable and that it had been used for agriculture for a long time . land occupation was calculated based on yield and cultivation time per kg of product ( si , section 3 ) . one of the upstream processes of vegetable growing is the production of seedlings , which are young plants to be bedded out . we assumed an average size of 20 cm per pot with an estimated weight of 20 g. based on the yield and number of seedlings planted per ha , the amount of peat and the transported weight per kg of product from the mining site were calculated . seedling production in switzerland or further north is generally assumed to take place in heated greenhouses over five weeks . for heating oil consumption , the data for eggplants were assumed for all vegetable seedlings because of similar temperature requirements . the nutrients , extracted by the plants , eroded and leached to water , have to be replaced by soil fertilization . here we considered effective fertilization with macronutrients using the ecoinvent processes ammonium nitrate , single superphosphate as p2o5 and potassium sulphate ( si , section 5 ) . individual pesticide production data were not available . in such cases , the generic pesticide process field emissions of pesticides are often farm - specific and models like in can be used to estimate such emissions accurately . the ecoinvent data set fertilizing by broadcaster with middle intensive fuel consumption was used as a proxy for horticultural machinery . data on the number of machinery operations and the working hours for running the machines were used to quantify the amount of machinery input per kg of crop ( si , section 8) . greenhouse production implies electricity use , for example , for lighting and irrigation pumps . the electricity demand was estimated using information from swiss cost calculation sheets assuming a price of 0.15 chf / kwh for industrial companies . the average european electricity mix ( entso - e , former ucte ) of low voltage was used for all crops except those originating from the americas , to which the u.s .- mix was applied . to be independent from outdoor temperature , greenhouses are built to provide the appropriate climate . to show the variability of fuel consumption related to seasonality , a time - dependent heating energy model for greenhouse production was developed and applied . this model considers the type of greenhouse ( heat transmission properties ) , the building dimensions , the difference in outside and inside temperature required by the specific crop , solar irradiation and the yield . for details if the sourcing season was unknown , an annual average amount of heating oil ( fossil fuel ) per crop was used for one growing period . all productions in switzerland and further north were modeled as heated and nonheated to approximate a winter and a summer production respectively . irrigation is needed in regions where rainfall is less than the amount of water required to grow a specific crop , where rainfall is seasonally unevenly distributed or if crops are cultivated in greenhouses . the amount of water irrigated depends on the culture as well as on soil and different climate parameters like temperature , wind and rainfall . the different amounts of irrigation water for all the crops grown in switzerland are available from elsewhere . short - term crops ( like lettuce and radish ) and open field crops use 400800 m / ha / growing cycle , long - term greenhouse crops use 30006000 m / ha / growing cycle . the irrigation inventory for imported crops was calculated according to pfister et al . as only the country of origin was known , a production weighted average amount was used , taking into account the geographical distribution of each crop within a country . domestic production covers 40% and 49% of the fruit and vegetable consumption respectively , whereas the rest is imported . the most important production sites in a country were identified for each product and the most evident transportation routes and means were chosen according to the scheme in table s4 ( si , section 11 ) . it was assumed that trucks from industrial countries are euro 4 or 5 standard with cargo weight > 32 t , except for distribution in switzerland , which was modeled with a specific fleet average truck of > 28 t. truck - transportation in emerging economies was simulated with an euro 3 standard for cargo weight > 32 t. by sea route the products are transported by freight ship and in the air by an intercontinental freight aircraft . the corresponding ecoinvent processes were employed and distances were measured with online tools ( si , section 11 ) . crops need to be cooled in order to avoid decay before arriving at the point of sale and to elongate the storage life . transportation was assumed to take place in fully loaded iso - containers with independent cooling aggregates . according to wild one teu (= twenty - foot equivalent unit ) is the size of a little standardized container with an average load of 10 t. furthermore , the transportation time ( si , section 12 ) was needed to model the consumed cooling energy with the ecoinvent data set diesel electric generating set . several crops ( asparagus , bananas , carrots , celery root , cucumbers , iceberg lettuce , lettuce , radish , spinach , and zucchini ) need to be cleaned after harvesting . it was assumed that 0.4 l of tap water is used per kg of crop , except for bananas which use 4.4 l per kg . energy consumption depends on storage time , outside temperature , ideal storage temperature ( crop specific ) ranging from 2 to 13 c and packing density , which is generally assumed to be 300 kg / m . nitrate and phosphorus - emissions into different compartments were modeled generically , because no site - specific values of the productions sites ( slope , soil , machine type , weather etc . ) is emitted into the air as ammonia ( nh3 ) , 1.7% as nitric oxide ( no ) and the same amount as nitrous oxide ( n2o ) into the air as well , whereas 35% is estimated to be leached as nitrate ( no3 ) into the soil . constant values of phosphate emission into groundwater ( 0.07 kg phosphate / ha / a ) and of phosphorus emission into surface water ( 0.245 kg phosphorus / ha / a ) were assumed . assumptions and data about mulch film application and flame treatment are documented in the si , section 6 and 7 . the elaborated lci data can be coupled with any lcia method . in this paper , we show selected results for the impact categories climate change and water stress . results in terms of a lcia method using multiple impact categories were calculated with recipe and are shown in the si , section 14 . human toxicity impacts due to pesticide use , if applied properly , were shown to be relatively small in relation to other impacts like gwp and were excluded in this study . in order to efficiently identify improvement potentials , crops were first ranked according to the impact caused by the total sales volume of a crop ( isc , total in eq 1):1where isc , i , j is the specific impact score per kg of crop c from origin i and produced with mode of production / transportation j , and mc , i , j is the respective mass of crop c sold by the retailer . in addition to the total impact , the sales - amount weighted average impact per kg of product and the variation in specific impact across different origins , production techniques and mode of transportation were also taken into consideration . priority crops for an in - depth investigation were selected by quantifying the maximal ( not necessarily realistic ) improvement potential per crop according to eq 2:2where ic is the maximal improvement potential for crop c ( in % of total current impact ) , mc total is the total mass of crop c sold , isc , average is the sales - amount weighted impact score per kg of crop c and isc , min the minimal specific impact for crop c found in the considered origins and mode of production / transportation . those crops for which the sum of the improvement potentials was larger than one - third of the current co2-footprint were selected for in - depth analysis . figure 2 shows the co2-footprint of fruit and vegetable sales , calculated according to eq 1 ( figure 2a ) and the specific co2-footprint with its variation ( figure 2b ) . asparagus , lettuce and cucumbers were selected for in depth investigation , to derive high - leverage recommendations for a reduction in environmental impact . switching to the respective production alternative with minimal impact for these three crops would achieve a reduction of more than one - third of the current overall co2-footprint caused by the sale of all crops considered ( table 1 ) . other crops like bananas , pears , apples , citrus fruits , and potatoes also cause a relatively large total co2-footprint because of large amounts sold , but due to their small specific impact the potential for improvement is limited . relative global warming potential ( gwp ) in % of the total gwp generated by all considered fruits and vegetables sold in 2007 ( ordered from top to bottom , 2a ) and sales - amount weighted impact per kg of product ( 2b ) . the error bars denote the minimum and maximum specific impact over all options assessed ( varying origin , means of transportation , production modes , etc . ) . asparagus was clearly the most important crop to be analyzed according to the ranking scheme applied . figure 3 shows that the main load of the gwp originates from air transport from mexico and peru . the carbon footprint of different origins and transportation options differs by a factor of 1619 , respectively , from the lowest ( produced locally in switzerland ) to the highest ( imported by airplane from mexico ( green asparagus ) and peru ( white asparagus ) ) . therefore , a recommendation to reduce air transport and to encourage seasonal production from near regions was derived . gwp of green and white asparagus imported to switzerland from different countries of origin . for the remaining crops , classified as high priority to reduce the carbon footprint , the main driver of impact was greenhouse heating with fossil fuels during production out of season . for example , a comparison between swiss cucumber production from unheated and heated greenhouses shows a gwp - difference by a factor of more than 10 ( figure 4 ) . a large difference between heated and nonheated production can also be observed for eggplants ( factor of 6 ) , tomatoes and peppers ( both factor of 4 ) and lettuce ( factor of 10 ) . emissions including those from fossil fuel - heating are not evenly distributed over the whole season . the results of the gwp combined with the seasonal heating energy model are shown for a swiss lettuce production in figure 4 . gwp of cucumbers grown either unheated or in ( with an annual average amount of heating oil ) fossil fuel heated greenhouses ( a ) . gwp of lettuce at harvesting time produced in a greenhouse for a year - round production ( b ) . energy demand for cool storage induces less gwp than import by ship from southern countries . for example comparing kiwis imported from italy and new zealand , import from italy is always less co2-eq . intensive , even when considering 36% higher yields , which have been reported for new zealand . different scenarios of the total gwp of the fruits and vegetables assessed reveal a reduction potential of 42% changing from the scenario with air - freighted oversea - asparagus and vegetables produced in heated greenhouses in northern europe to a supply without air transport and fossil fuel heated greenhouse productions . without air transport , asparagus alone bears a gwp - reduction potential of 20% . a similar reduction ( 22% ) can potentially be achieved by avoiding vegetables from heated greenhouses and sourcing them from southern countries during winter and spring , or , even better , from heated greenhouses with waste heat from other industrial processes . in figure 5b , the water consumed during the production of selected fruits of different origins is weighted by the water stress index ( wsi ) . differences in the environmental impact are mostly caused by water scarcity of a specific region and the ratio of irrigated water consumed to the yield . the impact is clearly visible for the asparagus and avocado production ( figure 5b ) , whereas for the other fruits and vegetables it is not . in some cases , a good water performance can be in contradiction to a good gwp performance , as in the case of citrus fruits from israel ( si , section 15 ) . in other cases , both indicators are in accordance , such as in the case of seasonal production of fruits and vegetables from switzerland , which have a low impact with respect to both indicators . fraction of water stress ( in % and ordered from top to bottom ) caused by the sales volume in 2007 normalized by the sum of water stress of all crops ( 5a ) and sales - amount weighted water stress ( irrigation water ( m)wsi ) per kg of ( 5b ) . several measures have been implemented to reduce the large impact due to air transport . products transported by air freight are declared with a label by air and the emissions are fully compensated through offsetting schemes . through efficient logistics and improved storage techniques the amount of white asparagus transported from overseas by ship was increased from 5090% from 2007 to 2009 . however , green asparagus is still not transported by ship from overseas due to substantial losses . to lower the impact of the green asparagus imported by air - freight the retailer decided not to sell this product at discount prices anymore since spring 2009 . with this measure it was possible to reduce the emissions from air - transported asparagus by 75% from 2008 to 2009 . in addition , a new production site in taroudant , morocco is being established to avoid air transport dependency . furthermore , the results of the study were communicated to the purchasing staff ( in the forms of a report , a leaflet and a calculation tool ) to enable an environmentally informed supply chain management for all products . airplane transport dominated the carbon footprint of fruits and vegetables , that is , asparagus and papaya . a decision recommendation for consumers could be , for instance , that seasonal consumption of local foods is to be preferred over out - of - season fruits and vegetables that are imported by plane . for retailers it is recommended to avoid long - distance transports or to prefer transport by ship whenever possible . these results are in accordance with the studies of jungbluth et al . or sim et al . , but differ from weber et al . , who conclude that foodmiles in the u.s . another general result is that greenhouse heating may be a key process for vegetables that are grown out of season in colder climates . in many cases , heating greenhouses with fossil fuels was more important than ground transport , even if distances were long ( e.g. , south spain to switzerland ) . thus , during winter and spring it is often better to purchase vegetables that are grown in greenhouses from southern countries , where no heating is needed , while during summer or fall , local production is often better than imports . however , there is often a trade - off between the relatively low carbon footprint of winter and spring production in southern countries and the water stress induced in these countries , a situation that needs to be carefully assessed case by case . the use of heating systems with nonfossil energy and particularly waste heat could be a solution which may reduce both carbon footprint and water stress impacts . some greenhouses functioning with waste heat are already in operation , for example , the greenhouse attached to a municipal solid waste incineration in hinwil , and the tropical centers in frutigen and wolhusen , switzerland , which are heated with geothermal heat ( warm water effluent from a tunnel ) and waste heat from a gas concentration unit respectively . the decision recommendation for food producers would thus be to search for such alternative heat energy sources or to avoid heating as much as possible . the latter is already standard practice for organic producers in switzerland , as heating is only permitted to avoid harvest losses from freezing temperatures according to the standards of bio suisse . emissions by sourcing their greenhouse - grown products locally during the season . in winter and spring they should look for imports from warmer locations , provided that there are no adverse effects such as water stress ( and further impacts not investigated here ) . retailers are suggested to use results from lca studies , to decide where to source each fruit and vegetable from , and which aspects to improve in collaboration with the producers in each case . they could also label best - practice products , although the communication of lca - results to consumers is a challenging task and consumer organizations already warn against too much and too complex information on products . finally , consumers should buy seasonal products or local products that can be stored over the season as much as possible to avoid both long - distance and air transport , as well as greenhouse heating . moreover , it is desirable that crops with low specific impact are consumed in large amounts , as is already the case for pear , grape , potato , melon , carrot , etc . to enable such decisions , policy makers should ensure that retailers label the origin , transportation , and mode of production of their products . storage energy is in some cases significant , and efficient cooling technologies are fairly important . nevertheless , local production combined with long storage tends to perform better than long - distance imports from countries like new zealand , which is for certain crops , such as kiwi and apple , a relevant country for imports into switzerland . our results are in accordance with blanke et al . , but in contradiction withmil i canals , who considered 540% loss for apples which are stored for 410 months . the latter assumption is justified for apples consumed in european spring . in many purchasing decisions , retailers or consumers can generate significant savings in environmental impacts by following simple guidelines as outlined above . although the study has been made for a swiss retailer , the lci data are adaptable to assortments of other retailers worldwide . some key pieces of information about the supply chain like crop , origin , transportation mode , and sales numbers were provided by the retailer . the inventory data are based on this information and use generic data for the production processes , for example , swiss averages from the horticultural association , which produces according to gap . however , it should be noted that variability is large between regions and even between farms . for example , eutrophying emissions are a function of many parameters including climatic factors . thus , our average data is rather uncertain and may need to be revised particularly for countries without gap - tradition in the field of fertilization , yield and machinery use and in case the data is applied to retailers which do not make sure that gap is followed by all suppliers . the storage lives of the analyzed products vary from 10 days to half a year , something which has , among other factors , an influence on the amount of food losses . food losses may be significant and should be assessed , although we were not able to collect representative data within this study . data on food losses are specific for each retailer , supply chain and crop . thus , such data should be added to the inventory data when performing lca studies . in the particular case of the commissioner of this study , it was decided that the highest leverage decisions can be taken on the levels of purchasing decisions of the retailers and communication to producers . the rational was that only sustainable products should be offered ( also for social standards which are not discussed in this paper ) , so that the consumers can buy any product without violating minimum standards and the vast majority of customers is covered . additionally , consumer information such as origin and mode of production of all fruits and vegetables are provided so that environmentally educated consumers have the chance to choose the environmentally friendliest product among those offered . the results of the implemented measures shows that the reduction potential identified by a lca - analysis and implemented into daily business can lower the overall impact without substantially compromising the company economically . it also demonstrates the opportunities of retailers for reducing environmental impacts of food consumption . food products are known to have significant environmental impacts other than climate change and water use impacts . those other potential impacts should be covered in a lca complementing the carbon and water evaluation to avoid problem shifting . further environmental effects of concerninclude impacts from land use , eutrophication and toxic effects . while for some of these impacts ( e.g. , ecotoxicity and eutrophication ) standard assessment methods exist , methodological developments are needed for others ( e.g. , soil fertility , erosion , salinization , and biodiversity impacts ) . a complete lcia including these impact categories is also needed for a fair comparison between organic and intensive production systems . furthermore , the assessment could be expanded to an analysis from cradle to grave , including the use phase ( transport from the store to where it is consumed , preparation like e.g. cooking , etc . ) and especially the food losses over the whole chain .

food production and consumption is known to have significant environmental impacts . in the present work , the life cycle assessment methodology is used for the environmental assessment of an assortment of 34 fruits and vegetables of a large swiss retailer , with the aim of providing environmental decision - support to the retailer and establishing life cycle inventories ( lci ) also applicable to other case studies . the lci includes , among others , seedling production , farm machinery use , fuels for the heating of greenhouses , irrigation , fertilizers , pesticides , storage and transport to and within switzerland . the results show that the largest reduction of environmental impacts can be achieved by consuming seasonal fruits and vegetables , followed by reduction of transport by airplane . sourcing fruits and vegetables locally is only a good strategy to reduce the carbon footprint if no greenhouse heating with fossil fuels is involved . the impact of water consumption depends on the location of agricultural production . for some crops a trade - off between the carbon footprint and the induced water stress is observed . the results were used by the retailer to support the purchasing decisions and improve the supply chain management .

Introduction Materials and Methods Results Discussion Outlook

PMC2664636

such molecules are found among screening hit lists , biological reagents , and even marketed drugs . these aggregates have the unusual property of nonspecifically inhibiting enzyme targets , leading to false positive hits in biochemical assays , a problem that is now well - recognized , particularly in high - throughput screening . still , exactly how aggregates cause inhibition remains poorly understood.(21 ) here we revisit the specific mechanism of nonspecific inhibition by investigating the structural changes that are induced in the enzyme upon binding to the aggregate . in 2003 mcgovern et al . observed three mechanistic features of small molecule aggregates that guided our investigation.(22 ) first , inhibition occurs via the direct binding of enzyme to aggregate , as shown by ( 1 ) the ability to sediment proteinaggregate complexes with centrifugation , ( 2 ) the punctate fluorescence observed by microscopy in mixtures of aggregates with green fluorescent protein ( gfpa ) , aabbreviations : gfp , green fluorescent protein ; hdx ms , hydrogendeuterium exchange mass spectrometry ; esi lcms , electrospray ionization liquid chromatographymass spectrometry ; maldi , matrix - assisted laser desorption / ionization ; cac , critical aggregation concentration . and ( 3 ) the direct observation of proteinaggregate complexes by transmission electron microscopy . second , aggregate - based inhibition can be rapidly reversed by the addition of a nonionic detergent such as triton x-100 , indicating that enzyme can quickly ( within tens of seconds ) regain activity from aggregate - based inhibition . last , several experiments appeared to be inconsistent with denaturation as a potential mechanism of action . for example , it seemed unlikely that enzyme could rapidly refold into its active state upon the addition of detergent if it were completely denatured when bound to the aggregate . it seemed equally unlikely that gfp could retain its fluorescence if it were completely denatured while bound to an aggregate . two other experiments suggested that inhibition was not due to denaturation : ( 1 ) additional denaturants such as guanidinium or urea did not increase inhibition by aggregates ( if anything , inhibition was decreased ) and ( 2 ) a destabilized mutant appeared to be no more sensitive to aggregate - based inhibition than its wild type counterpart . abbreviations : gfp , green fluorescent protein ; hdx ms , hydrogendeuterium exchange mass spectrometry ; esi lcms , electrospray ionization liquid chromatographymass spectrometry ; maldi , matrix - assisted laser desorption / ionization ; cac , critical aggregation concentration . as a result of mcgovern s work , we considered three possible mechanisms of action that might explain aggregate - based inhibition ( figure 1 ) . although we did not believe that there was large scale unfolding of the enzyme , it still seemed reasonable that there might be small - scale or local unfolding , which has also been proposed by ryan et al.(23 ) on the other hand , aggregate binding may have the opposite effect : instead of increasing flexibility , it may rigidify it , restricting those dynamic motions necessary for catalysis . finally , aggregates may physically sequester enzyme away from substrate . to explore these potential mechanisms , we chose to use hydrogendeuterium exchange mass spectrometry ( hdx ms ) , a technique widely used to measure changes in solvent accessibility for processes such as enzyme unfolding or proteinprotein interactions . hdx ms relies on the different exchange rates of the backbone amide protons with a deuterated solvent , which are measured by the change in mass as deuterium replaces hydrogen . to investigate changes in solvent accessibility , we quantified deuterium exchange of ampc -lactamase over 8 h in the presence or absence of an aggregating inhibitor , rottlerin . the differences in solvent accessibility were not localized to specific regions ( given the nonspecific nature of aggregate - based inhibition , we did not expect to see peptide - specific interactions ) ; rather , we observed a general trend across all peptides . the differences in solvent accessibility that we observed by mass spectrometry suggested that we may also see differences in protease sensitivity , which we investigated by gel electrophoresis of tryptic digests of our model enzyme in the presence or absence of several known aggregating inhibitors . combined , these experiments suggest small scale enzyme unfolding as a molecular mechanism for aggregate - based inhibition . ( c ) the aggregate physically blocks the active site and sequesters enzyme away from substrate . to examine the structural changes that occur in an enzyme when bound to a small molecule aggregate , we began by measuring changes in solvent accessibility using hdx ms . the experiments were conducted with ampc -lactamase , which is perhaps the enzyme best characterized for aggregate - based inhibition . rottlerin was chosen as a model aggregator because of the large concentration range between its critical aggregation concentration ( cac ) and when it begins to precipitate and because of its relatively high potency as an aggregator ( low micromolar ic50 vs ampc ) . -lactamase was incubated in deuterated mops buffer between 10 min and 8 h , in either the presence or absence of rottlerin . after exchange , the aggregates were disrupted with detergent and the exchange reaction was quenched by the addition of cold acid . these peptides were then separated and analyzed by electrospray ionization liquid chromatographymass spectrometry ( esi lcms ) . two modifications were necessary to accommodate the presence of aggregates in the hdx ms experiment . first , aggregateenzyme complexes had to be exchanged in the deuterated buffer at a lower concentration and then concentrated before further sample preparation . aggregates are a phase between soluble , free small molecule and precipitant , the latter of which does not typically inhibit enzymes , so there is an upper limit to the concentration that we can take an aggregating inhibitor : the concentration when it ceases to form more aggregates and instead begins to precipitate . these hdx ms experiments required micromolar concentrations of protein , and if a corresponding concentration of inhibitor were used , we would need an inhibitor that was soluble at millimolar concentrations . as aggregation is a form of insolubility , there are very few examples of molecules that have such high solubility and these molecules are often weak aggregators and unsuitable for this analysis . since we did not have an aggregator that could be used at millimolar concentrations , we instead reduced the concentration of the enzyme so that it would be mostly inhibited by a preprecipitant colloidal form of the aggregator ( 100 m for rottlerin ) . we relied on the fact that aggregates and aggregate - bound enzyme can be pelleted and therefore concentrated by centrifugation . although incubation was performed at a lower concentration of enzyme , we could concentrate the enzymeaggregate complexes prior to analysis by collecting the pellet and removing the supernatant . this has the additional benefit of guaranteeing that predominantly aggregate - bound complexes would be analyzed , as free enzyme would not be pulled down by centrifugation and would be many - fold lower in concentration . it was necessary to add triton mainly to release bound enzyme from the aggregates so that the complexes were not pulled down again when centrifugation was used to remove the immobilized pepsin . this resulted in a delicate balance between using enough detergent to release the enzyme for analysis but not so much that the detergent signals overwhelmed the peptide signals in the mass spectra , where triton signals were both numerous and strong . to increase separation between the peptide and detergent signals , we used esi lcms rather than matrix - assisted laser desorption / ionization ( maldi , which does not require peptide separation ) . as a result of the presence of aggregates , the inhibited samples generally showed much weaker signal intensities compared to uninhibited samples . often , the peptide signals were so weak that they could not be analyzed . the peptides that we reproducibly observed covered 41% of the -lactamase sequence ( figure 2 ) , representing several regions of the enzyme spanning both buried and exposed regions . although our results are not strong enough to determine whether specific areas experienced more exchange than others , it was also not our goal to do so . given that aggregates are nonspecific inhibitors , we were searching for a global effect , a mechanism that could explain inhibition of many enzymes and that was not restricted to specific residues or peptide sequences . the results suggested such a trend . across all of the peptides that we measured , enzymeaggregate samples showed deuterium incorporation greater than or equal to the deuterium incorporation of the enzyme alone ( table 1 ) . levels of deuteration were very low in both samples with and without inhibitor ; however , the trend of higher deuterium content in the aggregate - containing samples is consistent across all of the peptides . we suspect the low levels of incorporation are due to increased back exchange occurring during the protein concentration and chromatography steps . many peptides incorporated levels of deuterium that were not , in themselves , entirely convincing ; however , even here , each showed more deuterium exchange in the presence of aggregates than for the free enzyme . we never observed a peptide that had reduced deuterium exchange in the presence of aggregates . two peptides that did show significant exchange are shown in figure 3 ( a complete list of peptides is available in the supporting information ) . again , we observed very low deuterium incorporation , but the time points repeatedly indicated a significant difference between samples with and without aggregates . the higher deuterium incorporation of aggregate - bound enzyme across all 10 peptides suggested that the enzyme may be unfolded when bound to the aggregate . amino acid sequence and structure of ampc -lactamase : ( a ) enzyme sequence showing the peptic fragments that could be quantified reproducibly in the mass spectrum both with and without the aggregator rottlerin ; ( b ) two orientations of the structure of ampc -lactamase indicating the location of the peptic fragments that were observed in the mass spectrum ( fragments shown in purple , active site residues shown in yellow).(39 ) equal to number of amide bonds + 1 , excluding prolines . deuterium incorporation is calculated by subtracting the mass of the control undeuterated centroid from the centroid of the deuterated sample . deuterium incorporation is calculated as the ( deuterated mass charge charge ) minus ( undeuterated mass charge charge ) with the standard deviation of three replicate measurements . average ratio of side chain surface area to random coil values per residue calculated using getarea.(38 ) greater than 50% is considered exposed , and less that 20% is considered buried . mass envelopes and corresponding deuterium incorporation plots for two fragments from the mass spectrum of -lactamase in the presence ( dotted line ) or absence ( solid line ) of the aggregator rottlerin . the spectra have been expanded to show the isotopic distribution of the ions of interest : ( a ) the peptide containing residues 132146 ( monoisotopic m / z = 908.4 , + 2 charge state ) and ( b ) the peptide containing residues 291322 ( monoisotopic m / z = 813.7 , + 4 charge state ) . for each peptide , the isotopic distributions are shown for ( i ) the undeuterated sample , ( ii ) the sample that was deuterated for 4 h in the absence of rottlerin , and ( iii ) the sample that was deuterated for 4 h in the presence of rottlerin . if aggregates were unfolding bound enzyme , we might expect aggregate - bound enzyme to be more susceptible to proteolytic degradation than free , uninhibited enzyme . if an enzyme is even slightly unfolded , it should be measurably more sensitive to digestion by proteases ( figure 4 ) . given recent results showing the very slow off rate of enzyme from the aggregate and that the aggregate can be saturated with enzyme and so maintain a large excess of uninhibited protease , this experiment seemed feasible . we chose five known aggregators : rottlerin , congo red , eriochrome blue black b , nicardipine , and l-755,507 . -lactamase was preincubated with or without aggregates before the addition of trypsin to avoid competition with trypsin for binding space on the aggregate . free or aggregate - bound enzyme was then incubated with trypsin between zero min and 4 h , and the digestion was monitored by gel electrophoresis . if the enzyme is unfolded on the aggregate , it should be more susceptible to proteolysis compared to enzyme in the absence of aggregates ( a ) . if the enzyme is in its native state when bound to the aggregate , identical to unbound enzyme , it should be degraded at a comparable rate ( b ) . although some trypsin may be bound to the aggregate , trypsin is used in excess to ensure that there is free , active trypsin in solution : ( + ) indicates that aggregates are present ; ( ) indicates free enzyme . for all of the aggregators , the presence of aggregates had no effect on the band representing -lactamase in the absence of trypsin ( figure 5 , lanes 2 and 3 or 1 and 3 ) . in contrast , when we added trypsin , we observed significant digestion in the presence of aggregates but not in the absence ( figure 5 ) . for example , in figure 5a , the -lactamase band is present in both the aggregate - inhibited and free enzyme samples at zero minutes ( lanes 6 and 7 ) , but as time progresses , the -lactamase band in the presence of rottlerin becomes weaker and weaker until it is completely gone by 4 h ( lane 15 ) . conversely , -lactamase in the absence of rottlerin appears almost as strong after 4 h with trypsin as it did after zero min ( lane 14 ) . for congo red and eriochrome , the -lactamase band is present in both samples at zero min of trypsin incubation ( figure 5b , c , lanes 6 and 7 ) , but after only 15 min the -lactamase band is gone in the presence of aggregates ( lane 9 ) . again , -lactamase in the absence of aggregates remains undigested by trypsin even after 4 h ( lane 14 ) . although the effect was less pronounced , two other aggregators , nicardipine and l-755,507 , also increased sensitivity to trypsin ( figure 5d , e ) . in many of these samples , it is also possible to observe the formation of a large degradation product of -lactamase running below the trypsin band ( figure 5 ) . intriguingly , trypsin also appears to experience more self - degradation in the presence of aggregators ( figure 5a , c , lanes 4 and 5 ) . sdspage and silver stain of tryptic digests of -lactamase in the presence ( + ) or absence ( ) of aggregating inhibitors . the inhibitors are ( a ) 100 m rottlerin , the same inhibitor used in the hdx ms experiments , ( b ) 250 m congo red , ( c ) 250 m eriochrome blue black b , ( d ) 100 m l-755,507 , and ( e ) 250 m nicardipine . for gels b , c , and e , lane 1 is a molecular weight ladder . for gels a and d , lane 2 is a molecular weight ladder . lanes 2 and 3 for gels b , c , and e or lanes 1 and 3 for gels a and d show -lactamase without trypsin , in the absence ( ) or presence ( + ) of aggregator . lanes 4 and 5 contain trypsin ( no -lactamase ) in the absence and presence of aggregator , respectively . lanes 615 contain tryptic digests of -lactamase in the absence or presence of aggregator for digestion times of 0 min , 15 min , 30 min , 1 h , and 4 h ( from left to right ) . trypsin was used at a concentration of 0.01 mg / ml for ( a ) , 0.025 mg / ml for ( b ) , ( c ) , and ( d ) , and 0.05 mg / ml for ( e ) . a suspected degradation product of ampc is indicated with an arrow and labeled dp . the key result to emerge from this study is the mechanism by which association with colloidal aggregates leads to enzyme inhibition : partial protein denaturation . the increased hydrogendeuterium exchange of -lactamase when bound to rottlerin aggregates suggests that the enzyme s backbone amides are more exposed to solvent when bound to an aggregate , indicating at least partial enzyme denaturation ( figure 3 ) . consistent with this view is the increased susceptibility of the enzyme to trypsin degradation when bound to the characteristic aggregators rottlerin , congo red , eriochrome blue black b , nicardipine , and l-755,507 ( figure 5 ) . that proteins directly associate with aggregates has been established for some time and is supported by the precipitation of aggregate - bound protein on centrifugation , by the detection of aggregate binding with surface plasmon resonance,(20 ) and by imaging of aggregateprotein complexes by transmission electron microscopy and fluorescence microscopy.(22 ) what remained perplexing was why such association should lead to inhibition . proteins can be attached to solid supports , as in chromatography , without compromising their function , and so association , per se , seemed unlikely to lead to inhibition . an early hypothesis was that the aggregates were acting as denaturants , but several lines of evidence seemed to tilt against this . first , gfp retained fluorescence while bound to aggregate particles , which should not be true if gfp was substantially denatured on aggregate association . second , destabilized and up - stabilized mutants of the same enzyme were inhibited to the same extent by colloidal inhibitors , indicating that liability to denaturation did not increase susceptibility to aggregate - based inhibition . third , classical denaturants such as urea or guanidinium failed to potentiate aggregate - based inhibition ; rather , inhibition was actually reduced in the presence of these denaturants . finally , disruption of the aggregates by detergent returned enzyme activity within the dead time of the experiment , less than 15 s , suggesting that no substantial refolding was occurring . whereas none of these experiments were in themselves conclusive , taken together they alluded to another , unknown mechanism behind aggregate - based inhibition , and our understanding has remained at this point for the past 5 years . we can reconcile the earlier and present observations by noting that the previous study only seemed to exclude large scale protein denaturation . more local denaturation would not be expected to extinguish gfp fluorescence(36 ) nor would substantial refolding be necessary for the enzyme to regain activity upon aggregate disruption by detergent . as to the reduced inhibition observed in the presence of classical denaturants , we have since discovered that urea and guanidinium themselves disrupt colloidal aggregates , preventing any additive denaturant effect . finally , our ability to interpret the inhibitory effects of colloidal aggregates on destabilized and up - stabilized mutant enzymes was confounded by the cac of the colloidal particles and their unusually tight binding to proteins . neither of these phenomena were understood in 2003 , but recent studies have shown that aggregates undergo a cac phase transition , above which their affinities for proteins are in the picomolar dissociation range or better . given micromolar cac values and picomolar kd values for the proteinaggregate interaction , one would not expect to see differential inhibition of the mutant enzymes that were more or less stabilized by no more than 5 kcal / mol . returning to our three models ( figure 1 ) , we have ruled out reduced dynamics and physical sequestration , as both would have resulted in reduced solvent accessibility ( less deuterium exchange ) and protection from proteolysis ; rather , the reverse is observed in both cases . taken together , our previous studies and current experiments suggest that on association with colloidal , promiscuous inhibitors , proteins undergo partial denaturation , which manifests itself as enzyme inhibition . in such a mechanism , hydrogendeuterium exchange would increase , as would proteolysis , but the enzyme would not be so denatured that it could not rapidly return to its active conformation or that gfp would not retain fluorescence . two important caveats to these conclusions are the weak peptide signals in the mass spectra and the low overall deuterium incorporation . the presence of the aggregates interfered with the hdx ms , reducing signal - to - noise and experimental reproducibility . we suspect that we were only partially successful at disrupting aggregateenzyme complexes , and so lost peptide during sample preparation . typically this problem would be addressed by the addition of further triton x-100 , but this posed its own hazards for mass spectrometry because the detergent peaks easily overwhelmed our peptide signals . detergents better tolerated by mass spectrometry , such as -octaglucoside , were ineffective at disrupting the aggregates to free the peptides for analysis . the levels of deuterium incorporation in both the aggregate - inhibited and uninhibited samples were disconcertingly low , which we attribute to high back exchange . combined with the complicating factors of low peptide signal strength and overwhelming detergent signals , the chromatographic run time had to be increased to allow separation of the peptide and detergent peaks , thus leading to more back exchange . we suspect that the somewhat erratic incorporation of deuterium as a function of time is due to the low deuterium content ( which is more subject to signal - to - noise interference ) . because of the low signal - to - noise and low deuterium incorporation , we draw no conclusions about which parts of the enzyme are more exposed to solvent upon aggregate binding , as would ordinarily be tempting to do with this technique . that admitted , every single peptide that we observed had greater hydrogendeuterium exchange in the aggregate - bound protein compared to the free protein . this suggests that whereas we can not resolve the specific parts of -lactamase that are more denatured , the enzyme is being partially denatured . inhibition by each of five different aggregators increased susceptibility of -lactamase to proteolysis to the point where degradation was apparent within 15 min of trypsin addition , whereas digestion of uninhibited enzyme took place over several hours ( figure 5 ) . partial protein denaturation on association with colloidal aggregates offers the first molecular mechanism for the inhibitory effects of these particles . this mechanism is attractive in that it is general , relying on no particular feature of enzyme or aggregate structure . all that would be required are the nonpolar , buried residues that all enzymes possess , which might be expected to associate with the nonpolar surfaces that the colloidal aggregates inevitably feature . it also suggests the opportunity for more detailed , biophysical studies of the sort that have long been used to characterize protein stability and hydrophobic binding . as an aside , we note that as denaturants , colloidal aggregates are peculiar . their denaturation - by - sequestration mechanism on large isolatable particles may be unique and may lend itself to pragmatic uses . these might include the ability to sequester proteins in a relatively concentrated , inactive form and from a medicinal chemistry perspective to affect the adsorption and distribution of drugs and reagents.(4 ) this would be an interesting twist for a species that until now , owing to its pervasiveness in early drug and reagent discovery , has been only a great and recurring problem . at this time , particle formation appears to be a common feature of organic molecules in aqueous buffer : it is ubiquitous in early drug discovery and particularly problematic in high - throughput screening . as has been known for some time , small molecule colloidal aggregates inhibit protein via direct binding , an association that we now believe is surprisingly tight , picomolar or better . the low dissociation constant results in little to no measurable exchange between aggregate - bound and free protein.(33 ) conversely , the aggregates themselves are in dynamic equilibrium with their monomer small molecule components and will rapidly dissociate when diluted below their threshold of formation , the cac.(35 ) as an aside , we note that the cac of an organic molecule in a particular buffer will be relatively invariant . whether this molecule , above its cac , will observably inhibit depends on other variables , including the stoichiometric ratio of the protein it is sequestering ; if unrecognized , this can lead to confusion in reconciling the behavior of a particular molecule from assay to assay . the aggregates appear to be solid , densely packed particles , the larger of which can bind on the order of 10 000 protein molecules . although we have yet to conclusively determine that protein is not absorbed within the aggregate , adsorption to the surface has been observed by microscopy(22 ) and our calculations indicate that the particle surface has more than sufficient binding capacity to accommodate all bound protein.(35 ) the final insight to this picture is the conclusion that we have drawn here : that the aggregateprotein interaction results in partial denaturation and subsequent inhibition . this model appears to be true for a number of aggregating molecules , suggesting it may be general , but it remains unclear whether there may be different subtypes of aggregates . evidence of differing detergent sensitivity , size , and binding strength suggest that there are aggregates that behave uncharacteristically and may operate by additional mechanisms . regardless , we now have a far clearer understanding of these so often perplexing particles . ampc -lactamase was expressed and purified as previously described.(37 ) rottlerin , congo red , eriochrome blue black b , nicardipine , trypsin from porcine pancreas , and trifluoroacetic acid were purchased from sigma - aldrich . ampc -lactamase ( 215 m stock in 50 mm kpi , ph 7.0 ) was delivered to the deuterated solvent ( 50 mm mops buffer in d2o , ph 7.0 ) to a final concentration of 16 m for uninhibited enzyme or 0.16 m for samples containing the inhibitor rottlerin . the total volume of samples without rottlerin ( including the undeuterated and 100% deuterated controls ) was 10 l , whereas the total volume for each sample with rottlerin was 1 ml . the concentration of rottlerin in these samples was 100 m ( 0.5% dmso ) delivered from a 20 mm stock in dmso . the difference in -lactamase concentration and total volume was to accommodate for the limited solubility of rottlerin . after incubation in the deuterated solvent for 5 min or 1 , 2 , 4 , or 8 h , the samples containing rottlerin were centrifuged for 5 min at 16000 g to pull down and concentrate aggregateenzyme complexes and the top 990 l amount was discarded . the bottom 10 l volume was considered the pellet and were treated identically to the 10 l samples without rottlerin from this point on . to disrupt the aggregates , 1 l of 3% triton x-100 in 50 mm deuterated mops buffer was added to every sample and the samples were mixed vigorously by pipetting . immediately after , the samples were quenched with 90 l of ice cold 0.1% trifluoroacetic acid in water , ph 2.5 . samples were kept on ice with occasional mixing for 5 min , and then the pepsin beads were sedimented by centrifugation for 1 min at 16000 g at 4 c . the supernatant was collected and frozen with liquid nitrogen . the mass spectrometry data were acquired on an applied biosystems qstar pulsar hybrid lc / ms / ms system . chromatography was performed using an eldex micropro pump equipped with an in - house splitter capable of producing column flow rates of 0.31.0 l / min at pump flow rates of 5080 l / min . a phenomenex onyx monolithic column ( 0.100 mm 150 mm ) samples were injected manually using a valco c2 - 1006 injection valve with a 1.0 l loop . solvent a was 0.1% formic acid in water , and solvent b was 0.1% formic acid in acetonitrile . the identities of the peptides formed by digestion with pepsin were determined by lc / ms / ms analysis using the following conditions : column flow rate was 0.4 l / min , and a gradient was run from 3% to 50% solvent b in 35 min . the data obtained were searched against a database consisting only of the ampc sequence using protein prospector . for analysis of deuterated peptide samples , the same chromatography system was used . to minimize back exchange , the column flow rate was raised to 1.0 l / min and the gradient used was from 0% to 70% solvent b in 5 min . furthermore , the injector , injection syringe , sample loop , column , and all transfer lines were maintained in an ice bath . all data analysis was performed with the standard analyst 1.1 software native to the qstar . the average number of deuterons incorporated for each peptide was determined from the mass spectra . the centroid of the isotopic peak cluster for the undeuterated control was subtracted from the centroid of the isotopic peak cluster for each deuterated sample . the experimental conditions were the same in each analysis , and only the difference in deuterium incorporation between identical peptides was measured , so it was not necessary to correct for back exchange . solvent accessible surface area was calculated using getarea ( version 1.0 beta ) available at http://curie.utmb.edu/getarea.html , using a radius of 1.4 and default atomic radii and atomic solvent parameters.(38 ) the average percent accessibility was calculated as the average of the ratios of each side chain surface area to the random coil value . a sample of ampc -lactamase ( 0.5 m ) was incubated in 50 mm kpi , ph 7.0 , for 1 h with or without each aggregating inhibitor : 100 m rottlerin , 250 m congo red , 250 m eriochrome blue black b , 250 m nicardipine , or 100 m l-755,507 ( with a final concentration of 1% dmso in all samples , with or without inhibitor ) . samples of trypsin alone ( 0.01 or 0.025 mg / ml ) were prepared with or without each of the inhibitors and incubated for 1 h at room temperature in 50 mm kpi , ph 7.0 . tryptic digests of -lactamase were prepared by first adding 0.5 m of the enzyme to a solution with or without each inhibitor . after 5 min , trypsin was added to the solution and the digests were incubated at room temperature for 0 min , 15 min , 30 min , 1 h , or 4 h. the total volume of all samples was 25 l . to prepare the samples for sdspage , 5 l of loading buffer ( containing 0.25% bromophenol blue , 0.25% xylene cyanol ff , 30% glycerol by volume , and 5 mm dithiothreitol ) was added to each sample and the samples were boiled for 2 min .

one of the leading sources of false positives in early drug discovery is the formation of organic small molecule aggregates , which inhibit enzymes nonspecifically at micromolar concentrations in aqueous solution . the molecular basis for this widespread problem remains hazy . to investigate the mechanism of inhibition at a molecular level , we determined changes in solvent accessibility that occur when an enzyme binds to an aggregate using hydrogendeuterium exchange mass spectrometry . for ampc -lactamase , binding to aggregates of the small molecule rottlerin increased the deuterium exchange of all 10 reproducibly detectable peptides , which covered 41% of the sequence of -lactamase . this suggested a global increase in proton accessibility upon aggregate binding , consistent with denaturation . we then investigated whether enzymeaggregate complexes were more susceptible to proteolysis than uninhibited enzyme . for five aggregators , trypsin degradation of -lactamase increased substantially when -lactamase was inhibited by aggregates , whereas uninhibited enzyme was generally stable to digestion . combined , these results suggest that the mechanism of action of aggregate - based inhibitors proceeds via partial protein unfolding when bound to an aggregate particle .

Introduction Results Discussion Experimental Section

PMC2999716

many studies have shown a significant role for thyroid hormones in the development and maturation of the mammalian central nervous system.1,2 these hormones regulate axonal and dendritic growth and synapse formation.3 growth retardation and severe cognitive impairment are known complications following thyroid hormone deficiency in the prenatal and neonatal periods.4 it has been shown that hypothyroidism is associated with changes in gene expression in both the central and peripheral nervous system.5 the inability to produce long - term potentiation ( ltp ) in the rat hippocampus and impaired learning and memory in both rats and humans are among the functional consequences of hypothyroidism.6 other studies have suggested that hypothyroidism affects behavioral conditions and is accompanied by emotional symptoms , including lethargy and dysphoria.7 - 11 the results of other studies have shown that several cognitive deficits , including attention and memory processing deficits , general intelligence and visual - spatial skills , are induced by hypothyroidism.9,10 hypothyroidism has a lesser effect on auditory attention , motor skills , language and set - shifting.7,10,12 in addition , there is evidence indicating that even subclinical hypothyroidism is associated with depressive symptoms and cognitive impairment.13 nitric oxide ( no ) , a free radical gas , is known to play critical roles in biologic systems;14 it acts like a diffusible intercellular signaling molecule in the brain and spinal cord.15 no synthase ( nos ) is the enzyme that produces no from l - arginine . no can act as an important mediator in synaptic plasticity , ltp and the consolidation of ltp.16 - 18 in addition , there are reports that suggest a relationship between nmda ( n - methyl - d - aspartate)receptors and the no system in learning and memory.19,20 several studies have shown that nos inhibitors impair the consolidation of memory21,22 and block the induction of ltp.21 - 24 there is , however , some evidence showing that l - arginine , an no precursor , improves memory formation and reverses the effect of nos inhibition.25 there are also reports indicating that no donors activate both ltp and long - term depression.23,26 these results suggest the involvement of no in learning and memory processes . the relationship between thyroid hormones and the no system has been well documented.23,26 - 28 there is evidence that thyroid hormones are involved in regulating no synthase gene expression in the brain . considering the aforementioned findings , this study aimed to investigate the effect of hypothyroidism induced during the neonatal and juvenile periods on learning and memory of offspring and on no metabolite concentrations in the hippocampus . twenty pregnant female wistar rats ( 8 weeks old and weighing 200 20 g ) were kept in separate cages at 22 2 c in a room with a 12 h light / dark cycle ( light on at 7:00 am ) . offspring were randomly divided into two groups and treated according to the experimental protocol from the first day after birth through the first two months of life . rats in the control group received normal drinking water , whereas the second group received the same drinking water supplemented with 0.03% methimazole ( sigma , usa ) to induce hypothyroidism.29 after 60 days , seven male offspring of each group were randomly selected and tested in the morris water maze ( mwm ) . in the methimazole group , hypothyroidism was confirmed by testing serum thyroxine concentration levels using the radioimmunoassay method ( daisource , t4- ria - ct ) . animal handling and all related procedures were carried out in accordance with the rules set by the mashhad university of medical sciences ethical committee . a circular black pool ( 136 cm diameter , 60 cm high , 30 cm deep ) was filled with water ( 2024 c ) . a circular platform ( 10 cm diameter , 28 cm high ) was placed within the pool and was submerged approximately 2 cm below the surface of the water in the center of the southwest quadrant . outside the maze , fixed visual cues were present at various locations around the room ( i.e. , a computer , hardware and posters ) . an infrared camera was mounted above the center of the maze and an infrared led was attached to each rat for motion tracking . before each experiment , each rat was handled daily for 3 days and habituated to the water maze for 30 sec without a platform . the animals performed four trials on each of the eight consecutive days , and each trial began with the rat being placed in the pool and released facing the side wall at one of four positions ( the boundaries of the four quadrants , labeled north ( n ) , east ( e ) , south ( s ) and west ( w ) . the rat was allowed to swim until it found and remained on the platform for 15 seconds . if 60 seconds had passed and the animal had not found the platform , it was guided to the platform by the experimenter and allowed to stay on the platform for 15 sec . the rat was then removed from the pool , dried and placed in its holding bin for 5 min . the time latency to reach the platform and the length of the swimming path were recorded by a video tracking system.29,32 - 34 on the ninth day , the platform was removed , and the animals were allowed to swim for 60 s. the time spent in the target quadrant ( q1 ) and the traveled path were compared between groups . after the last session of the mwm test , blood samples were taken from all rats to determine hypothyroidism status and to measure the no metabolites no2 and no3 ( griess reagent method ) . the animals were then sacrificed , and hippocampi were removed and submitted to no metabolite measurements in the tissue . the griess reaction was adapted to assay nitrates as previously described.35 briefly , standard curves for nitrates ( sigma , st . louis , missouri , usa ) were prepared , and samples ( 50 l plasma and 100 l tissue suspension ) were added to the griess reagent . the proteins were subsequently precipitated by the addition of 50 l of 10% trichloroacetic acid ( sigma ) . the contents were then vortex - mixed and centrifuged , and the supernatants were transferred to a 96-well flat - bottomed microplate . absorbance was read at 520 nm using a microplate reader , and final values were calculated from standard calibration plots.36 swim time latency and the length of the traveled path over the eight training days were analyzed by repeated measures analysis of variance anova . the time spent in the target quadrant ( q1 ) and the length of the swimming path in this quadrant was compared using unpaired t - tests . comparison of serum thyroxin levels and plasma levels of no metabolites was carried out using unpaired t - tests . twenty pregnant female wistar rats ( 8 weeks old and weighing 200 20 g ) were kept in separate cages at 22 2 c in a room with a 12 h light / dark cycle ( light on at 7:00 am ) . offspring were randomly divided into two groups and treated according to the experimental protocol from the first day after birth through the first two months of life . rats in the control group received normal drinking water , whereas the second group received the same drinking water supplemented with 0.03% methimazole ( sigma , usa ) to induce hypothyroidism.29 after 60 days , seven male offspring of each group were randomly selected and tested in the morris water maze ( mwm ) . in the methimazole group , hypothyroidism was confirmed by testing serum thyroxine concentration levels using the radioimmunoassay method ( daisource , t4- ria - ct ) . animal handling and all related procedures were carried out in accordance with the rules set by the mashhad university of medical sciences ethical committee . a circular black pool ( 136 cm diameter , 60 cm high , 30 cm deep ) was filled with water ( 2024 c ) . a circular platform ( 10 cm diameter , 28 cm high ) was placed within the pool and was submerged approximately 2 cm below the surface of the water in the center of the southwest quadrant . outside the maze , fixed visual cues were present at various locations around the room ( i.e. , a computer , hardware and posters ) . an infrared camera was mounted above the center of the maze and an infrared led was attached to each rat for motion tracking . before each experiment , each rat was handled daily for 3 days and habituated to the water maze for 30 sec without a platform . the animals performed four trials on each of the eight consecutive days , and each trial began with the rat being placed in the pool and released facing the side wall at one of four positions ( the boundaries of the four quadrants , labeled north ( n ) , east ( e ) , south ( s ) and west ( w ) . for each trial , the rat was allowed to swim until it found and remained on the platform for 15 seconds . if 60 seconds had passed and the animal had not found the platform , it was guided to the platform by the experimenter and allowed to stay on the platform for 15 sec . the rat was then removed from the pool , dried and placed in its holding bin for 5 min . the time latency to reach the platform and the length of the swimming path were recorded by a video tracking system.29,32 - 34 on the ninth day , the platform was removed , and the animals were allowed to swim for 60 s. the time spent in the target quadrant ( q1 ) and the traveled path were compared between groups . after the last session of the mwm test , blood samples were taken from all rats to determine hypothyroidism status and to measure the no metabolites no2 and no3 ( griess reagent method ) . the animals were then sacrificed , and hippocampi were removed and submitted to no metabolite measurements in the tissue . the griess reaction was adapted to assay nitrates as previously described.35 briefly , standard curves for nitrates ( sigma , st . louis , missouri , usa ) were prepared , and samples ( 50 l plasma and 100 l tissue suspension ) were added to the griess reagent . the proteins were subsequently precipitated by the addition of 50 l of 10% trichloroacetic acid ( sigma ) . the contents were then vortex - mixed and centrifuged , and the supernatants were transferred to a 96-well flat - bottomed microplate . absorbance was read at 520 nm using a microplate reader , and final values were calculated from standard calibration plots.36 swim time latency and the length of the traveled path over the eight training days were analyzed by repeated measures analysis of variance anova . the time spent in the target quadrant ( q1 ) and the length of the swimming path in this quadrant was compared using unpaired t - tests . comparison of serum thyroxin levels and plasma levels of no metabolites was carried out using unpaired t - tests . the serum thyroxine concentration in methimazole - treated animals was significantly lower compared to that of control animals ( p<0.01 ; fig . 1 ) . in addition , the time latency and the length of the swimming path over the eight training days were significantly higher in offspring of the methimazole group ( p<0.001 ; figs . 2 and 3 , respectively ) . in the probe trial , the time spent in the target quadrant ( q1 ) by the offspring of the methimazole group was significantly lower compared to controls ( p<0.001 ; fig . 4a ) . furthermore , the length of the traveled path in q1 by animals in the methimazole group was shorter than for those in the control group ( p<0.001 ; fig . 4b ) . there were no significant differences in the time spent or the length of the swim path in the other quadrants between the two groups . in addition , there were no significant differences between the plasma concentrations of no2 or no3 ; however , the concentration of no metabolites in the hippocampi of the methimazole group offspring was higher than that of the control animals ( p<0.05 ; figs . 5a and 5b , respectively ) . neonatal hypothyroidism has been clearly shown to be related to cognitive dysfunction.9 - 11,37,38 for example , studies in humans have shown that a temporal deficiency in thyroid hormones during developmental periods impairs cognitive functions such as attention , learning and memory.39 experimental hypothyroidism in developing rats has also been shown to result in impaired learning and memory.40,41 in this study we induced hypothyroidism in rats , resulting in impaired learning and memory during the neonatal and juvenile periods . hypothyroidism impairs hippocampal - dependent learning and short- and long - term memory.42,43 in addition , hypothyroidism impairs early and late phases of ltp.44 the exact mechanisms underlying the induced deficits in memory and ltp have not been elucidated . many studies have demonstrated that cognitive dysfunction in hypothyroidism is likely due to abnormal brain development , diminished inter - neuronal connectivity and , in particular , impaired synaptic plasticity in the hippocampus.45 it has been shown that maturation and function of the hippocampus are dependent on thyroid hormones.43 transient hypothyroidism has even been shown to impair synaptic transmission and plasticity in the adult hippocampus.46,47 it has also been reported that impairment of ltp and memory due to hypothyroidism correlates with changes in c - jun and c - fos protein expression and extracellular signal regulated kinases ( erks ) levels in the hippocampus during crucial periods of brain development.48,49 changes in the expression of other proteins such as synapsin i , synaptotagmin i , and syntaxin may also be involved in hypothyroidism - induced memory deficits.50 it has also been suggested that thyroid hormone deficiency results in changes in brain regions such as the dorsal hippocampal - mpfc pathway and changes in the glutamate release , which can lead to cognitive disturbances.51 - 53 moreover , hypothyroidism has been shown to induce oxidative stress in the hippocampus.54 the relationship between oxidative stress , neuronal damage and cognitive dysfunction has been well documented.55 - 58 in addition , it is well known that neonatal hypothyroidism is accompanied by a delay in myelinogenesis and a decrease in the number of myelinated axons.59,60 in the present study , we have shown that a deficiency of thyroid hormones during lactation and in the neonatal period could impair learning of offspring in the mwm test . the animals in the hypothyroid group had significantly higher time latency to find the platform during every day of training ( fig . 2 ) , indicating a deleterious effect of the lack of thyroid hormones on spatial learning processes . the results presented here also confirm that methimazole - induced hypothyroidism during the neonatal and juvenile periods impairs spatial memory given that the animals in the hypothyroid group spent less time in the target quadrant compared to the control group when tested on the ninth day ( probe trial ; fig . the results of the present study are consistent with findings from our previous study , in which we found an impairment in the mwm in adult rats with methimazole - induced hypothyroidism over a 180-day period.29 it has also been reported that treatment of thyroidectomized adult rats with thyroxine improved radial arm water maze tasks and ltp in the ca1 area of the hippocampus.61 these researchers showed that cyclic - amp response element binding protein ( creb ) and mitogen - activated protein kinases ( mapks ) may contribute to the impairment of hippocampal - dependent learning and memory ; however , the authors suggested that calcium - calmodulin - dependent nos and brain - derived neurotrophic factor ( bdnf ) may not have roles in this process.61 the free radical gas no has been associated with different forms of learning and memory and in several forms of synaptic plasticity thought to be involved in memory formation . this association has been widely confirmed via pharmacological studies , which have utilized a variety of substances and methods to inhibit no synthase . results from knockout studies in mice have revealed that mice deficient in enos and nnos expression exhibit impaired ltp.20,61 - 65 it has also been reported that nitrergic neurons , the neurons that produce no , increase in number after spatial learning in rats , which can be interpreted as upregulation induced by behavioral training.63 this evidence implies that no participates in the memory process.63 in contrast to these results , which indicate a positive role for no in learning and memory , it has been shown that inhibition of nos does not prevent the induction of ltp or impair spatial memory . thus , these findings suggest that no does not play an important role in memory and learning.66 here , we have examined the relationship between no and hypothyroidism . we found that both hypothyroidism and impaired mwm performance were present in the methimazole group . we also found a significant increase in hippocampal no metabolites in the methimazole - treated group . the association between hypothyroidism and increased no levels in the brain has been previously investigated . one study indicated that maternal thyroid hormone deficiency during the early gestational period resulted in a significant elevation in neuronal nitric oxide synthase ( nnos ) expression with associated neuronal death in the embryonic rat neocortex.67 it has also been shown that nnos acts as a negative regulator of neurogenesis.67 - 69 the inhibitory effect of no on neurogenesis may be due to a reduction in the proliferative potential of neural precursor cells70 or a defect in the survival of newly generated neurons after differentiation.71 there is evidence showing that thyroid hormone deficiency increases cell death.67 the relationship between nnos and poor survival of neurons and thyroid hormone deprivation has also been suggested.67 it has been shown that even a moderate and transient decrease in maternal thyroxine significantly increases nnos expression , which is reversible by hormone replacement.67,72 in contrast , another report showed that propylthiouracil ( ptu)-treated animals exhibited reduced nos activity that could be rescued by t4 administration.72 it has also been shown that hypothyroidism changes the pattern of nos activity in a tissue - dependent manner . nos activity was significantly increased in both the right and left ventricles , but it was significantly reduced in the aorta , whereas in the vena cava , renal cortex and medulla , the enzyme activity was non - significantly higher compared to controls.73 the results of the present study also showed no difference in no metabolites between the hypothyroid group and controls in peripheral blood samples . it might be suggested that an increase in no in the hippocampus is due to stimulatory effects of thyroid hormone deprivation on nnos activity . although some studies have suggested no to be a neurotransmitter that plays an important role in enhancing memory , others have claimed that its excess in the hippocampus may result in memory deficits.74 no has been shown to have bidirectional effects . under normal physiological conditions , it acts as an important neuronal messenger ; however , when no increases to high concentrations , it has a toxic effect that can lead to neuronal death.75 - 77 a common example of the toxic effect of no in the cns is due to glutamate neurotransmission and the activation n - methyl - d - aspartate ( nmda ) receptors , which leads to significant increases in intracellular calcium , followed by stimulation of neuronal nos.77,78 finally , other findings have suggested selective enhancement of reactive oxygen species ( ros ) and lipid peroxidation in the amygdala and hippocampus of rats after three weeks of treatment with methimazole . these authors showed a significant increase in nos activity,54 suggesting a correlation between increased nos activity and elevated oxidative stress . it may therefore be suggested that increased no production in the hippocampus during hypothyroidism acts as an oxidative stressor , which may explain the deficits in learning and memory observed here . in this study , we found that hypothyroidism induced during the neonatal and juvenile periods resulted in impaired learning and memory of rats tested in the morris water maze . in addition , we found an increase in no metabolites in the hippocampus , suggesting that the impairment of memory in hypothyroidism may be due to increases in nos and no . we would like to thank the vice chancellor of research of mashhad university of medical sciences for financial support .

introduction : severe cognitive impairment follows thyroid hormone deficiency during the neonatal period . the role of nitric oxide ( no ) in learning and memory has been widely investigated.methods:this study aimed to investigate the effect of hypothyroidism during neonatal and juvenile periods on no metabolites in the hippocampi of rats and on learning and memory . animals were divided into two groups and treated for 60 days from the first day of lactation . the control group received regular water , whereas animals in a separate group were given water supplemented with 0.03% methimazole to induce hypothyroidism . male offspring were selected and tested in the morris water maze . samples of blood were collected to measure the metabolites of no , no2 , no3 and thyroxine . the animals were then sacrificed , and their hippocampi were removed to measure the tissue concentrations of no2 and no3.discussion:compared to the control group 's offspring , serum thyroxine levels in the methimazole group 's offspring were significantly lower ( p<0.01 ) . in addition , the swim distance and time latency were significantly higher in the methimazole group ( p<0.001 ) , and the time spent by this group in the target quadrant ( q1 ) during the probe trial was significantly lower ( p<0.001 ) . there was no significant difference in the plasma levels of no metabolites between the two groups ; however , significantly higher no metabolite levels in the hippocampi of the methimazole group were observed compared to controls ( p<0.05).conclusion : these results suggest that the increased no level in the hippocampus may play a role in the learning and memory deficits observed in childhood hypothyroidism ; however , the precise underlying mechanism(s ) remains to be elucidated .

INTRODUCTION MATERIALS AND METHODS Animals and treatments Morris water maze apparatus and procedures Biochemical assessment Statistical analysis RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS

PMC4569820

parasitological study - laboratory breeding scheme for snails - the snails used in this study were originally obtained from water channels running in alexandria governorate ( egypt ) and were maintained for two years in the parasitology department , faculty of medicine , alexandria university , egypt . the snails were housed in glass aquaria containing snail - conditioned water and were fed lettuce leaves , tetramine fish food and calcium carbonate , under suitable environmental laboratory conditions , using the method described by eveland and haseeb ( 2011 ) . source and maintenance of the parasite - s. mansonicercariae were originally harvested from shedding b. alexandrinasnails naturally infected with an egyptian strain of s. mansoni and collected from alexandria water channels . maintenance of the s. mansoni life cycle was conducted between our laboratory snails and swiss strain albino mice ( el naga et al . groups of 10 snails were placed in 200 ml beakers containing aged de - chlorinated tap water ( dtw ) under direct sunlight for 2 h. each cercarial suspension was carefully shaken to obtain an even mixture and the average number of cercariae per ml was estimated . mice were infected with 100 cercariae / mouse using the paddling tail - immersion technique according to the method described by dettman et al . all work with laboratory animals was conducted in accordance with the egyptian national animal welfare standards and was approved by the ethical committee of the faculty of medicine , alexandria university . at seven weeks post - infection ( p.i . ) , mice were sacrificed and their livers were used as a source of parasite eggs . the snails were exposed individually to eight-10 vigorously swimming freshly hatched miracidia under direct sunlight for 3 - 4 h and then were kept in the dark and maintained under previously described conditions ( el naga et al . 2010 , eveland & haseeb 2011 , mostafa & el - dafrawy 2011 ) . selection of susceptible and resistant snail stocks - separation of resistant and susceptible isolates was achieved according to zanotti - magalhes ( 1997 ) . for the selection of resistant individuals , snails that remained uninfected after exposure to infection susceptible individuals were selected by isolation of snail progeny that yielded high infection frequencies ( f1 ) and were then reared singly for self - reproduction ; these progeny were selected as the experimental susceptible group ( zanotti - magalhes et al . experimental design - two groups ( f2 ) were used in our experiments : 400 susceptible snails ( group i ) and 400 resistant snails ( group ii ) . each group was further subdivided equally into two subgroups : 200 young susceptible ( subgroup ia ) and 200 young resistant ( subgroup iia ) snails were infected at the age of two months ( before egg laying was initiated ) , at the size of 3 - 4 mm in diameter . another 200 adult susceptible ( subgroup ib ) and 200 adult resistant ( subgroup iib ) snails were infected at the age of four months ( after egg laying was initiated ) , at the size of 8 - 10 mm in diameter . afterwards , the infected snails of each subgroup were returned to separate plastic aquaria and kept in darkness . two weeks later , 100 snails from each experimental subgroup were subjected to enzyme assay testing . the other 100 snails in each subgroup were used for the determination of parasitological parameters , starting from the 28th day post - exposure , twice weekly for three weeks . 1:a scheme showing the followed experimental design with regards to the parasitological parameters . 2:a scheme showing the followed experimental design with regards to cytosolic superoxide dismutase enzyme assay . determination of susceptibility and resistance of individual snails of the four subgroups - at four weeks post - exposure to the parasite , the snails from each experimental subgroup were individually checked for cercarial shedding twice weekly , repeatedly for three weeks . the susceptible and nonshedding snails of each subgroup were separated and returned to different containers . on the first day of detecting cercariae , the susceptible snails in each subgroup were isolated individually in small transparent plastic containers , each in 100 ml of aged dtw , and were kept in darkness throughout the shedding period . each of the containers contained only one susceptible snail and was labelled with the experimental subgroup , the snail number , the pre - patent period ( ppp ) and the number of shed cercariae at each shedding assessment ( zanotti - magalhes et al . nonshedding individuals from each subgroup were returned together into a separate aquarium and were tested twice weekly for cercarial shedding until the 49th day p.i . at the end of the three weeks dedicated to shedding , nonshedding snails were examined under the dissecting microscope to make sure there are no arrested developmental stages present . moreover , if any snail died during the ppp , it was squeesed between two slides , entirely dissected and inspected under a stereo - dissecting microscope to detect developing sporocysts , which appear as opaque discrete masses within the snail tissue ( corra & paraense 1971 , niemann & lewis 1990 ) . each shedding snail in all experimental subgroups was investigated for the following parasitological parameters . ppp - ppp is defined as the period from the day of snail exposure to miracidia to the day immediately before the first shedding of cercariae ( haroun 1996 , yousif et al . starting from the 28th day post - exposure , all subgroups were examined twice weekly until the 49th day post - exposure ; each snail was tested individually . infection rate ( ir ) - the snail ir was calculated by dividing the number of shedding and positive crushed snails by the number of exposed snails . the percentage of susceptible and resistant snails in each subgroup was then determined ( frandsen 1979 , yousif et al . the snail ir was calculated at the end of the experiment using the following equation : mean cercarial output ( mco ) - on the first day of cercariae detection , positive snails were separated individually and placed in plastic cups . the cercariae shed from each susceptible snail were counted under a stereomicroscope twice weekly for three successive weeks ( chu & dawood 1970 , frandsen 1979 ) . mortality rate ( mr ) - the number of dead snails for each subgroup was counted starting from the day of exposure to the parasite until the 49th day p.i . the number of dead snails in the breeding aquaria was recorded twice weekly . according to yousif ( 1998 ) , the mr was calculated using the following equation : sod1 enzyme assay - one hundred snails from each experimental subgroup were used for the enzyme assay . each snail was cleaned gently , dissected and examined under a stereomicroscope to detect developing sporocysts . snails that did not show any sporocysts under the microscope were considered to be resistant . two control groups were used in the biochemical study : uninfected young snails and uninfected adult snails . the soft tissue of the dissected snail was perfused with normal saline , blotted on tissue paper and weighed . the tissue was homogenised with phosphate - buffered saline ( ph 7.2 ) by sonication for 2 min in a glass test tube ; to prevent temperature elevation to protect the enzymes present , the tube was placed in a beaker containing ice . the homogenised tissue was centrifuged at 500 g for 4 min and the supernatant containing the proteins was stored in aliquots at -20c until use . the collected supernatants were used for an sod1 enzyme assay . the protein concentration for each homogenate supernatant the sod1 activity was determined by adding the substrate of the enzyme to the snail tissue homogenate and measuring the increase in absorbance over time during the reaction with a spectrophotometer according to todd and gomez ( 2001 ) . a sod1 enzyme assay kit , purchased from biodiagnostic ( egypt ) , was used and relies on the ability of sod1 to inhibit the phenazine methosulphate - mediated reduction of nitroblue tetrazolium . calculation : where a control = the change in absorbance at 560 nm over 5 min following the addition of 5-methylphenazinium methyl sulfate ( pms ) ( r4 ) to the reaction mixture in the absence of the sample , a sample = the change in absorbance at 560 nm over 5 min following the addition of pms to the reaction mixture in the presence of the sample . statistical analysis - data were analysed using the ibm spss software package v.20.0 ( kirkpatrick & feeney 2013 ) . qualitative data are described using a number and percent and quantitative data are described using the range ( minimum and maximum ) , mean , standard deviation and median . comparisons between the different studied subgroups were tested using the chi - square test . when more than 20% of the cells showed an expected count of less than 5 , correction for chi - square was performed using monte carlo correction . the distributions of quantitative variables were tested for normality using the kolmogorov - smirnov test , the shapiro - wilk test and the dagstino test . when a normal data distribution was revealed , parametric tests were applied ; if the data were abnormally distributed , nonparametric tests were used . for normally distributed data , comparisons between the different studied subgroups were analysed using the f - test ( anova ) and the post - hoc test ( scheffe ) for pairwise comparisons . the significance of the obtained results was judged at the 5% level ( kotz et al . ethics - all work with laboratory animals was conducted in accordance with the egyptian national animal welfare standards and was approved by the ethica committee of the faculty of medicine , alexandria university . parasitological study - ppp - snails of each subgroup were individually examined for cercarial shedding twice weekly , starting from the 28th day after exposure to infection until the 49th day ( table i ) . table inumber of shedding snails in each of the examined pre - patent period ( ppp ) days in the studied subgroupsppp ( n = 100)day 28th32nd36th40th44th49thia372716500iia2 3 5 79 11 ib11 14 16 17 9 7 iib0 0 0 0 0 0 79.451 45.965 23.200 < 0.001 < 0.001 p < 0.001 p < 0.001 a : significance with subgroup ia ( young susceptible ) ; b : significance with subgroup iia ( young resistant ) ; c : significance with subgroup ib ( adult susceptible ) ; d : statistically significant at p 0.05 ; iib : adult resistant subgroup ; mc : monte carlo test ; n : number of members in each subgroup ; : value for chi - square test . a : significance with subgroup ia ( young susceptible ) ; b : significance with subgroup iia ( young resistant ) ; c : significance with subgroup ib ( adult susceptible ) ; d : statistically significant at p 0.05 ; iib : adult resistant subgroup ; mc : monte carlo test ; n : number of members in each subgroup ; : value for chi - square test . the maximum number of shedding snails on the 28th day among the four subgroups was observed in the young susceptible subgroup ( ia ) . these differences were demonstrated among the susceptible young ( ia ) and susceptible adult ( ib ) subgroups at all studied time points , except on the 36th day . in contrast , there were no significant differences between the young resistant ( iia ) and adult resistant ( iib ) subgroups at the earlier time points . significant differences were found at the 40th , 44th and 49th days . additionally , significant differences between the young subgroups ia and iia at all the time points , except on the 40th day , were also observed . furthermore , significant differences were found between the susceptible and resistant adult subgroups ( ib and iib ) on all days of shedding . ir % - the percentage of susceptible and resistant snails in each subgroup was determined . irs were 92% , 74% , 37% and 0% in the subgroups ia , ib , iia and iib , respectively . statistically significant differences among the susceptible and resistant groups at different snail ages and among young and adult subgroups with different compatibility patterns were evident ( table ii ) . table iiinfection rate ( ir ) among the studied subgroups iaiiaibiib pir ( % ) 9237740200.315 < 0.001 sigia - iia , ia - ib , ia - iib , ib - iia , ib - iib , iia - iib -- a : statistically significant at p 0.05 ; ia : young susceptible subgroup ; ib : adult susceptible subgroup ; iia : young resistant subgroup ; iib : adult resistant subgroup ; sig : significance between subgroups using chi - square test ( ) . a : statistically significant at p 0.05 ; ia : young susceptible subgroup ; ib : adult susceptible subgroup ; iia : young resistant subgroup ; iib : adult resistant subgroup ; sig : significance between subgroups using chi - square test ( ) . total cercarial production ( tcp ) and mco per susceptible snail - the total number of cercariae produced by the susceptible snails in subgroups ia , ib and iia was counted twice weekly over a period of three weeks . the mean number of cercariae shed by each susceptible snail in the different studied subgroups was calculated and is presented in tables iii , iv . the young susceptible subgroup ( ia ) yielded the highest mco , at 298.1 132.13 ( tables iii , iv ) . table iiicercarial roduction in the studied subgroupssubgroupsshedding snails ( n)tcpmcoia85151,002298.1 132.13iia379,87744.49 32.35ib7441,73294.13 44.17iib000.0 0.0ia : young ceptible bgroup ; ib : adult ceptible bgroup ; iia : young resistant subgroup ; iib : adult resistant subgroup ; mco : mean cercarial output ( mean number of cercariae / snail / shedding time ) ; tcp : total number of cercariae / subgroup . ia : young ceptible bgroup ; ib : adult ceptible bgroup ; iia : young resistant subgroup ; iib : adult resistant subgroup ; mco : mean cercarial output ( mean number of cercariae / snail / shedding time ) ; tcp : total number of cercariae / subgroup . table ivmean cercarial shedding over three successive weeks per susceptible snail among the studied subgroups iaiiaibiibmin - max97 - 59418 - 17320 - 2140 - 0mean sd298.1 132.144.5 32.3594.13 44.20.0 0.0median271.033.5090.500.0f test234.333 p < 0.001 sigia - iia , ia - ib , ia - iib , iia - ib , ib - iib , iia - iib a : statistically significant at p 0.05;b : statistically significant at p 0.001 ; ia : young susceptible subgroup ; ib : adult susceptible subgroup ; iia : young resistant subgroup ; iib : adult resistant subgroup ; sig : significant difference between subgroups using post - hoc test ( scheffe ) . normally distributed data were expressed in mean standard deviation ( sd ) and was compared using f test ( anova ) . a : statistically significant at p 0.05;b : statistically significant at p 0.001 ; ia : young susceptible subgroup ; ib : adult susceptible subgroup ; iia : young resistant subgroup ; iib : adult resistant subgroup ; sig : significant difference between subgroups using post - hoc test ( scheffe ) . normally distributed data were expressed in mean standard deviation ( sd ) and was compared using f test ( anova ) . mr % - the number of dead snails for each subgroup was counted from the day of exposure to infection until the 49th day p.i . the mr for each subgroup was 47% , 35% , 27% , 14% for subgroups a positive correlation between both infection and mrs of each subgroup was demonstrated , with mortality increasing with infection ( table vi ) . table vmortality rate ( mr ) among the studied subgroups at the different studied pre - patent periods with compatibility status of the dead snailsmr ( % ) day 28th32nd36th40th44th49thia ( 47)7 ( 7s)02 ( 2s)5 ( 5s)14 ( 14s)19 ( 19s)iia ( 27)2 ( 2r)3 ( 3r)3 ( 1r , 2s)5 ( 2r , 3s)6 ( 2r , 4s)8 ( 4r , 4s)ib ( 35)0 03 ( 3s)6 ( 6s)11 ( 10s , 1r)15 ( 13s , 2r)iib ( 14)1 ( 1r)1 ( 1r)2 ( 2r)3 ( 3r)3(3r)4(4r) 27.085 11.897 0.4101.059.368 13.461 p < 0.001 p = 0.015 p = 1.000 p = 0.8740.025 0.004 a : significance with subgroup ia ( young susceptible ) ; b : significance with subgroup ib ( adult susceptible ) ; c : statistically significant at p 0.05 ; iia : young resistant subgroup ; iib : adult resistant subgroup ; mc : monte carlo test ; r : resistant ; s : susceptible ; : value for chi - square test . a : significance with subgroup ia ( young susceptible ) ; b : significance with subgroup ib ( adult susceptible ) ; c : statistically significant at p 0.05 ; iia : young resistant subgroup ; iib : adult resistant subgroup ; mc : monte carlo test ; r : resistant table vicorrelation between infection rate ( ir ) and mortality rates ( mr ) in the studied subgroupscorrelationir ( % ) mr ( % ) pia9247 < 0.001 iia37270.130ib7435 < 0.001 iib014 < 0.001 r ( p)0.983 ( 0.017)- a : statistically significant at p 0.05 ; ia : young susceptible subgroup ; ib : adult susceptible subgroup ; iia : young resistant subgroup ; iib : adult resistant subgroup ; p : p - value for chi - square test ; r : pearson coefficient . a : statistically significant at p 0.05 ; ia : young susceptible subgroup ; ib : adult susceptible subgroup ; iia : young resistant subgroup ; iib : adult resistant subgroup ; p : p - value for chi - square test ; r : pearson coefficient . enzyme assay - the sod1 enzyme activities in the noninfected ( young and adult ) subgroups were 0.26 0.04 and 0.30 0.03 u / g tissues , respectively . in the infected subgroups , the highest level of sod1 enzyme activity was obtained in the adult resistant subgroup ( iib ) , with a mean of 0.90 0.07 u / g tissues , whereas the lowest enzyme activity was in the susceptible individuals of the young susceptible subgroup ( ia ) , at 0.32 0.03 . the enzyme activity was higher in the resistant snails , regardless of their age , in comparison to susceptible and noninfected snails . furthermore , the sod1 activity was higher in the adult subgroups than in the young subgroups ( table vii ) . table viithe level of cytosolic superoxide dismutase enzyme ( sod1 ) per snail s gram tissue in u / g tissues among the studied subgroupssod1 ( u / g)cacbia sia riia siia rib sib riibmin - max0.20 - 0.310.25 - 0.350.25 - 0.350.43 - 0.630.34 - 0.450.66 - 0.830.21 - 0.440.61 - 0.730.79 - 0.99mean sd0.26 0.040.30 0.030.32 0.030.55 0.070.40 0.030.76 0.050.34 0.070.68 0.040.90 0.07median0.260.300.320.580.400.770.360.670.91f - - - - 209.018 - - - - p - - - - < 0.001 - - - - sig--- b - d b , c , e b - f e , g b - f , h b - i a : statistically significant at p 0.05;b : significance with group uninfected young;c : significance with group uninfected adult ; d : significance with subgroup ia ( young susceptible members);e : significance with subgroup ia ( young resistant members ) ; f : significance with subgroup iia ( young susceptible members ) ; g : significance with subgroup iia ( young resistant members ) ; h : significance with subgroup ib ( adult susceptible members);i : significance with subgroup ib ( adult resistant members ) ; ca : young noninfected ; cb : adult noninfected ; ia r : young susceptible subgroup ( resistant members ) ; ia s : young susceptible subgroup ( susceptible members ) ; ib r : adult susceptible subgroup ( resistant members ) ; ib s : adult susceptible subgroup ( susceptible members ) ; iia r : young resistant subgroup ( resistant members ) ; iia s : young resistant subgroup ( susceptible members ) ; sig : significance between subgroups was done using post - hoc test ( scheffe ) . normally distributed data were expressed in mean standard deviation ( sd ) and was compared using f test ( anova ) . a : statistically significant at p 0.05;b : significance with group uninfected young;c : significance with group uninfected adult ; d : significance with subgroup ia ( young susceptible members);e : significance with subgroup ia ( young resistant members ) ; f : significance with subgroup iia ( young susceptible members ) ; g : significance with subgroup iia ( young resistant members ) ; h : significance with subgroup ib ( adult susceptible members);i : significance with subgroup ib ( adult resistant members ) ; ca : young noninfected ; cb : adult noninfected ; ia r : young susceptible subgroup ( resistant members ) ; ia s : young susceptible subgroup ( susceptible members ) ; ib r : adult susceptible subgroup ( resistant members ) ; ib s : adult susceptible subgroup ( susceptible members ) ; iia r : young resistant subgroup ( resistant members ) ; iia s : young resistant subgroup ( susceptible members ) ; sig : significance between subgroups was done using post - hoc test ( scheffe ) . normally distributed data were expressed in mean standard deviation ( sd ) and was compared using f test ( anova ) . infection of biomphalaria species by the larval stage of s. mansoni leads to an intimate , long - term association that largely challenges the snail s physical integrity and severely compromises its biological fitness ( sandland et al . although the infective stage ( miracidia ) is small relative to the host , once inside the host s body , miracidia initiate major developmental changes to support their extensive growth and transformation to more specialised larval stages ( de jong - brink 1995 ) . based on a postulation of the short life span of miracidium in water , which at the same time gives rise to hundreds of cercariae , schistosome development inside its snail intermediate host can be considered the weakest link in the parasite s life cycle . additionally , although a given parasite strain can only develop within a compatible snail , no such discrimination occurs in the human host . therefore , snail control is highly effective in eliminating schistosomiasis and is also easier than changing human habits . for these reasons , searching for snail factors that prevent parasite development will help in designing tools that can be utilised for controlling schistosomiasis ( knight et al . 2014 ) . genetic background and the ids are snail - related factors that are known to affect the compatibility of biomphalaria to s. mansoni ( richards 1975 , negro - corra et al . 2007 ) . age - dependent variability in susceptibility to s. mansoni has been studied in b. glabrata(richards 1984 , spada et al . 2010 ) and it was found that the number of genes controlling resistance differs with the age of the snail , which in turn affects the compatibility pattern ( richards 1977 , richards et al . moreover , the number of genes controlling resistance varies amongbiomphalaria species ( richards 1977 , richards et al . 2005 ) , which highlights the importance of studying the effect of age on the genetic outcome of b. alexandrina . these cells produce the enzyme cytosolic sod1 , an important enzyme that catalyses the conversion of o2- to h2o2 . the h2o2 produced by biomphalaria snails kills s. mansoni through its potential oxidative damage , thereby affecting snail susceptibility ( bender et al . goodall et al . ( 2006 ) found that sod1 enzyme expression in b. glabrata differs among susceptible and resistant snails . however , the effect of the snail s age on the activity of this enzyme has not been evaluated . within this context , the current work examined the effect of the age of b. alexandrina snails on their genetic outcome and on the activity of sod1 ( as a representative of ids ) . the present work was conducted using approaches involving both parasitological and biochemical techniques and was achieved by including 200 snails in each of four experimental subgroups : young susceptible ( ia ) , adult susceptible ( ib ) , young resistant ( iia ) and adult resistant ( iib ) . in each subgroup , 100 snails were dedicated for parasitological examination that began four weeks post - exposure to miracidia and continued for the following three weeks . the other 100 snails were used for the biochemical study ( sod1 enzyme assay ) , which was performed at two weeks post - exposure to infection . moreover , the effect of the interaction of snail age , genetics and internal defence on the outcome of infection was also examined . our results revealed that younger snails , belonging to either a susceptible or resistant group , showed higher susceptibility when compared to adult snails . young susceptible snails exhibited the shortest range of ppp , with the majority of shedding snails being recorded by the 28th day , at 37 of 85 shedding snails . moreover , the remainder of shedding snails in the same subgroup showed their first shedding before the 40th day p.i . : 27 snails shed cercariae for the first time on the 32nd day , 16 snails shed on the 36th day and five snails shed on the 40th day . regarding the adult susceptible subgroup ( ib ) , of 74 shedding snails , only 11 shed for the first time on the 28th day p.i . the maximum number of shedding snails was 16 and 17 recorded by the 36th and the 40th days , respectively . ( 2011 ) stated that high snail compatibility to trematode infection is characterised by a short ppp . the significant differences observed between subgroups ia and ib with regard to shedding durations indicate that subgroup ia ( young susceptible ) carries higher susceptibility to s. mansoni infection , accompanied by rapid development of the parasite inside the snail . as for subgroup iia ( young resistant ) shedding members , the maximum number of shedding snails for the first time was 11 that were recorded by the 49th day with only two snails shed for the first time by the 28th day . conversely , the snails belonging to the adult resistant subgroup iib displayed only the resistant phenotype , with no recoded ppp . subgroup iia ( young resistant ) showed a significant difference compared to subgroup iib ( adult resistant ) , which contained only resistant individuals , indicating higher susceptibility of the former . these significant differences between subgroups ia and ib and between iia and iib can be attributed to the effect of age . it was not surprising that the compatibility pattern also affected the outcome of infection , as evidenced by the significant differences between subgroups ia and iia and between subgroups ib and iib . moreover , the significant differences recorded between young resistant , susceptible snails and adult susceptible , susceptible snails ( iia and ib ) may be attributed to the effect of the interaction between age and genetics on compatibility patterns . this was represented by a longer ppp in the susceptible individuals in subgroup iia than in snails of the aforementioned adult susceptible subgroup . in the current study , some individuals of subgroups iia and ib displayed a ppp of 49 days . the delay in parasite development in these snails indicates their lower susceptibility to infection . a comparison between the young resistant ( iia ) subgroup and adult susceptible ( ib ) subgroup revealed that 11 snails shed for the first time on the 49th day in the former group , but only seven in the latter . however , this difference was not statistically significant , indicating that both age and compatibility pattern are important in determining the outcome of infection . richards ( 1984 ) stated that genetic factors determining b. glabrata juvenile nonsusceptibility operate throughout the snail s life , masking the presence of the factors for adult susceptibility and , thus , unfortunately complicating studies on the influence of age on snail susceptibility . the highest ir in the present study was exhibited by the snails belonging to the young susceptible subgroup ia . in total , 92% of subgroup ia ( young susceptible ) , 74% of subgroup ib ( adult susceptible ) and 37% of subgroup iia ( young resistant ) were susceptible . it should be noted that even the snails that died during the course of the experiment were included in the statistical analysis ; these snails were examined for developing sporocysts . although our experiment was carried out on snails resulting from self - reproduction , some resistant snails were obtained in the progeny of susceptible individuals , indicating the dominance of the resistance character in b. alexandrina . in their studies , both shoukry et al . ( 2010 ) observed the appearance of resistant snails originating from cross of susceptible parents . a probable explanation for the resistant members obtained in the susceptible subgroups is that , although resistance alleles are sometimes not expressed in the snail phenotype , they can accumulate through generations to produce a resistant phenotype that appears in later progeny . that is , the parents of these resistant snails harboured unexpressed resistance genes , which subsequently accumulated among successive generations ( el naga et al . similarly , the resistance character in b. glabrata snails is inherited in a dominant manner ( richards & merritt 1972 , lewis et al . abdel - hamid et al . ( 2006 ) and el - nassery et al . ( 2013 ) found that both susceptibility and resistance in b. alexandrina are hereditary characters and are genetically controlled . in contrary to lewis et al . ( 2002 ) , who found that susceptible b. glabrata parents did not give rise to any resistant progeny , susceptible subgroups in the current study resulted in 8 - 26% resistant individuals in the two age groups . ( 2010 ) that a susceptible population of b. alexandrina may contain more resistance alleles than b. glabrata , thus accounting for the appearance of resistant progeny originating from completely susceptible parents . although the two susceptible subgroups contained resistant snails , the significant difference noted between them suggests that the resistant alleles obtained from f1 parents were potentiated by the impact of age , resulting in the appearance of more resistant individuals in the adult susceptible subgroup ( ib ) . in the current work , this result was also observed in other studies using different species ( lewis et al . however , 37 susceptible members were recorded in our young resistant subgroup ( iia ) , which reinforces our assumption on the effect of the age of b. alexandrina on snail compatibility pattern . the appearance of susceptible individuals in the young resistant subgroup could also be explained by its weaker internal defence when compared to the adult resistant subgroup . this was proven by the significant difference noted in sod1 enzyme activity between resistant snails in the young and adult resistant subgroups , at 0.68 0.04 and 0.90 0.07 u / g tissues , respectively . anderson et al . ( 1982 ) attributed the lower irs of adult snails compared to younger snails to a number of different factors . first , older snails possess physical barriers to penetration imposed by their tough thickened body covering . additionally , they produce greater quantities of mucus , which replete with chemicals that confuse searching miracidia . older snails also move faster and are thus more difficult to locate by miracidia . furthermore , egg - laying capacity could be among the factors causing more resistance in adult snails , wherein the snail expends all its energy on reproduction , as stated by richards ( 1977 ) . our main goal was to ascertain whether resistant alleles transmitted from susceptible parent snails to their young progeny are more functional at the adult stage and our results suggest that they are . we propose that resistance alleles in the susceptible population may become more functional through the snail aging process , which is responsible for the lower susceptibility in adult b. alexandrinasnails . the highest susceptibility in the young susceptible subgroup was also evidenced by the highest tcp among the four studied subgroups , at 151,002 . this was followed by the adult susceptible subgroup , which produced 41,732 cercariae over the three weeks of shedding . according to the frandsen classification , the three subgroups can be categorised in classes 4 , 2 and 1 ( well compatible , poorly compatible and not very compatible , respectively ) . regarding the adult resistant subgroup , no cercariae were produced at all , placing this subgroup in frandsen class 0 or resistant snails ( frandsen 1979 ) . the recorded mrs in the present study were 47% , 35% , 27% and 14% in subgroups ia , ib , iia and iib , respectively and were directly proportional to the irs in the different subgroups . the higher mrs noted in the susceptible compared to the resistant groups could be explained by the devastating effects of s. mansoni on snail health . s. mansoni is considered a parasite that exerts a selective pressure on the susceptible population . during its development , s. mansoni sporocysts invade biomphalaria s digestive gland and the adjacent gonads , leading to destruction of these organs ( lemos & andrade 2001 ) . thus , infection becomes an element of natural selection , which is highly negative for infected snails ( rosa et al . moreover , the disturbance in metallic ion concentrations due to trematode infection is considered another cause of mortality in parasitised snails ( mostafa & dajem 2010 ) . furthermore , the exposure of susceptible snails to a high dose of infection ( 8 - 10 miracidia ) per snail at the same time may explain the higher mortality recorded in the laboratory , as the majority of these miracidia are thought to develop without much restraint . in contrast , under natural conditions , the snail is not likely to be exposed to such a high number of miracidia at a given time ( corra & paraense 1971 ) . the mrs recorded in the two resistant subgroups were 35% and 14% , subgroups iia ( young resistant ) and iib ( adult resistant ) , respectively . the mortality of the resistant snails can be attributed to a high concentration of the sod1 enzyme , which produces h2o2 , a highly ros . although these reactive species are beneficial for combating the invading parasite , they also seriously damage biological membranes in the host tissue , the so - called " cost of resistance " ( bender et al . 2007 , bonner et al . 2012 ) ; hence , ros are considered a double - edged sword ( beckman & ames 1998 ) . ( 2010 ) stated that exposure alone of b. alexandrina snails to s. mansoni miracidia has a negative impact on snail survival rates . in the present work , we functionally investigated the activity of the sod1 enzyme in a whole - snail soft tissue homogenate at two weeks after exposure to infection . for the control noninfected snails , the results were 0.26 0.04 and 0.30 0.03 u / g of sod1 activity in the noninfected young and adult groups , respectively . cytosolic sod1 is present during the course of the snail lifetime to protect cells against damage by the free radicals that are generated due to normal metabolism ( bender et al . 2005 ) . regarding the results of the experimental subgroups in the current study , the combined effects of both age and compatibility pattern resulted in the highest sod1 activity in the adult resistant subgroup , with a mean of 0.90 0.07 u / g tissue . in contrast , the lowest enzyme activity was found in the susceptible snails of the young susceptible subgroup , at 0.32 0.03 u / g tissue . furthermore , as shown intable vii , the sod1 activities were higher in the resistant individuals of the studied subgroups than in the corresponding susceptible individuals . this indicates the importance of this enzyme in determining a snail s compatibility status . our results of higher sod1 activity levels in resistant vs. susceptible snails are in agreement with hahn et al . ( 2004 ) , mahmoud and rizk ( 2004 ) , bender et al . ( 2005 ) and bayne ( 2009 ) , who reported an increased number of haemocytes generating superoxide in s. mansoni resistant snails . the resulting free radicals were efficient for encapsulating or eliminating the parasite in resistant snails , but failed in the case of susceptible snails ( mahmoud & rizk 2004 ) . it is worth mentioning that studies on the genes of a resistant b. glabrata strain have revealed that polymorphisms in the gene encoding cytosolic sod1 are associated with susceptibility / resistance ; such results reinforce the role of sod1 in schistosome resistance ( goodall et al . the results of the present work showed that the age of b. alexandrina snails affects sod1 activity levels . significant differences in sod1 activity levels between young and adult snails obtained from the same ( f1 ) parent were observed , with a higher activity at the adult stage . goodall et al . ( 2006 ) reported thatbiomphalaria sod1 is encoded by three alleles and one was found to be significantly associated with resistance . furthermore , some alleles controlling compatibility were found to be affected by snail age ( richards 1977 , richards & shade , richards et al . , it would be interesting to assess whether the genes encoding sod1 are among the other genes that are modified by snail age . in summary , the significant differences in all parasitological and biochemical parameters between young and adult snails in the same group were attributed to the age effect , which made resistance alleles more functional in adults , with higher sod1 enzyme activity . moreover , the complex interaction between age , genetic background and ids between the susceptible and resistant subgroups results in great variability in compatibility patterns . this interaction led to the appearance of the highest significant difference between the young susceptible ( ia ) and adult resistant ( iib ) subgroups . the results presented herein can have potential epidaemiological implications inbiomphalaria control . by determining the age at which a snail is most susceptible to s. mansoni infection , the optimal timing for the application of molluscicides could be identified . this in turn would increase the efficacy of the applied method , potentiating schistosomiasis control . moreover , identification of the most resistant snails is important for use in later biological snail control , after studying the compatibility of successive generations . investigations of genes encoding enzymes involved in the respiratory burst are recommended . in particular , the genes responsible for the production of h2o2 should be targeted as potential candidates for genetic loci causally related to the susceptibility / resistance ofb .

of the approximately 34 identified biomphalaria species , biomphalaria alexandrina represents the intermediate host of schistosoma mansoni in egypt . using parasitological and sod1 enzyme assay , this study aimed to elucidate the impact of the age of b. alexandrina snails on their genetic variability and internal defence against s. mansoni infection . susceptible and resistant snails were reared individually for self - reproduction ; four subgroups of their progeny were used in experiment . the young susceptible subgroup showed the highest infection rate , the shortest pre - patent period , the highest total cercarial production , the highest mortality rate and the lowest sod1 activity . among the young and adult susceptible subgroups , 8% and 26% were found to be resistant , indicating the inheritance of resistance alleles from parents . the adult resistant subgroup , however , contained only resistant snails and showed the highest enzyme activity . the complex interaction between snail age , genetic background and internal defence resulted in great variability in compatibility patterns , with the highest significant difference between young susceptible and adult resistant snails . the results demonstrate that resistance alleles function to a greater degree in adults , with higher sod1 activity and provide potential implications for biomphalaria control . the identification of the most susceptible snail age enables determination of the best timing for applying molluscicides . moreover , adult resistant snails could be beneficial in biological snail control .

MATERIALS AND METHODS RESULTS DISCUSSION

PMC5139929

resin composites are the most commonly used aesthetic restorative materials due to their universal usage , resemblance to tooth structure in color and mechanical properties , requiring minimal removal of tooth structure and ease of chair side use . today , the common problem of aesthetic dentistry is discoloration and color mismatch of tooth - colored restorations after consumption of chromogens . the color change of composite resins depends on intrinsic and extrinsic factors ; intrinsic factors involve changes in the filler , matrix and matrix / filler interface ; extrinsic factors involve absorption or adsorption of dyes from external sources , such as coffee , tea , nicotine and mouthrinses like chlorhexidine . in the recent years , prescription of mouthrinses for the control of caries and periodontal disease has become more common . in mentally retarded cases or after periodontal surgeries , plaque control with mechanical methods is not properly possible ; thus , chemical methods like the use of chlorhexidine mouthrinse as an antiseptic solution are required . in comparison to common antibiotic therapies , chlorhexidine is a well - known biguanide compound with effective anti - plaque action and chromogenic potential causing brown staining of teeth , tongue , silicate and resin restorations . various staining mechanisms have been described for chlorhexidine including degradation of chlorhexidine to release parachloraniline , non - enzymatic browning reactions ( the maillard reactions ) , protein denaturation by chlorhexidine with metal sulfide formation and precipitation of anionic dietary chromogens by cationic antiseptics . lamba et al , reported that immersion of composite , glass ionomer cement and compomer in mouthrinses can significantly change the color of these materials . celik et al , investigated the effect of three chlorhexidine mouthrinses on composites and reported that all composites showed acceptable discoloration . in the recent years , considerable improvements have occurred in formulation of composite materials , mainly by use of nanotechnology . nanofilled composites are new materials containing inorganic nanofillers scattered in the resin matrix and have improved mechanical strength , optical properties , wear resistance , elasticity modulus and color stability . microhybrid composites are dominant in dental markets , thus , in this study , we compared the color stability of new efficient composites with each other . instrumental techniques decrease subjective interpretation as compared with the visual methods . in comparison of these two methods , spectrophotometer is a more accurate method to detect color changes of dental restorations . a spectrophotometer uses cie system , which is based on human perception and was developed by the commission intrnationale de leclairage in 1978 . color change is shown with e in this system , which is total color change based on l * , a * and b * values . the l * value refers to lightness and a * and b * values refer to chromaticity coordinates . positive a * values show a shift towards red and negative values show a shift towards green ; positive b * values show a shift towards yellow and negative values show a shift towards blue ; when e is less than or equal to 1 unit , it is not visible to the human eye ; when e is between 1 and 3 units , the color change is visually perceptible to the experienced examiner ; when e is greater than or equal to 3.3 units , the color change is clinically unacceptable . discoloration of aesthetic restorations is problematic , wasting time and cost for replacement of restorations due to dissatisfaction of patients . but prescription of chlorhexidine mouthrinses for a minimum of two weeks after periodontal surgeries is a common practice , which may cause discoloration of aesthetic composite restorations of patients . the aim of this study was to evaluate the effects of chlorhexidine mouthrinses available in the iranian market on color stability of microhybrid and nanofilled composites . in this in - vitro study , two composite resins and seven types of chlorhexidine mouthrinses were used . artificial saliva ( hypozalix spray , biocodex , france ) was used as the control group . a ph meter ( labtron , tehran , the details of materials used in this study are presented in tables 1 and 2 . mouthrinses used in this study composites used in this study ( shade : a2 ) bisphenol a - glycidyl methacrylate bisphenol a ethoxylated dimethacrylate urethane dimethacrylate triethylene glycol dimethacrylate polyethylene glycol dimethacrylate one - hundred and sixty disc - shaped specimens , 80 from each composite , were made in prefabricated celluloid molds with dimensions of 7x2 mm . composites were placed in molds and compressed with a 1 mm thick glass slab for 30 seconds to obtain a uniformly smooth surface . each specimen was light - cured with valo light curing unit ( ultradent , south jordan , usa ) with a light intensity of 800 mw / cm for 20 seconds on both sides . a radiometer ( kerr , demetron , orange , ca , usa ) the bottoms of specimens were coded from one to 10 to facilitate the color measurement of each specimen . the upper surface of each specimen was ground with 600 , 800 , 1000 , and 2000-grit silicon carbide papers ( matador , cologne , germany ) successively under running water . all specimens were placed at the bottom of a container in such a way that they were completely dipped in the solution . after polishing , the specimens were immersed in artificial saliva at 37c for 24 hours in an incubator in a closed container , allowing post - polymerization . each specimen was then blotted dry with a filter paper and then subjected to color measurement by placing the probe tip perpendicular to the specimen surface . the initial color values ( l * , a * , b * ) were measured using vita easy shade advance ( vita zahnfabrik , bad sckingen , germany ) against a white background before insertion of samples in the solutions . the spectrophotometer was calibrated after color measurement of every three samples by placing the probe tip against the calibration block . the specimens were immersed in mouthrinses once a day for two minutes ( n=10 ) , which was equivalent to two rinses with mouthwash per day for one minute . after two minutes of immersion in the mouthrinses , the specimens were immersed in artificial saliva in an incubator at 37c . this process was repeated for two weeks to simulate the use of chlorhexidine mouthrinses for two weeks , which is commonly prescribed after dental treatments like periodontal surgeries . after one and two weeks of immersion , color measurements were repeated with the same spectrophotometer , and these measurements were made under the same conditions and in the same manner described for initial color measurement . the total color change ( e * ) was calculated using the following formula : e=(l*)2+(a*)2+(b*)22 the collected data were statistically analyzed with spss version 22 using two - way anova to evaluate the interaction effects of the material type and mouthrinse on color change . one - way anova , tukey 's post hoc test , t - test and paired t - test were used to analyze data at a significance level of 0.05 . one - hundred and sixty disc - shaped specimens , 80 from each composite , were made in prefabricated celluloid molds with dimensions of 7x2 mm . composites were placed in molds and compressed with a 1 mm thick glass slab for 30 seconds to obtain a uniformly smooth surface . each specimen was light - cured with valo light curing unit ( ultradent , south jordan , usa ) with a light intensity of 800 mw / cm for 20 seconds on both sides . a radiometer ( kerr , demetron , orange , ca , usa ) the bottoms of specimens were coded from one to 10 to facilitate the color measurement of each specimen . the upper surface of each specimen was ground with 600 , 800 , 1000 , and 2000-grit silicon carbide papers ( matador , cologne , germany ) successively under running water . all specimens were placed at the bottom of a container in such a way that they were completely dipped in the solution . after polishing , the specimens were immersed in artificial saliva at 37c for 24 hours in an incubator in a closed container , allowing post - polymerization . each specimen was then blotted dry with a filter paper and then subjected to color measurement by placing the probe tip perpendicular to the specimen surface . the initial color values ( l * , a * , b * ) were measured using vita easy shade advance ( vita zahnfabrik , bad sckingen , germany ) against a white background before insertion of samples in the solutions . the spectrophotometer was calibrated after color measurement of every three samples by placing the probe tip against the calibration block . the specimens were immersed in mouthrinses once a day for two minutes ( n=10 ) , which was equivalent to two rinses with mouthwash per day for one minute . after two minutes of immersion in the mouthrinses , the specimens were immersed in artificial saliva in an incubator at 37c . this process was repeated for two weeks to simulate the use of chlorhexidine mouthrinses for two weeks , which is commonly prescribed after dental treatments like periodontal surgeries . after one and two weeks of immersion , color measurements were repeated with the same spectrophotometer , and these measurements were made under the same conditions and in the same manner described for initial color measurement . the total color change ( e * ) was calculated using the following formula : e=(l*)2+(a*)2+(b*)22 the collected data were statistically analyzed with spss version 22 using two - way anova to evaluate the interaction effects of the material type and mouthrinse on color change . one - way anova , tukey 's post hoc test , t - test and paired t - test were used to analyze data at a significance level of 0.05 . two - way anova revealed that the interaction effects of composite type and mouthrinses on color change were statistically significant ( p=0.049 ) . the mean values and standard deviations of l , a and b color parameters for each group of composite resin after seven and 14 days of immersion in chlorhexidine mouthrinses are shown in table 3 . the mean l , a and b values and standard deviation of filtek z250 compared to filtek z350xt in different solutions after seven and 14 days the mean values and standard deviations of color change ( e ) for each group of composite resin after seven days of immersion in chlorhexidine mouthrinses are shown in table 4 . the mean e values and standard deviation ( sd ) of filtek z250 compared to filtek z350xt in different solutions after seven and 14 days statistically significant difference between the two time points in each group and composite at significance level of 0.05 . -the similar superscripted uppercase letters in the same column show no significant difference between groups at each time point ( p>0.05 ) -the similar superscripted lowercase letters in the same row show no significant difference between two composites at each time point ( p>0.05 ) column chart showing the mean e17 values and standard deviation of filtek z250 compared to filtek z350xt in different solutions filtek z250 in vi - one mouthrinse showed maximum discoloration ( e=3.051.49 ) after seven days , which had a statistically significant difference with the control group ( p=0.004 ) but it did not show significant differences with other groups . filtek z350xt in behsa mouthrinse showed maximum discoloration ( e=1.740.7 ) after seven days , which had a statistically significant difference with the control ( p=0.000 ) , najo ( p=0.004 ) and epimax ( p=0.014 ) mouthrinses . the e values after 14 days revealed that all groups had acceptable color change ( e<3.3 ; fig . the mean values and standard deviations of color change ( e ) of each group of composite resin after 14 days of immersion in chlorhexidine mouthrinses are shown in table 4 . column chart showing the mean e114 values and standard deviation of filtek z250 compared to filtek z350xt in different solutions filtek z250 in najo mouthrinse ( e=2.651.46 ) and after that in vi - one ( e=2.432.23 ) showed maximum discoloration ; although it did not have significant differences with other groups ( p>0.05 ) . filtek z350 xt in behsa showed maximum discoloration ( e=2.340.78 ) , which had significant differences with other groups ( p<0.05 ) . the e values in filtek z350xt after 14 days in comparison to seven days showed higher discoloration in all groups and the discoloration was significant in all groups except for chlorhexidine and najo . the e values in filtek z250 after 14 days in comparison to seven days showed higher discoloration in epimax , hexodine , najo and behsa groups and lower discoloration in other groups , and the discoloration was only significant in the control and epimax groups . the present study evaluated the effects of seven commercially available chlorhexidine mouthrinses in the iranian market on color stability of two types of resin - based composites . the results revealed that the color change of composites after seven and 14 days of immersion in mouthrinses was clinically acceptable ( e<3.3 ) . with regard to color parameters ( l*a*b * ) , the results showed that l values were negative , which indicated that the specimens became darker in all groups . in a * axis , the specimens color shifted towards red in all groups , and in b * axis , the color of microhybrid composite shifted towards yellow and that of nanofilled composite shifted towards blue . in addition , filtek z250 ( microhybrid ) showed less color change than filtek z350xt ( nanofilled ) . after the first and second weeks , the color change ( e value ) of filtek z250 with larger filler particles was greater than that of filtek z350xt in all groups . filtek z250 contains filler particles with an average size of 0.6m ; while filtek z350xt contains filler particles ranging from 5 to 20 nm . larger particles create a rough surface while polishing and smaller particles create a smooth surface ; thus , composites with larger fillers are more susceptible to discoloration . in addition , composites with larger filler particles are more susceptible to water sorption by the polymer network and the effect of coloring agents on the quality of the bond between the matrix and filler can cause discoloration . water sorption may cause micro - cracks by expanding and laminating the resin component and hydrolyzing the silane and decrease the functional life of composite resins ; thus , micro - crack formation and interfacial gaps between the filler and matrix allow soluble dyes of mouthrinses to penetrate and cause discoloration ; these reasons can explain the greater color stability of filtek z350xt . our findings are in accordance with those of al - qaisi and alrahman and festuccia et al . after seven days of immersion in mouthrinses , vi - one mouthrinse showed greater discoloration after alcohol - based mouthrinses . weak acids like citric acid have a softening effect on polymer matrix and decrease the microhardness of composites in addition to alcohol . kin , epimax , and vi - one mouthrinses have citric acid in their composition but in this study , vi - one showed greater discoloration . the manufacturers of kin and epimax claim that these mouthrinses have color preservers in their composition that inhibit discoloration of teeth and composites ; therefore , the greater discoloration of vi - one may be due to the absence of color preserver in its formulation . after 14 days of immersion in mouthrinses , microhybrid composite in najo with 10% alcohol ( e=2.65 ) and nanofilled composite in behsa with 11.6% alcohol ( e=2.34 ) showed greater discoloration . alcohol concentration in mouthrinses varies from 0 to 27% , which is comparable to the alcohol percentage of beer ( 4% ) and wine ( 12% ) . ethanol is a bipolar molecule , which enhances the dissolution of hydrophobic and hydrophilic components . alcohol in mouthrinses does not have any other therapeutic effect except as a solvent . due to this reason , in the clinical setting , alcohol - free mouthrinses have been proven to have the same effect as alcohol - based mouthrinses with fewer side effects . our findings are similar to those of villalta et al , asmussen , and stober et al , who reported that alcohol had a softening effect on composites , so the chromaticity of composites increases in alcohol - based solutions . comparison of discoloration between e17 and e114 revealed that filtek z350xt had significantly greater discoloration after 14 days in comparison to seven days in all groups except for two ( chlorhexidine and najo ) ; it means that all specimens showed a significant progressive increase in color change with increasing number of rinses in all chlorhexidine mouthrinses except for two groups , and filtek z250 showed a significant discoloration only in two groups ( control and epimax ) after 14 days in comparison to seven days ; this result may be due to the higher potential of nanofilled composites for discoloration as mentioned above and showed that increasing the frequency and duration of rinses can affect the color stability of nanofilled composites more than microhybrid composites . in this study , despite the low ph of epimax ( ph=4.3 ) , it did not show greater discoloration than other groups . we did not find a correlation between ph and discoloration of composites , which is in accord with the findings of diab et al , who reported that the ph of test solutions did not have an effect on discoloration of restorative materials . in clinical situations , different factors may affect the color stability of restorative materials such as the presence of saliva , salivary pellicle , and the effect of different foods and beverages , which are difficult to simulate in an in - vitro setting . this result may be due to the absence of these chromogenic materials , which could intensify the chromogenic potential of chlorhexidine mouthwashes . the results of the present study indicated that composites showed an acceptable color change after two weeks of immersion in chlorhexidine mouthrinses . mouthrinses , which contained alcohol and citric acid showed greater discoloration ; thus , it is better to limit their prescription .

objectives : this study aimed to evaluate the effects of chlorhexidine mouthrinses on color stability of nanofilled and micro - hybrid resin - based composites.materials and methods : in this in - vitro study , 160 disc - shaped specimens ( 7x2 mm ) were fabricated of filtek z250 and filtek z350xt enamel ( a2 shade ) . the samples of each group were randomly divided into eight subgroups ( n=10 ) . the specimens were incubated in artificial saliva at 37c for 24 hours . the baseline color values ( l * , a * , b * ) of each specimen were measured according to cie lab system using a reflection spectrophotometer . after baseline color measurements , the control samples were immersed in saliva and the test groups were immersed in kin ( cosmodent ) , vi - one ( rozhin ) , epimax ( emad ) , hexodine ( donyaye behdasht ) , chlorhexidine ( shahrdaru ) , najo ( najo ) and behsa ( behsa ) mouthrinses once a day for two minutes . the specimens were then immersed again in saliva . this process was repeated for two weeks . color measurements were made on days seven and 14 . two - way and one - way anova and tukey 's post hoc test , t - test and paired t - test were used to analyze data at a significance level of 0.05.results:all specimens displayed color change after immersion in the mouthrinses . significant interactions were found between the effects of materials and mouthrinses on color change.conclusions:all composite resins tested showed acceptable color change after immersion in different mouthrinses . filtek z350xt showed less color change than filtek z250 . mouthrinses containing alcohol ( behsa and najo ) and citric acid ( vi - one ) caused greater discoloration of composites .

INTRODUCTION MATERIALS AND METHODS Preparation of samples: Baseline color measurement: Immersion of specimens in chlorhexidine mouthrinses: Second and third color measurements: RESULTS DISCUSSION CONCLUSION

PMC4635724

treating cognitive impairment , along with motor recovery and improving performance in daily activities , is one of the key focus areas of stroke rehabilitation . as cognition is closely related to patient motivation and motor function improvement , implementing cognitive function treatment in a timely and effective manner is crucial for the overall success of poststroke rehabilitation . previous reports showed 25%-40% of patients suffer from dementia by three months after stroke , and the number increased to 50%-75% when patients with minor symptoms of cognitive impairment are included [ 2 - 4 ] . among patients with brain lesions , memory disturbance is commonly a critical challenge in rehabilitating patients to enable them to return to active participation in society and the workforce . research has suggested that rehabilitation programs designed to maximize neuroplasticity are effective for improving memory functions in these patients . recently , transcranial direct - current stimulation ( tdcs ) and repetitive transcranial magnetic stimulation ( rtms ) , noninvasive methods for stimulating the cerebral cortex , have been intensively studied as a treatment for brain injury . while both modalities are used to treat brain injury , tdcs is the preferred method , as it allows simultaneous application of brain stimulation and conventional rehabilitation . concluded that applying tdcs to the brain region improved motor functions and increased motor cortex excitability . recent studies also suggested that tdcs was useful in treating cognitive dysfunction , and improving attention , memory , and executive function [ 7 - 9 ] . however , there are still insufficient data to confirm its effect on the cognitive functions of stroke patients . thus , in this study , we determined the effectiveness of tdcs in cognitive recovery in acute and subacute stroke patients . we selected stroke patients with cognitive impairment and a score of 27 on the korean version of mini mental state exam ( k - mmse ) from patients treated at the department of rehabilitation medicine , asan medical center , from october 1 , 2010 to march 30 , 2012 . the guardians of the patients indicated that the patients had experienced their first stroke , and had no previous history of abnormal cognitive functions . patients who were classified as acute or subacute , within 6 months from their stroke , and who showed no damage to their temporal lobe on magnetic resonance imaging , were selected . subjects were then included when they were willing and available for five treatment sessions per week . patients with apraxia , aphasia , and neglect were excluded , given that these factors may have an impact on the outcome of cognitive function tests . for the safety of patients , we excluded those who had a history of craniectomy or seizure . we used the double - blind method to randomly divide the subjects into three test groups : ( 1 ) the left fronto - temporal anode stimulation ( left - ftas ) group , ( 2 ) the right fronto - temporal anode stimulation ( right - ftas ) group , and ( 3 ) the sham group . patients in each group received tdcs treatment for 30 min , five times a week , for 3 weeks . the treatments were performed using a phoresor ii auto model pm850 ( iomed , salt lake city , ut , usa ) , an fda - approved device , with 5 5 cm sponge electrodes attached to the patient s scalp . the intensity of the current was set at 2 ma , which was confirmed as safe by iyer et al . tdcs anodal electrodes were placed according to the 10 - 20 international eeg system , at t3 for the left - ftas group and at t4 for the right - ftas group . for the sham group , sponge electrodes were attached using the same method as for the left - ftas group , but without any current running . the cognitive rehabilitation program used in the study was comcog ( maxmedica inc . , seoul , korea ) , with the focus on attention and memory improvement for cognitive disorder patients . the subjects were evaluated before the treatment and after completing the 3-week program , using the k - mmse and computerized neurocognitive function tests ( cnt , maxmedica , seoul , korea ) to assess cognitive function , and the korean version of the modified barthel index ( k - mbi ) to assess activities of daily living . the cnt included visual and auditory continuous performance tests ( cpts ) to evaluate continuous concentration on visual and auditory stimuli . the kruskal - wallis test was used to test the homogeneity among the three groups , and the wilcoxon signed - rank test was used to analyze the preand post - treatment data for each test group . the kruskal - wallis test was used to compare the three test groups , and the mann - whitney test was used as the follow - up test . we selected stroke patients with cognitive impairment and a score of 27 on the korean version of mini mental state exam ( k - mmse ) from patients treated at the department of rehabilitation medicine , asan medical center , from october 1 , 2010 to march 30 , 2012 . the guardians of the patients indicated that the patients had experienced their first stroke , and had no previous history of abnormal cognitive functions . patients who were classified as acute or subacute , within 6 months from their stroke , and who showed no damage to their temporal lobe on magnetic resonance imaging , were selected . subjects were then included when they were willing and available for five treatment sessions per week . patients with apraxia , aphasia , and neglect were excluded , given that these factors may have an impact on the outcome of cognitive function tests . for the safety of patients we used the double - blind method to randomly divide the subjects into three test groups : ( 1 ) the left fronto - temporal anode stimulation ( left - ftas ) group , ( 2 ) the right fronto - temporal anode stimulation ( right - ftas ) group , and ( 3 ) the sham group . patients in each group received tdcs treatment for 30 min , five times a week , for 3 weeks . the treatments were performed using a phoresor ii auto model pm850 ( iomed , salt lake city , ut , usa ) , an fda - approved device , with 5 5 cm sponge electrodes attached to the patient s scalp . the intensity of the current was set at 2 ma , which was confirmed as safe by iyer et al . tdcs anodal electrodes were placed according to the 10 - 20 international eeg system , at t3 for the left - ftas group and at t4 for the right - ftas group . for the sham group , sponge electrodes were attached using the same method as for the left - ftas group , but without any current running . the cognitive rehabilitation program used in the study was comcog ( maxmedica inc . , seoul , korea ) , with the focus on attention and memory improvement for cognitive disorder patients . the subjects were evaluated before the treatment and after completing the 3-week program , using the k - mmse and computerized neurocognitive function tests ( cnt , maxmedica , seoul , korea ) to assess cognitive function , and the korean version of the modified barthel index ( k - mbi ) to assess activities of daily living . the cnt included visual and auditory continuous performance tests ( cpts ) to evaluate continuous concentration on visual and auditory stimuli . the kruskal - wallis test was used to test the homogeneity among the three groups , and the wilcoxon signed - rank test was used to analyze the preand post - treatment data for each test group . the kruskal - wallis test was used to compare the three test groups , and the mann - whitney test was used as the follow - up test . the subjects for this study included : ( 1 ) six male and nine female patients in the left - ftas group , ( 2 ) seven male and eight female patients in the right - ftas group , and ( 3 ) seven male and eight female patients in the sham group . the mean age of patients in each group was as follows : ( 1 ) 60.9 12.9 years for the left - ftas group , ( 2 ) 58.915.0 years for the right - ftas group , and ( 3 ) 68.514.6 years for the control group , with no significant differences between the groups . the mean time elapsed from the onset of stroke to the start of the treatment program was : ( 1 ) 42.231.9 days for the left - ftas group , ( 2 ) 38.127.0 days for the right - ftas group , and ( 3 ) 39.529.6 days for the sham group , with no significant differences between the groups ( table 1 ) . the results from post - treatment cnt suggested that the left - ftas group showed significant improvement in the backward digit span test , the verbal learning test - delayed recall , a visual span test , and the k - mmse . all three groups showed significant improvement in k - mbi scores ( table 2 ) . comparison of pre- and post - treatment data for each group suggests that the left - ftas group showed significant improvement in auditory memory , compared to the other groups . however , there were no significant differences between the groups in terms of visual memory and attention . there were no differences between the groups in terms of k - bmi ( table 3 ) . the subjects for this study included : ( 1 ) six male and nine female patients in the left - ftas group , ( 2 ) seven male and eight female patients in the right - ftas group , and ( 3 ) seven male and eight female patients in the sham group . the mean age of patients in each group was as follows : ( 1 ) 60.9 12.9 years for the left - ftas group , ( 2 ) 58.915.0 years for the right - ftas group , and ( 3 ) 68.514.6 years for the control group , with no significant differences between the groups . the mean time elapsed from the onset of stroke to the start of the treatment program was : ( 1 ) 42.231.9 days for the left - ftas group , ( 2 ) 38.127.0 days for the right - ftas group , and ( 3 ) 39.529.6 days for the sham group , with no significant differences between the groups ( table 1 ) . the results from post - treatment cnt suggested that the left - ftas group showed significant improvement in the backward digit span test , the verbal learning test - delayed recall , a visual span test , and the k - mmse . all three groups showed significant improvement in k - mbi scores ( table 2 ) . comparison of pre- and post - treatment data for each group suggests that the left - ftas group showed significant improvement in auditory memory , compared to the other groups . however , there were no significant differences between the groups in terms of visual memory and attention . there were no differences between the groups in terms of k - bmi ( table 3 ) . the objective of this study was to determine the effect of tdcs treatment on the temporal lobe in terms of cognitive recovery of poststroke patients . to date , only a small number of studies have been done on tdcs as a treatment method for cognitive impairment . while montie et al . suggested that cathode stimulation to the left fronto - temporal lobe resulted in improvement in naming in aphasia , other studies concluded that anode stimulation to the left perisylvian region resulted improvement in word memory in healthy patients . boggio et al . also concluded that anode stimulation to the left fronto - temporal lobe helped to improve memory performance in alzheimer disease . the results of our study suggested that anodal stimulation to the left fronto - temporal lobe improved auditory memory , confirming the conclusion of earlier studies , although the treatment did not affect visual memory . cnt test showed that the treatment had been positively effective in all areas , but other three groups , excluding auditory memory , showed no statistical difference . only few studies have been done on the effect of tdcs on cognitive function in stroke patients . while kang et al . suggested that anode stimulation at the dorso - lateral prefrontal cortex improved attention , kim et al . concluded that rtms on the same region was only effective in treating depression , and there have been two reports on patients with stroke who experienced linguistic improvement , but these studies only involved 10 and 13 patients , respectively . no studies have clearly defined the effects of noninvasive brain stimulation on the cognitive function of stroke patients . studies have discovered that tdcs affects the brain by activating n - methyl - d - aspartic acid ( nmda ) receptors and suppressing the cerebral cortex via gamma - aminobutyric acid ( gaba ) receptors . the effect of tdcs is known to last for approximately 90 minutes , during which the cerebral cortex remains excited , but there are too few studies to be able to draw conclusions on the accumulated effect . this report showed that marked improvements are noted after 3 weeks of tdcs treatment , but the accumulated effect could not be determined because serial cognitive function was not assessed . the findings from this study may have been limited by the small number of patients in each test group . cerebral infarction and hemorrhage were both included and lesions varied largely , so that spontaneous recovery could not be fully excluded . as well as excluding cathode stimulation from the treatment . additionally , the subjects were not tested for depression , which may have affected the outcome . as it has been suggested that depression is prominent among stroke patients and as depression may affect attention and memory , the possibility remains that the study may have included patients with depression , whose depression symptoms were affected by tdcs , which then subsequently resulted in improved cognitive function . furthermore , poststroke apathy can affect cognitive function , but this could not be addressed in this study . since the subjects were evaluated for cognitive performance at 24 hours after completion of the 3-week treatment program , the long - term effect of tdcs treatment remains undetermined . moreover , we excluded patients with abnormal cognitive function based on the guardian s statement , but we had not tested the cognitive function of patients before they suffered their stroke , which may have an impact on the study result . nevertheless , this study confirmed that tdcs is effective in treating poststroke cognitive impairment . we were also able to conclude that stimulation on the temporal lobe , as well as on the prefrontal lobe , as suggested by earlier studies , is helpful in cognitive recovery . the results from this study confirmed that applying tdcs to the left temporal lobe effectively improved auditory memory of patients with poststroke cognitive impairment . we concluded that tdcs can be employed as a treatment sub - protocol in a cognitive treatment program . however , further studies with a larger subject pool and various stimulation methods should be conducted to confirm these findings .

background and purposeto investigate whether transcranial direct - current stimulation ( tdcs ) can improve cognition in stroke patients.methodsforty-five stroke patients ( 20 males and 25 females , average age : 62.7 years ) with cognitive dysfunction were included in this prospective , double - blinded , randomized case control study . all patients were right - handed and the mean elapsed time after stroke was 39.3 days . three different treatments groups were used : ( 1 ) anodal stimulation of the left anterior temporal lobe , ( 2 ) anodal stimulation of the right anterior temporal lobe , and ( 3 ) sham stimulation . tdcs was delivered for 30 minutes at 2 ma with 25 cm2 electrodes , five times / week , for a total of 3 weeks , using a phoresor ii auto model pm 850 ( iomed ) . the evaluation of cognitive impairment was based on a computerized neuropsychological test ( cnt ) , korean mini - mental state examination ( k - mmse ) . the korean version of the modified barthel index ( k - mbi ) was used to assess activities of daily living functionality . these evaluations were conducted in all patients before and after treatment.resultseach group included 15 patients . pre - treatment evaluation showed no significant differences between the three groups for any of the parameters . there was significant improvement in the verbal learning test on the cnt in the left anodal stimulation group ( p < 0.05 ) . there were , however , no significant differences in the k - mmse or k - mbi scores among the three groups.conclusionsthese results demonstrated the beneficial effects of anodal tdcs on memory function . thus , tdcs can successfully be used as a treatment modality for patients with cognitive dysfunction after stroke .

Introduction Methods Subjects Treatments Statistical analysis Results General characteristics of the subjects Pre- and post-treatment comparison Comparison between the three groups Discussion Conclusions

PMC4610300

molecular dynamics ( md ) has had a long and important history in computational biology and has made many recent contributions toward understanding a variety of biological phenomena , including protein folding , protein ligand binding , and conformational change of large biomolecules . though running a large - scale simulation has historically been a demanding task , recent advancements in both software and hardware have made simulations of complex systems possible . nonetheless , because of the high - dimensional nature of the resulting time series , the generation of many md trajectories is not sufficient for providing scientific insight . in fact , data analysis has increasingly become a major barrier in the application of md simulations . one analysis technique attempts to estimate the slowly equilibrating collective motions , or eigenprocesses , contained in a simulation ( e.g. , proteins folding from unfolded conformations , ligands binding to a target , enzymes shifting from an inactive to active state , etc . ) . these eigenprocesses are useful as they can be measured experimentally , but by constructing them from a simulation , we can provide a level of detail that is unavailable to experiment alone . markov state models ( msms ) are one such method that has been useful in a variety of contexts . briefly , an msm describes continuous dynamics in phase space as a jump process over a finite number of states that partition the space . practically , the construction of an msm is an active area of research as there are significant hurdles that must be overcome in order to build a model that accurately describes the dynamics contained in a simulation . for example , an msm requires the definition of a state decomposition that is , a partition of phase space into discrete states . the accuracy of the model relies heavily on the quality of this state decomposition , but although there have been a number of improvements to this process , there is no consensus on the best method for defining a state space . an msm , however , is merely one way to estimate a system s eigenprocesses from a simulation . therefore , we can hope to overcome the practical challenges faced when constructing an msm by approximating the dynamics within a different scheme . toward this end , we introduce an extension of time - structure based independent components analysis ( tica ) using the kernel trick for estimating the characteristic eigenprocesses contained in a simulation without using an msm . first , we lay the theoretical foundation for the method , then formulate the problem and derive its solution , and finally , we use the method to analyze several toy models as well as md simulations of the fip35 ww domain from shaw et al . in this section , we briefly discuss markovian dynamics and the transfer operator approach but leave most details to other works . we also note that , for clarity , most of our notation is borrowed from prinz et al . we are interested in discrete - time dynamics in some phase space that are markovian , ergodic , and time - reversible . this density is properly normalized such that dx (x ) = 1 . let x(t ) denote a markov process in . because of the markov property , there exists a transition probability density such that p(x , y;)dy is the probability of being in some volume ( dy ) around y after time given that the system started at x. the dynamical progression of the markov chain can be described via a propagator operator , which maps a probability density at time t , pt(x ) , to a new density at time t + later , pt+(x):1additionally , there exists a corresponding operator called the transfer operator defined such that2 the functions ut(x ) are related to probability densities by3(while the propagator provides a more intuitive operation , the transfer operator s eigenfunctions will be the eigenprocesses we actually estimate in the following methods . ) the action of the transfer operator can be decomposed into a series of relaxation processes , which correspond to its eigenfunctions , i(x ) , and their eigenvalues , i:4where5 the eigenfunctions of the transfer operator are orthogonal and normalized such that6as a result of this decomposition , we can model the dynamics of the markov chain directly if we have an estimate of the eigenfunctions of the transfer operator . in fact , an msm provides an estimate of this transfer operator by estimating a finite number of the eigenfunctions as a sum of indicator functions , where the indicator functions define the state decomposition in phase space . however , the msm is not the only technique that can approximate the transfer operator . the eigenvalues of the transfer operator correspond to a relaxation time scale , ti , which can be measured via the autocorrelation function of a trajectory { x0,x1, ... ,xt , ... } projected onto the corresponding eigenfunction:7 in recent work from no and nske , it was shown that a variational principle can be derived for the eigenvalues of the transfer operator , meaning that given a mean - centered , integrable function , , the autocorrelation of f at time is always less than the autocorrelation of the first nonstationary eigenfunction , 1 , of the transfer operator:8 this is powerful because it implies that one can find the slowest dynamical processes in a simulation without building an explicit estimate of the operator itself , but by directly estimating its eigenfunctions . have successfully constructed estimates of the transfer operator s top eigenfunctions in the span of a prespecified library of basis functions by leveraging this variational bound . here , we take a different approach that does not require a predefined basis set . in what follows , we are working in the space and will assume that it is a euclidean space with some dimension , d. therefore , if x and y are in , then xy = xy denotes the inner product and x y = xy the outer product between the two length - d column vectors . time - structure based independent component analysis ( tica ) is an unsupervised learning method for finding projections that decorrelate slowly . given a markov chain in phase space : { xt } , tica attempts to find a vector v such that the autocorrelation of xt projected onto v is maximized ( eq 9).9here , and is some lag time greater than zero . to compute the expectation values in eq 9 , assume we can sample m pairs of points in phase space , { ( xt ,yt ) } , where xt is sampled from the equilibrium distribution , ( ) , and yt from the transition probability density , p(xt, ; ) . then , we can estimate the time - lag correlation matrix , covariance matrix , and mean vector using a maximum likelihood approach with a multivariate normal approximation ( see si section let1011where xt = xt , with mean given by12 given these matrices , eq 9 becomes13it can be shown that the solution to eq 13 is also a solution to the generalized eigenvalue problem:14 furthermore , the remaining eigenvectors from eq 14 are the slowest projections constrained to being uncorrelated with the previously found solutions . this method has proven to be very useful for constructing msms that reproduce the slowest time scales in a simulation . however , more importantly , since eqs 7 and 9 are the same , the solutions to the tica problem ( tics ) are also solutions to the variational problem and therefore provide estimates of the slowest eigenfunctions of the transfer operator . these estimates , however , are crude since the tics are constrained to be linear functions of the input coordinates , but if we can extend the tica problem to include nonlinear degrees of freedom then we will be able to estimate the true eigenfunctions more accurately . the simplest approach to extending the tica problem beyond linear combinations of coordinates in is to use a mapping function , : v , which expands the original representation of the data into a higher - dimensional feature space , v. we can then calculate the linear tics in the feature space , v , but since is arbitrary , these tics can in fact be nonlinear functions of the coordinates in . the dimensionality of v , however , could be enormous ( or perhaps infinite ) , and so , this scheme is impractical except in very simple cases . under certain conditions , however , it is possible to perform analysis in v without ever actually calculating the representations , (x ) , by employing what is known as the kernel trick . for illustration , we reproduce a simple example from schlkopf et al . of the kernel let and define the mapping function between phase space , , and feature space , v , to be (x ) = ( x12 , , x22 ) . because of this definition , it is easy to show that15equation 15 indicates that we can calculate the dot product in v in one of two ways . we can either compute (x ) and (y ) and explicitly calculate their dot product or we can simply square the dot product of the vectors in . either route gives us the same result , but the latter has the advantage of being much easier in cases where the feature space s dimension is very large imagine using a polynomial of degree much greater than two in eq 15or infinite . kernels are used commonly in machine learning when extending linear problems to provide nonlinear solutions . any problem that can be rewritten entirely in terms of inner products can be extended to use the kernel trick , and in the following , we show how to rewrite the tica problem in this way . remember that the tica problem searches for a projection that maximizes the autocorrelation function of a projection onto a vector v. our goal is to rewrite the optimization problem ( eq 13 ) in terms of only inner products and then apply the kernel trick to solve the tica problem in high - dimensional feature spaces . this assumption is only violated when one coordinate can be written as a linear combination of the other coordinates . in these cases , we need only project the data into a nonredundant space and continue the analysis there . we start with a set of m pairs of points in phase space , { ( xt , yt ) } , where xt is sampled from the equilibrium distribution , ( ) and yt from the transition probability density , p(xt, ; ) . consider a function : v that maps points in phase space , , into a higher - dimensional feature space , v. now we want to solve the tica problem in this new space v. the solution , v v , is a solution to the eigenvalue problem given in eq 14 . however , the covariance and time - lag correlation matrices are calculated in terms of the vectors in v : here , we have used (x ) (y ) to denote the outer product of the two vectors (x ) , (y ) v. for now , assume that the (xt ) values and (yt ) values are centered ( see supporting information ( si ) centering data in the feature space ) . it can be shown that the solution , v , is in the span of the xt values and yt values , which means that we can write v as a linear combination of the input data points ( see si let such that16we will rewrite the tica problem in v in terms of a gram matrix k , where k has 2 m rows and 2 m columns , defined as17where (x)(y ) is the inner product between the two vectors (x),(y ) v. in other words , the gram matrix can be broken into four blocks:18such that each block is an m m matrix and19202122let imm denote the m - dimensional identity matrix and 0mm denote an m by m matrix of all zeros . then , let r be a 2 m 2 m matrix given by eq 23.23 in the si derivation of the ktica solution , we show that the numerator and denominator of the rayleigh quotient in eq 9 can be rewritten in terms of and k:2425and so the tica problem in the feature space v can be written totally in terms of inner products:26which is itself a rayleigh quotient and so the solutions satisfy the generalized eigenvalue problem:27 however , solving such problems can be challenging because the matrix kk can have zero eigenvalues , which correspond to directions in v that have zero variance associated with them . these directions are either due to redundant coordinates ( in v ) or under sampling and so are not desirable solutions to the tica problem . however , these zero variance directions lead to infinite eigenvalues in eq 27 . this issue is not exclusive to the kernel tica problem and is faced in most kernel learning methods . several solutions have been reported in the cca , lda , and svm literature that introduce a regularization term . we use the method applied to kernel - cca in mika et al . but realize that another form of regularization may be even more useful . the result is a modified objective function that penalizes the solutions according to the l2 norm on .28 this new rayleigh quotient can be written as the solution to a modified generalized eigenvalue problem:29 for large enough , the result is a positive definite matrix , kk+i2m2 m , which allows us to compute the solutions reliably . we refer the reader to other kernel methods that use a similar regularization scheme for a discussion , as well as the results section two - dimensional brownian dynamics . as in the case of linear tica , there are many solutions to the ktica problem ( herein called ktics ) , where each is the slowest projection subject to being uncorrelated with all of the slower ktics . let v v denote the i ktic and let denote the i eigenvector of eq 29 corresponding to v : because of the variational principle , the ktics are direct estimates of the transfer operator s right eigenfunctions : the first eigenfunction ( 0(x ) = 1 x ) is not estimated by ktica as it is stationary , and so the second eigenfunction is estimated by the first ktic . however , to directly compare to the eigenfunctions of the transfer operator , we need to ensure that the solutions to the ktics are normalized correctly . the eigenfunctions of the transfer operator are orthonormal with respect to the inner product defined in eq 5 . since the first eigenfunction is equal to one for all points x , this means that the eigenfunctions have these properties : 1 . all dynamic eigenfunctions ( i > 0 ) have zero mean ( ) . since the gram matrix is centered in the feature space ( see si section centering data in the feature space ) , the ktics have zero mean . the eigenfunctions are uncorrelated and are normalized such that they have unit variance ( cov(i(x ) , j(x ) ) = i , j = ij ) . as shown in the si section derivation of the ktica solution , the variance of the i ktic , is given by so we can normalize each such that the variance is one . arbitrary points in can be projected onto the ktics as this operation can be written entirely in terms of inner products:31we emphasize here that it is possible to evaluate the value of v(x)and therefore i+1(x)without ever specifying or v. in a sense , the ktics behave like a kernel density estimator , where the value at some point x depends on how close that point is to the training points . to solve the ktica problem , we need to calculate the gram matrix , k , given in eq 18 , which requires the choice of a kernel function that specifies inner products in the feature space . although there are a variety of kernel functions available , for this work we chose the commonly used gaussian kernel:32here , x and y correspond to two conformations of a molecule . technically , these vectors are points in phase space and represent the positions and momenta of all atoms in the simulation . however , in most bimolecular simulations , we do not care to describe the rotation and translation of the molecule . moreover , because we only have finite sampling , if we attempt to describe rotation and translation in our models , then we will likely do so only by sacrificing accuracy in describing the conformational change that we are interested in . for this reason , for each application we select an internal set of coordinates ( e.g. , all dihedral angles or contact maps of a protein ) to use in eq 32 . these coordinates will depend on the particular system being studied , and so we leave their specification to the application sections below . with the kernel given in eq 32 , the gram matrix is defined as33 the solutions to eq 29 provide an explicit form of the estimates of each eigenfunction i+1:34 this expression highlights the analogy to a gaussian kernel density estimator , where we can evaluate i+1 at any point x based on the eigenvectors from eq 29 and how close x is to each point in the training set . in addition to the kernel , we must choose the correlation lag time , , and the regularization strength , . in previous studies , tics were largely unperturbed by the choice of ; hence , for each of our example systems , we fix a and ask for the best model given this choice . therefore , there are two free parameters we must specify before we can solve eq 29 : and . bayes theorem demonstrates that the probability of a model given the data is proportional to the probability of the data given the model , which means that we can select a model based on maximizing the model s likelihood of generating new data . here , a model is the set of ktics from eq 29 solved with particular values for and . therefore , our strategy will be to solve eq 29 over a range of values for and and then pick the model that assigns the highest probability to new data . as the ktics are estimates of the transfer operator s eigenfunctions , we can define a likelihood based on eq 4 . let k be a positive integer , then let { ( at ,bt )}t = 1n be pairs of test points that were not used in computing the ktics , where at is sampled from the true equilibrium distribution , ( ) , and bt from the true transition probability density , p(at, ; k ) . then , the probability of observing all of these transitions can be written in terms of the infinitely many eigenfunctions of the transfer operator : however , we do not have access to infinitely many eigenfunctions , so we instead truncate the expansion at the top m + 1 eigenfunctions to calculate an approximate probability , p.35 this approximation is better when m is large , or when { i : i > m } are very small . since the equilibrium probability is not dependent on either of and , we can ignore the ( ) terms when comparing models . this is especially clear when looking at the approximate log - likelihood:36 for all applications , we calculate a set of ktics for a variety of values of and on a subset of the full data set ( the training set ) . then , eqs 34 and 36 can be used to evaluate the approximate likelihood on { ( at , bt)}t = 1n the test set that was initially left out of the calculation . the values of and that are selected are those that are most likely according to eq 36 . one technical issue when calculating these probabilities is that since we truncate the infinite expansion at relatively few ktics , the resulting probability is not guaranteed to be positive . in practice , this can be difficult to handle but can be avoided by only testing pairs of points that are separated by many multiples of . in this way , the faster components we ignore in eq 36 have equilibrated and so the truncated sum is a better approximation of the true ( infinite ) expansion . admittedly , this problem likely indicates that defining such a probability based solely on the slowest eigenfunctions of the transfer operator is not possible . there may in fact exist a better scheme for evaluating these models ; however , in the results below , we show that , at least for simple systems , the likelihood approach does work . computationally , solving the ktica problem requires finding the solution to eq 29 , which is a generalized eigenvalue problem . however , the matrices on either side are 2 m 2 m , where m is the number of data points in the training set . this is a computational challenge for all kernel methods , as the solution becomes more difficult to compute as more data is used . here . however , there are many sophisticated solutions that have been developed for other kernel - learning methods , and these will certainly be useful in the future . we simulated a markov jump process on a one - dimensional , four - well potential as suggested in prinz et al . the potential given by37was discretized into 100 points between 1 and 1 , and a transition matrix was defined such that at each point , there was nonzero probability to move left , move right , or stay at the current point . in each case , the probability to move from point xi to xj was proportional to exp[(v(xj)v(xi ) ) ] . from the first 1000 pairs of transitions separated by 10 time steps were used in the ktica method . the euclidean distance between points was used in conjunction with the gaussian kernel defined in eq 32 to calculate the gram matrix . the approximate likelihood scores were calculated for 1000 pairs of points sampled from the second trajectory separated by 10 timesteps . in this simple case , there were many models that were roughly the same quality as judged by the likelihood scores ( figure 1 ) . approximate log likelihood evaluated on test data provided a similar score for most models we tested . models using a value of 0.01 for or a value of 10 for were given very low scores , while all other models seemed to be good enough . the best model according to the likelihood evaluated on test data correctly determined the top three eigenfunctions for this system . the ktics are shown in blue while the actual eigenfunctions are shown in red . it is illustrative to remember that the tica solution is trivial in this one - dimensional case . even in this simple case , the nonlinearity in the ktics prove to be essential for estimating the transfer operator s eigenfunctions . since the generating process was itself an msm , there are three slow eigenfunctions , corresponding to transitions across the three barriers on this system . the best model correctly determined the slowest three ktics and are illustrated in figure 1 . these results illustrate the value of nonlinearity in estimating the eigenfunctions of the transfer operator . in fact , since the dynamics take place in a one - dimensional space , there is only one solution to the regular tica problem , which only ( crudely ) estimates the slowest eigenfunction of the true transfer operator . we also investigated the mller potential , and simulated dynamics that were governed bywhere = 10 , kt = 15 , and r(t ) is a delta - correlated gaussian process with zero mean , and v(x ) was defined aswhere a = ( 1,1,6.5 , 0.7 ) ; b = ( 0 , 0 , 11 , 0.6 ) ; c = ( 10,10,6.5 , 0.7 ) ; a = ( 200,100,170 , 15 ) ; x = ( 1 , 0,0.5,1 ) ; y = ( 0 , 0.5 , 1.5 , 1 ) as suggested by mller and brown . using the euler maruyama method and a time step of 0.1 , we produced two trajectories of length 10 time steps . the initial positions were sampled via a uniform distribution over the box : [ 1.5,1.2 ] [ 0.2,2.0 ] . the ktica algorithm was used to analyze the first trajectory by ( regularly ) sampling 2000 pairs of transitions separated by 10 timesteps . the cross - validation scores were calculated for 1000 pairs of transitions separated by 50 timesteps using the top two ktics . as there are essentially three local minima in the mller potential , the top two eigenfunctions should correspond to population transfer between the three wells . the model selected by the likelihood criterion correctly estimates these eigenfunctions ( figure 2 ) while numerous other models do not . ktica models for the mller potential simulations are not as well - behaved as the one - dimensional case . there were a few models that assigned a nonpositive value for the likelihood of test data ( white regions above ) . however , the maximum likelihood model correctly identified the top two eigenfunctions , which separate the three major minima in the potential energy surface . these eigenfunctions are depicted in the bottom two panels , with the red and blue color corresponding to the value of the ktic . the solutions are highly dependent on the choice of the , which indicates the necessity of regularization . if is too small , then the ktics focus on only a few points that are not sampled often in the data set . when it is too large , the eigenvalues are underestimated and the eigenfunctions are distorted from the correct functions . the approximate likelihood evaluated on test data chooses an intermediate value of whose ktics correctly separate the three wells in the mller potential ( figure 3 ) . three models with = 0.5 but varying values are depicted above . these results illustrate the necessity of using regularization . when is too small ( top ) , the solutions focus on only a few points in the data set that are not sampled heavily , while when it is too large , the solutions are distorted ( bottom ) . fortunately , the likelihood based model selection criterion picks an intermediate value of ( middle ) whose ktics are better estimates of the actual eigenfunctions . a single trajectory of alanine dipeptide ( ala1 ) was simulated using the amber99sb - ildn force field in implicit solvent ( gbsa ) for 500 ns using openmm6.0 with a time step of 2 fs at 300 k using a langevin integrator . the trajectory was subsampled every 250 fs for analysis and split in half , with the first half serving as the training data and the second serving as the test data . the ktica calculation was performed by regularly sampling 2000 pairs of points separated by 1.25 ps from the training data . the gaussian kernel was used in conjunction with a distance metric defined on intra - atomic distances . for each conformation , the distance between all pairs of heavy atoms were computed , which formed a vector of length 45 . then , the distance between two conformations was the euclidean distance between these vectors . for the likelihood calculation , we regularly sampled 5000 pairs of points separated by 6.25 ps to ensure that the approximate likelihoods defined by the top two ktics were non - negative for most models . we should not expect this approximate probability to accurately describe motions at 1.25 ps but would hope that as one looks further in time the probability based on the top two eigenfunctions becomes more accurate . since alanine dipeptide has been the subject of many computational studies , it is known that the slowest two relaxations occur along the axes of the ramachandran plot of the two dihedral angles , and . the most likely model recapitulated these two slow processes ( figure 4 ) . we reanalyzed the two 100 s md trajectories of the fip35 ww domain performed by shaw et al . the ktica problem was solved by analyzing 10 000 pairs of transitions separated by 50 ns from the first trajectory . ( note that because of the size of the data set , the pairs of transitions overlapped one another . this is , in a sense , the same as using the sliding window approach for counting transitions when building an msm , which is common practice . ) the gaussian kernel defined in eq 32 was used with a distance metric defined on the protein s contact map . each conformation was represented as a 528-element vector of intramolecular residue residue distances . where all pairwise distances were tracked between residues that were at least three amino acids away from each other . the distance between a pair of residues was computed by taking the distance between the two closest heavy atoms in the respective residues . the likelihood defined by the slowest ktic alone was evaluated on 1000 pairs of transitions separated by 500 ns . this time was long enough to assign non - negative likelihoods to a majority of the models we tested . as in the one - dimensional case , there are several models that are very good according to the cross - validation scores , while models with small values of and perform poorly . alanine dipeptide has been studied extensively , and it is known that the slowest two relaxations occur along the axes of the ramachandran plot . the projection of the test data set into these axes shows that the top two ktics correctly identify the slow motions in the alanine dipeptide simulations . likelihood defined by only the slowest ktic was evaluated for the ww domain test data . again , small values of and produced likelihoods that were often negative for test data ; however , there is a region in which all likelihoods were positive . the maximum likelihood model s slowest ktic corresponded to the folding transition , as it was correlated with rmsd to the native state . the time scale associated with this ktic was 4.4 s , which agrees with previous analysis of this same data set . the slowest eigenfunctions of the transfer operator for the ww domain are not known , but for the maximum likelihood model , the slowest ktic is physically reasonable as it separated folded and unfolded states in the simulation ( figure 5 ) . additionally , the ktic had a corresponding time scale of 4.4 s , which is consistent with previous analysis of this data set . though the tica method has proven to be remarkably successful for building state decompositions for msm analysis , its solutions are limited because they are constrained to be linear functions of the input coordinates . for this reason , the extension to nonlinear functions by using the kernel trick represents a substantial improvement that allows ktica to provide estimates of the transfer operator s eigenfunctions directly , without the use of an msm . we have shown here that the method can accurately calculate the slowest eigenprocesses of simple systems but admit that there is still work to be done before it can be routinely used for analyzing arbitrarily complex dynamics . computationally , the matrices in eq 29 scale with the amount of data used , which means that as the systems become larger and more complicated and more data is needed , the eigenvalue problem will become increasingly difficult to solve . however , this problem is not unique to ktica and , in fact , is ingrained in all kernel learning methods . for this reason , we expect significant improvements to be made rapidly as these methods have been extensively studied in machine learning .

the allure of a molecular dynamics simulation is that , given a sufficiently accurate force field , it can provide an atomic - level view of many interesting phenomena in biology . however , the result of a simulation is a large , high - dimensional time series that is difficult to interpret . recent work has introduced the time - structure based independent components analysis ( tica ) method for analyzing md , which attempts to find the slowest decorrelating linear functions of the molecular coordinates . this method has been used in conjunction with markov state models ( msms ) to provide estimates of the characteristic eigenprocesses contained in a simulation ( e.g. , protein folding , ligand binding ) . here , we extend the tica method using the kernel trick to arrive at nonlinear solutions . this is a substantial improvement as it allows for kernel - tica ( ktica ) to provide estimates of the characteristic eigenprocesses directly without building an msm .

Introduction Theory Results Conclusions

PMC4488252

goldman and benedek described the structure of shark ( spin dogfish ) , squalus acanthias , and determined the relationship between morphology and nonswelling properties of the cornea . the authors described the morphology of the corneal stroma which consisted of parallel running lamellae which are interconnected across by sutures . a comparative study of collagen in the cornea and sclera of young and adult spiny fish showed that corneal sutural fibrils contained collagen type i , whereas the scleral cartilage matrix contained type ii . the immunofluorescence labelling of collagen type i was observed on the thick fibrils but not on the thin fibrils . praus and goldman studied the nature of glycosaminoglycans of the shark dogfish and reported that the shark cornea contained 75% galactosaminoglycans of the total glycosaminoglycans present . recently scott mccall et al . used the shark dogfish as a model to study collagen cross - linking . the distribution of corneal layers in the fish cornea varies depending on the aquatic environment . s. p. collin and h. b. collin , in their review , describe the structure of the cornea in various fish . the florida gar fish ( lepisosteus platyrhincus ) cornea consists of the epithelium , bw , stroma , dm , and endothelium . the corneas of the sandlance fish ( limnichthyes fasiciatus ) , pipefish ( corythoichthys paxtoni ) , and salamander fish ( lepidogalaxias salamandroides ) are covered by the epithelium which contains goblet cells . the stroma is composed of a thin dermal stroma which contained aggregates of pigments and sutures , an iridescent layer , a mucous layer , and anterior sclera stroma . the posterior part of the cornea is covered by the dm and endothelium [ 79 ] . in ratfish ( hydrolagus collie ) ( cartilaginous fish ) , the cornea contains the conventional epithelium , bw , stroma with sutures , and dm . the deep - sea teleost [ coryphaenoides ( nematonurus ) armatus ] contains a very peculiar cornea . the fish cornea consists of an epithelium followed by a thick dermal stroma and mucous layer . they have the most primitive vertebrate cornea containing cfs organized in stromal lamellae which are crossed by fibrillar sutures . the primitive morphology of the cornea provides its nonswelling properties and maintains transparency . there have been no studies carried out on the organization of pgs and cfs of the stingray and shark . in this paper we investigated the ultrastructure of cfs , pgs , and sutures of the shark and stingray stroma . we were interested to see whether there would be any ultrastructural differences between these closely related species . three stingrays ( 6 - 7 year old ) ( species : dasyatis americana ) and three sharks ( 6 - 7 year old ) ( species : squalus acanthias ) were used for the study . the eyes were fixed in 4% paraformaldehyde within 60 minutes of the fish 's death . the eyes were kept in 4% paraformaldehyde for 12 hours and then washed in phosphate buffer ( 3 15 minutes ) . the corneas were removed from the eyes and were fixed in 2.5% glutaraldehyde containing 0.05% cuprolinic blue ( bdh ltd . , dorset ) using a critical electrolyte concentration mode sodium acetate + magnesium chloride buffer overnight at room temperature . the tissue was washed in 2.5% glutaraldehyde in sodium acetate + magnesium chloride ( 3 15 minutes ) followed by another wash in sodium tungstate ( 3 15 minutes ) . the tissue was then dehydrated through a graded ethanol series ( 50% to 100% ) 15 minutes each and 100% acetone ( 2 30 minutes ) . they were infiltrated in a mixture of acetone and taab 031 resin ( 1 : 1 ) for 8 hours . the tissue was further infiltrated in 100% taab 031 resin for 8 hours ( 3 ) . each quadrate was placed in green mold for embedding into blocks for sections to be cut . the central part of the cornea in each quadrate was oriented at the cutting surface of the block and polymerized in taab 031 resin for 8 hours at 70c . all the sections were cut at a 90 degree angle to the surface of the cornea ( cross section ) . semithin ( 1 m ) sections were collected on glass slides and stained using toluidine blue . the sections were stained with 2% uranyl acetate ( 10 minutes ) and lead citrate ( 10 minutes ) and observed using transmission electron microscopy jeol 1400 ( jeol ltd . , akishima , japan ) . all the digital images were taken from the central part of the cornea . to analyze the cfs and pgs , 3 digital images were taken from the 4 lamellae of the anterior stroma , 4 lamellae from the middle stroma , and 4 lamellae from the posterior stroma above descemet 's membrane . to observe the pgs distribution without the structure of cf the thickness of the lamellae , the minimum cf diameter , center - to - center spacing , and pg area were measured using the soft imaging system ( item , soft imaging system gmbh , mnster , germany ) analysis program . the electron micrographs of cfs were processed with the item program and color coded according to the size of the cfs and pgs . the mann whitney u test ( spss ) was used for statistical analysis because the data were not normally distributed . tissue procurement and use it was ethically approved by the local ethical committee , king saud university , saudi arabia , and king khalid eye specialist hospital , riyadh , saudi arabia . the corneal stroma of the stingray consisted of the lamellae which were running parallel to the surface of the cornea . electron microscopic observations showed that the bw consisted of randomly running cfs ( figure 1(b ) ) . in the posterior part of the bw , the cfs of the bw ran parallel to the corneal surface and blended ( arrowhead ) into the most anterior stromal lamellae ( figure 1(b ) ) . the anterior lamellae adjacent to the posterior part of the bw were very thin and some of them consisted of three to four cfs ( figures 1(b ) and 2(a ) ) . below these thin lamellae , occasionally , orthogonally running cfs were observed ( figures 1(b ) and 2(b ) ) . the thickness of the most anterior stromal lamellae was from 0.9 m to 2.33 m . the cf diameter in these lamellae was very small ( 20 nm ) ( figure 2(a ) ) . the whole cornea contained approximately 25 lamellae . under the electron microscope the lamellae thickness varied from the anterior to the posterior stroma . the thinnest lamellae were in the anterior stroma whereas the thickest lamellae were in the middle stroma . the mean thickness of the anterior stromal lamellae ( 19 ) , middle stromal lamellae ( 1020 ) , and posterior stromal lamellae ( 2126 ) was 2.62 m ( n = 27 ) , 8.65 m ( n = 30 ) , and 4.88 m ( n = 15 ) , respectively . the lamellae in the anterior stroma were not interlacing but instead were running parallel to each other ( figure 2(b ) ) . the cfs within the lamellae in the anterior , middle , and posterior stroma were running parallel to each other . the cross section of the cfs showed the distribution the cfs of variable diameters within the lamellae ( figure 2(d ) ) . the posterior part of the cornea was covered by the dm ( figure 2(f ) ) . the dm consisted of very fine , loosely interwoven fibrils ( figure 2(f ) ) . the keratocytes were distributed throughout the stroma and contained a large nucleus ( figure 3(a ) ) . throughout the stroma of the stingray , sutures were observed running obliquely to the lamellae ( figure 3(b ) ) . in some places the longitudinal section of the sutures showed that they consisted of two types of fibrils , thick cfs and thin microfibrils ( figure 3(c ) ) . the cross section of the suture showed that the thin microfibrils were surrounded by the thick cfs ( figure 3(d ) ) . the attachment of the pgs was not observed within the microfibrils ( figure 3(f ) ) . the cfs in the anterior and posterior part of the suture were randomly arranged and blended with the cfs of the lamellae ( figure 4(a ) ) . in some places the anterior and posterior end of the sutures also contained groups of microfibrils surrounded by the cfs which blended into the lamella ( figures 4(a ) and 4(b ) ) . at the anterior and posterior end of the sutures , pgs were attached to the cfs but not to the microfibrils ( figure 4(c ) ) . the longitudinally running microfilaments were occasionally observed in the stroma ( figure 4(a ) ) . the structure of the shark cornea was very similar to the structure of the stingray cornea except for the distribution of pgs around the cfs . in the shark , the sutures of the three or four lamellae were connected to each other and ran in straight lines unlike in the stingray . the pgs were small in size and closely arranged around the cfs ( figure 4(e ) ) . the cf diameter of the stingray and shark was analyzed by processing with an electron micrograph ( figures 5(a ) and 5(c ) ) using the item program . after processing , the images were displayed using color coding to demonstrate the distribution of variable diameters of cfs ( figures 5(b ) and 5(d ) ) . in the stingray , the mean diameters of the cfs in the anterior ( 22.06 3.22 nm , n = 263 ) , middle ( 22.12 4.40 nm , n = 255 ) , and posterior stroma ( 22.21 5.05 , n = 307 ) were very similar to each other ( table 1 ) . the interfibrillar spacing between the cf in the anterior stroma ( 32.74 8.19 nm , n = 263 ) was less than the interfibrillar spacing in the middle stroma ( 36.9 5.12 , n = 255 ) and posterior stroma ( 37.83 7.48 nm , n = 307 ) ( table 1 ) . in the shark , the cf diameters in the anterior ( 23.37 2.92 nm , n = 278 ) , middle ( 24.80 2.51 nm , n = 263 ) , and posterior stroma ( 24.59 2.44 nm , 303 ) were also very similar to each other . in the shark the interfibrillar spacing in the anterior ( 35.84 4.18 nm , n = 278 ) and posterior stroma ( 35.17 4.37 nm , n = 263 ) was less than in the middle stroma ( 37.86 3.93 nm , n = 303 ) . in the stingray , most of the cfs were in the range from 15 to 25 nm ( red and green ) and a few of them were in the range from 25 to 30 nm ( blue ) . in the shark a very few cfs were in the range from 15 to 20 nm ( red and green ) , and most of the cfs were in the range from 20 to 30 nm ( green and blue ) . the cf diameter of the stingray in the anterior , middle , and posterior stroma was significantly less ( p < 0.0001 ) than those in the shark ( table 1 ) . the interfibrillar spacing in the anterior , middle , and posterior stroma of the stingray was also significantly different ( p < 0.0001 ) from those of the shark ( table 1 ) . in the stingray , the different sizes of pgs were decorated around the cfs of the stroma ( figures 2(d ) , 2(e ) , and 2(f ) ) and around the cfs of the suture ( figures 3(d ) and 3(e ) ) . the structure of the pgs in the anterior , middle , and posterior stroma was identified in the electron micrographs taken from the corneal sections which were not stained with uranyl acetate and lead citrate ( figures 6(a ) , 6(c ) , and 6(e ) ) . the electron micrographs ( figures 6(a ) , 6(c ) , and 6(e ) ) were processed using the item program and the variable sizes of the pgs were demonstrated by color coding in the digital images ( figures 6(b ) , 6(d ) , and 6(f ) ) . the pgs in the anterior and middle stroma were rod shaped and their sizes ranged from 50 to 464 nm ( red and green color ) ( figures 6(b ) and 6(d ) ) . the pgs in the posterior stroma were very large and ranged from 465 to 1292 nm ( blue , yellow , and pink ) ( figure 6(f ) ) . the pgs in the posterior stroma were thick in the middle and thin at the edges . the mean pgs areas in the anterior and middle stroma were similar to each other and not significantly different . the pgs area in the posterior stroma ( 324.64 182.00 nm , n = 465 ) was significantly ( p < 0.001 ) higher compared to the mean pgs area of the anterior ( 224.54 82.64 nm , n = 384 ) and middle ( 225.52 80.86 nm , n = 460 ) stroma . the density of the pgs was higher in the middle and posterior stroma compared to the anterior stroma in the stingray ( table 2 ) . the electron micrographs of the pgs in the anterior , middle , and posterior stroma , shown in figures 7(a ) , 7(c ) , and 7(e ) , were color coded according to their variable sizes and are shown in figures 7(b ) , 7(d ) , and 7(f ) . the mean area size of the pgs in the anterior stroma ( 194.52 79.26 nm ) was less than those in the middle ( 209.09 92.04 nm ) and posterior ( 201.65 92.67 nm ) stroma ( table 2 ) . the density of the pgs was also less in the anterior stroma than in the middle and posterior stroma ( table 2 ) . the pgs analysis also showed that the mean pgs area of the anterior ( p < 0.001 ) , middle ( p < 0.003 ) , and posterior ( p < 0.001 ) stroma of the stingray was significantly higher compared to the mean pgs area of the anterior , middle , and posterior stroma of the shark . the density of the pgs in the stingray in the anterior , middle , and posterior stroma was less than those in the shark stroma ( table 2 ) . the spacing between the pgs in the shark was also significantly ( p < 0.001 ) less compared to the spacing between the pgs of the stingray ( table 2 ) . the normal human cornea is composed of five layers , epithelium , bowman 's layer ( bw ) , stroma , descemet 's membrane ( dm ) , and endothelium . it is composed of cfs lamellae which contain angular lamellae relative to the transverse lamellae ( fibres ) along the corneal surface . these lamellae ( fibres ) branch several times and connect to each other and in many cases interleave into the bowman 's layer . the highest degree of this interconnectivity was observed in the transverse lamellae in the anterior stroma . it is believed that transverse lamellae and their interconnectivity play an important role in determining corneal biomechanics and stress distribution , resisting the formation of regions with stress , which are more susceptible to deformation . it was also suggested that interconnectivity of the lamellae in the anterior stroma is likely to serve to stabilize corneal shape and prevent lamellar slippage [ 15 , 16 ] . the interconnectivity also provides rigidity to the anterior stroma and specifies corneal curvature and shape . our studies showed that the stroma of the stingray consisted of concentric lamellae lying in parallel to the corneal surface and interconnected across by sutures throughout the stroma . the structure of the stingray cornea was very similar to the structure of shark cornea described by goldman and benedek . the interconnectivity of lamellae by sutures in the stingray and shark can be compared with the interconnectivity of lamellae in the anterior human stroma described by winkler et al . . we believe that interconnectivity of the lamellae by sutures in the stingray and shark plays an important role in determining corneal biomechanics and stabilizing corneal shape , as has been observed in the anterior stroma of the human cornea by winkler et al . . these sutures were also observed in the stroma of salamander and shark fish [ 1 , 9 ] . the sutures in the shark run in straight lines continuously across 3 or 4 lamellae and are connected to each other . in the stingray the sutures of different lamellae were not connected to each other and did not run in straight lines . our observations showed that the sutures in the stingray and shark consisted of thick cfs and thin microfibrils . the thin filaments were surrounded by thick fibrils as previously reported by goldman and benedek . thick cfs were composed of collagen type i whereas the thin fibrils were not composed of collagen type i . the thin fibrils were never twisted whereas the thick fibrils were observed twisting and joining together . the thick fibrils of the sutures were decorated by pgs but not the thin fibrils . goldman and benedek reported that thin fibrils never stretched within the sutures and they inhibit the stretching of the sutures . it has been suggested that the cylindrical core of thin fibrils was the principal bundle of the suture . we presumed that the cylindrical core of thin fibrils provides mechanical strength and resistance to swelling of the cornea . if the stretching of sutural fibrils was inhibited then the thickness of the cornea might be limited by the length of the thin fibril bundles . occasionally orthogonally running cfs were observed suggesting that the fibrils might be entering into the early stages of the formation of a suture and blending with the lamellar cfs . this phenomenon might be providing mechanical strength to the lamellar cfs and might be restricting the separation of the cfs within the lamellae and the separation of the lamellae from each other . in the cornea , three core proteins ( lumican , keratocan , and mimecan ) bearing glycosaminoglycan ( gag ) chains of keratan sulfate and one protein ( decorin ) bearing gag chains of chondroitin / dermatan sulfate are present . it has been reported that lumican plays an important role in regulating the cf diameter and it was higher in concentration in the posterior part of the stroma compared with that in the anterior stroma . the cf diameters in the anterior , middle , and posterior stroma were not significantly different . the density of the cfs was higher in the posterior stroma . in the shark , the cf diameter and density were significantly smaller compared with those in the middle and posterior stroma . this could be due to higher concentrations of lumican in the posterior stroma . comparing the shark and stingray , the cf diameters of the stingray were significantly smaller compared to the cf diameters of the shark throughout the stroma . this could also be due to the reduced amount of lumican present in the stingray compared with that in the shark . goldman and benedek reported that in the shark the cfs diameters in the anterior stroma ( 32.7 2.1 nm ) were larger than in the posterior stroma ( 28.2 3.8 nm ) . in the present study , the means of minimum cf diameter of shark in the anterior ( 23.37 2.92 nm ) , middle ( 24.80 2.51 nm ) , and posterior ( 24.59 2.44 nm ) stroma were very similar to each other . the mean of minimum cfs diameters of the shark in our study was smaller than the cfs diameter of the shark reported by goldman and benedek . this could be due to different processing techniques or methods of calculating the diameter . in our experiments tissue was processed with glutaraldehyde containing cuprolinic blue in sodium acetate + magnesium buffer and taab 031 resin was used , whereas they processed tissue in glutaraldehyde and osmium tetroxide embedded in epon 812 . goldman and benedek reported that the density of cfs from anterior to posterior stroma of the shark was approximately in the range from 450/m to 540/m whereas our studies showed that the density of cfs from anterior stroma to posterior stroma was in the range of 547/m to 595/m . he reported that stained cs / ds filaments are ~70 nm long , whereas ks pgs are ~70 nm long . lewis et al . reported that pgs composed of cs / ds were very large ( about 300 nm ) whereas pgs composed of ks were small ( about 65 nm ) . in the stingray , it is believed that these large pgs in the posterior stroma of stingray are composed of cs / ds pgs . the presence of the large pgs in the posterior stroma may play an important role in regulating the hydration of the stingray cornea . the pgs analysis in the shark showed that there was no significant difference in the mean area of the pgs in the anterior , middle , and posterior stroma . the density of pgs in the posterior and middle stroma was significantly ( p < 0.001 ) higher compared to the density of the pgs in the anterior stroma . it is surprising that both the stingray and the shark belong to the same class , but the pgs mean areas in the stingray were significantly ( p < 0.001 ) larger compared to the pgs mean area in the shark in the anterior , middle , and posterior stroma . the density of the pgs however was significantly less in the stingray compared to the pgs density in the shark in all regions ( anterior , middle , and posterior stroma ) . pgs of stingray and shark , like those in bovine and mouse , show no specific order . the distance between the pgs in the shark is significantly smaller compared to those in the stingray . it could be possible that this close association of the high density of pgs in the shark may strengthen the stromal cfs which provide strength to their large sized cornea as previous studies showed that large animals such as camels have a large density of small pgs . it has been suggested that in comparison with the cornea of more advanced vertebrates , the nonswelling shark cornea contains large amounts of highly - sulfated chondroitin sulfate and small amounts of keratan sulfate which regulate hydration in the aquatic environment . scott mccall et al . suggested that interlamellar bonds increase the mechanical strength of the cornea because they could physically link the entire adjacent lamellae of the corneal stroma . these physical interactions could occur between separate collagen fibrils both within an individual lamella and in adjacent lamellae . these interactions between the cfs could arise via the terminal domains of the gag chains that extend from the sides of all collagen fibrils . in elasmobranch corneas , the sutures provide a physical interlamellar bond supporting the mechanical strength of the cornea and inhibiting the swelling of the cornea in the shark [ 1 , 24 ] . we suggest that the physical strength of the sutures might be provided by the interaction of the cfs which occurs via the terminal domain of the gag chains that extend from the side of the cfs . we believe that pgs play an important role in providing the physical strength of sutures that inhibit the swelling of the cornea in the stingray and shark . the presence of high levels of chondroitin sulfate and low levels of keratan sulfate in the shark and in the stingray may also contribute to the nonswelling properties of their corneas . further studies are required to investigate the role of keratan sulfate and chondroitin sulfate by using their specific antibodies and employing biochemistry , immunohistochemistry , and immunogold techniques .

we report here the ultrastructural organization of collagen fibrils ( cf ) and proteoglycans ( pgs ) of the corneal stroma of both the stingray and the shark . three corneas from three stingrays and three corneas from three sharks were processed for electron microscopy . tissues were embedded in taab 031 resin . the corneal stroma of both the stingray and shark consisted of parallel running lamellae of cfs which were decorated with pgs . in the stingray , the mean area of pgs in the posterior stroma was significantly larger than the pgs of the anterior and middle stroma , whereas , in the shark , the mean area of pgs was similar throughout the stroma . the mean area of pgs of the stingray was significantly larger compared to the pgs , mean area of the shark corneal stroma . the cf diameter of the stingray was significantly smaller compared to the cf diameter in the shark . the ultrastructural features of the corneal stroma of both the stingray and the shark were similar to each other except for the cfs and pgs . the pgs in the stingray and shark might be composed of chondroitin sulfate ( cs)/dermatan sulfate ( ds ) pgs and these pgs with sutures might contribute to the nonswelling properties of the cornea of the stingray and shark .

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

PMC4483443

cerebral palsy ( cp ) , defined as a nonprogressive injury to the brain , covers numerous neurological conditions , which result in abnormal development of movement and postural control . injury to the brain typically results in impairments in motor function , such as muscle weakness , spasticity , and decreased selective motor control , which are primary causes of children with cp limitations in the performance of activities of daily living and participation1 . children with cp have a decreased level of daily physical activity in comparison to their healthy peers . they do not use their physical reserves sufficiently during the day to achieve optimal levels of daily physical activity2 . children with cp need to maintain a higher level of physical fitness than healthy children in order to maintain the natural growth process as they grow older3 . the peak oxygen uptake ( vo2 peak ) , typically used as a measure of aerobic fitness , is lower in children with cp , as has been shown through exercise tests using cycle ergometry or a treadmill4 , 5 . the maximum aerobic physical fitness , vo2 peak , is an internationally acknowledged measure of the fitness of the cardiorespiratory system . three studies4,5,6 have studied vo2 peak in children and adolescents with cp during a maximal exercise test . these studies compared vo2 peak to the values observed in normally developing ( nd ) children and adolesents5 , 6 . the most functional and appropriate way to assess vo2 peak in children with cp who are able to walk independently is a progressive walking or running - based maximal exercise test4 , 7 . various researchers have developed treadmill walking maximal exercise tests to assess individuals with cp and to investigate the effectiveness of rehabilitation programs4,5,6 , 8,9,10,11 . however , these methods limit the generalization of the results to actual daily walking , because a variety of comfortable walking speeds are exhibited by children with different types of cp5 . therefore , it is important to test children under general conditions that are comparable to daily living conditions . for estimating the maximal aerobic capacity , an adapted cycle ergometer test specifically developed for cp can be used12 , 13 . this aerobic test has the advantage of reducing errors that are produced when walking with diverse patterns . currently , studies of ergometer aerobic tests14 have provided insufficient evidence of their effect12 , 15 ; furthermore , no direct cardiorespiratory results have been considered in the aerobic measurements16 . although unnitan et al.17 studied the effect of uphill walking on children with diplegic cp , and measured the of physical fitness of adult male cp subjects18 , their evaluations were performed using an arm ergometer . therefore , their score does not reflect changes in fitness resulting from a mainly leg - based training program . timed walking tests may offer a less expensive , simpler , and safer alternative to laboratory testing of children with cp10 , 19 . among such tests , the 6-min walk test ( 6mwt ) has been shown to predict cardiorespiratory fitness in both healthy and severely disabled children , whether used with or without gas collection20 , 21 . novac et al.22 systematically discussed the most effective and applicable interventions for children with cp . in their reports of the literature , the most recommended and applicable intervention was green light intervention , and they emphasized the importance of fitness training for aerobic fitness . before children with cp perform aerobic fitness tests , their aerobic ability should be measured . however , previous studies have reported in lacked consistent results and utilized various measurement methods and environments . thus , it is necessary to provide a precise method and measurement for base data to obtain accurate reference values . accordingly , this preliminary study aimed to determine the cardiorespiratory endurance of children with cp using a case series study in order to provide the reference data required for interventions appropriate for south korean cp sufferers , since aerobic ability evaluation and interventions for it for children with cp are not well recognized in south korea . six children participated in this study . four subjects ( 3 boys , 1 girl ) had a diagnosis of bilateral or unilateral spastic cp , and 2 subjects ( 1 boy , 1 girl ) were children with normal development . four children with cp gmfcs ( gross motor function classification system ) level i and ii and two normally developing children ( age : 715 years ) were studied ( table 1table 1.general characteristics of subjectssubjectstypegmfcs levelgender ( boys : 0 , girls : 1)age ( years)height ( cm)weight ( kg)nda--01215148b--1711522cpcspastic bilateral101214031dspastic bilateral2015155.446espastic unilateral21711632fspastic unilateral201013838cp : cerebral palsy ; nd ; normally developing children ; gmfcs : gross motor function classification scale ) . subjects with cp were recruited from a medical center in gyeonggi - do south . this study complied with the ethical prinsiples of the declaration of helsinki , and all the subjects and parents received explanations regarding the purpose and procedure of the study before voluntarily agreeing to take part . all parents signed an informed consent statement before the start of the measurements . before testing , a medical examination was completed and anthropometric data were collected . the inclusion criteria were gmfcs levels i to ii , uni- or bilateral spastic cp , age of 516 years , ability to walk continuously for 6 min , and ability to provide informed consent . potential subjects were excluded if they had received orthopedic surgery or neurosurgery and/or botulinum toxin injection within the previous 6 months , or had cardiac or respiratory conditions that could have been negatively affected by the proposed training . all the selected subjects were also free of intellectual problems that could have influenced their participation and/or motivation . cycle ergometer testing was performed with calf supports ( motomed viva cycle , reek medizinterchnik gm bh , betzenweiler , germany ) . the cycle ergometer test served to familiarize and habituate the subjects , and it also allowed us to check whether a given subject needed to have his / her feet attached to the pedals by velcro strapping23 . the concentration and volume of oxygen consumed ( vo2 ) , peak respiratory exchange ratio ( rer peak ) , and minute ventilation ( ve ) were measured simultaneously by portable metabolic measurement systems with prevent pneumotachs ( medgraphics vo2000-medical graphics , st . the ergometer test began with a 5-min warm up followed by 612 min of exercise with increasing intensity at a pedaling rate of 50 rpm as paced by a metronome . verbal encouragement was provided throughout the test to help each child achieve maximal effort . the subjects were also fitted with a neoprene face mask that could be used with the vo2000 . a polar hr strap was also fitted around the chest of the subjects ( polar electro oy , kempele , finland ) . gas exchange and pulmonary ventilation values were recorded using the breathbreath mode with 20-s average values printed during testing . cp : cerebral palsy ; nd ; normally developing children ; gmfcs : gross motor function classification scale the 6-min walk test was performed in the laboratory corridor between two lines set 20 m apart according to a standard procedure24 under the supervision of the same physiotherapist . the subjects were instructed to walk as far as possible in 6 min . the subjects received standardized and vigorous verbal encouragement , and they were advised of the distance covered and time remaining every 30 s. all subjects performed , the cycle - ergometer test first , and the 6-min walking test was conducted on the next day to avoid fatigue effects . the ages of the subjects were distributed as follows : two were aged 7 years , two were aged 12 years , one was aged 10 years , and one was aged 15 years ; i.e , the youngest and oldest subjects were 7 and 15 years of age . regarding the subjects gmfcs assessment , three were level ii , and one was level i. two normal children of 7 and 12 years age participated in this experiment to serve as controls for comparison with the children with cp . the vo2 peak ( ml / kg / min ) of child f ( cp ) was 57.4 , which was the highest , and that of child d was 22.6 , which was the lowest . the two normal children , a and b , had vo2 peaks ( ml / kg / min ) of 22.9 and 56.5 , respectively . in the 6mwt , child c ( cp ) walked 578 m , which was the longest distance , and child e walked 390 m , which was the shortest distance ( table 2table 2.subjects peak cardiorespiratory responses to ergometrytypesubjectsvo2 peakrer peakve peakhr peak6mwtnda22.90.8218.5105580b56.50.9723.8135450cpc25.91.3625.3145578d22.61.0222.9131527e44.50.8732.5129390f57.40.9759.5147560vo2 peak ( ml / kg / min ) : peak oxygen uptake ; rer peak : peak respiratory exchange ratio ; ve peak ( l / min ) : maximum ventilation per minute ; hr peak ( beats / min ) : peak heart rate ; 6mwt ( m ) : 6-min walk test ) . furthermore , the comparison of vo2 peak between normal child a and child c ( cp ) showed that the former had a value of 22.9 , which was lower than that of the latter . the comparison of vo2 peak between normal child b and child e ( cp ) showed that the latter had a value of 44.5 , which was lower than that of the former . the comparison of 6mwt between normal child a and child c ( cp ) and between normal child b and child e ( cp ) showed that the normal children walked further than the children with cp . vo2 peak ( ml / kg / min ) : peak oxygen uptake ; rer peak : peak respiratory exchange ratio ; ve peak ( l / min ) : maximum ventilation per minute ; hr peak ( beats / min ) : peak heart rate ; 6mwt ( m ) : 6-min walk test this preliminary study aimed to determine the cardiorespiratory endurance of children with cp using a case series study to provide the reference data required for interventions appropriate for south korean cp sufferers , since aerobic ability evaluation and interventions for it for children with cp are not well recognized in south korea . children with cp have lower vo2 peak values ( ml / kg / min ) as well as shorter walking distances in set times than normal children . numerous studies have reported the lower physical activities of children with cp in terms of aerobic abilities4 , 5 , 1725 , and some studies have reported that children and adolescents with cp have lower physical activity abilities than normal peers26 . verschuren et al.6 reported that comparisons with normative data sets suggest that adolescents with cp tend to participate in less structured and lower intensity physical activities than healthy adolescents . durstine et al.27 described a vicious circle in which persons with a chronic disease or disability experience less physical activity , which then leads to a cycle of deconditioning and further physical deterioration and reduction in activity . however , as seen in this study , the child with cp aged 7 ( subject e ) had a lower vo2 peak value than the normally developing child ( subject b ) , and the child with cp aged 12 ( subject c ) had a higher vo2 peak value than the normally developing child ( subject a ) . subject b ( nd ) had a lower vo2 peak than subject e ( cp ) , consistent with the results of previous studies6 , 13 . one study6 reported that a child with cp had a vo2 peak value of 42 ml / kg / min , which was close to our study result of 44.5 ml / kg / min ; however , the interpretation of the results of this previous are limited , because the subjects were aged from 7 to 17 years . with regard to the comparison of the results of subjects a and c , it is necessary to determine the reason for the higher vo2 peak value in children with cp than in normal children , considering of various possibilities . a child with gmfcs level i can perform gross motor skills such as running and jumping and participate in physical and dynamic activities . in contrast , a child with cp at gmfcs level ii can not participate in physical and dynamic activities such as playground activities owing to his / her limited abilities . despite examining only one case in this study , our results suggest children with cp who have small body mass and high functional level have even higher vo2 peak values than normal children . nsenga et al.13 reported that children with cp at gmfcs levels i and ii can have cardiorespiratory endurance similar to that of normal children of similar ages . the former showed higher values ( 41.4 ) than the latter ( 33.6 ) . this result was used as an explanation for the lower vo2 of children with cp . the present study found comparable values for children and adolescents with cp and normally developing subjects . the ventilation peak ( vepeak ) value for child c ( cp ) was higher ( 25.3 ) than that ( 18.5 ) of child a ( nd ) . similarly , the child e ( cp ) showed a higher value ( 32.5 ) than that ( 23.8 ) of child b ( nd ) . the distance walked strongly correlated with the 6mwt vo2 peak ( r = .948 ) and was related more modestly to the cycle - ergometer vo2 peak ( r = 0.625)19 . in this study , vo2 was not collected while performing the 6mwt . however , the results of the present study show that children with cp ( c and e ) walked shorter distances than normal children ( a and b ) . nsenga et al.19 reported that the 6mwt distance increased after aerobic training , showing a correlation between the cardiorespiratory capacity and 6mwt distance . although a previous study suggested there is no significant correlation between the ergometer vo2 peak and 6mwt distance , maanum et al.28 reported that the 6mwt distance was a significant predictor of cardiorespiratory capacity for children with spastic cp . peak rer is generally used as a secondary criterion for maximal effort ; however , large individual variations may be present depending on the test modes and protocols29 . the average peak rer values in the present study ( 0.871.36 ) were comparable to the average values reported in previous studies of children with cp ( 0.91.15 ) , and most studies have reported a peak hr of 190 bpm4 , 30 . these results does not support the finding that the children did not produce their maximal effort because of the lower peak hr values . however , the present study conducted ergometer tests and not treadmill tests ; furthermore , there was an age difference among the subjects . thus far , most studies of children with cp have focused on cycle ergometer or treadmill walking tests6 , 8,9,10 , seeking to verified the effectiveness of these tests12 , 13,14,15 , 17 . however , the available literature on the cardio - respiratory capacity of children with cp focuses on treadmill walking rather than cycle ergometry . verschuren et al.7 noted that treadmill protocols such as the bruce and balke tests are not appropriate for children with cp . furthermore , treadmill testing is difficult for many children with cp because they suffer from severe balance issues31 . it is difficult to compare the children from all studies that have provided information about vo2 peak for children and adolescents with cp . although many studies have recognized the importance of fitness for maintaining aerobic capacity , it is not easy to conduct aerobic tests with children with cp owing to the limitations described in the studies cited above . among studies with a high evidence level , the accuracy of the results may not be that high owing to the use of arm ergometry rather than a test of the lower extremities . other studies include a case study15 with a low evidence level and studies with a small number of subjects12,13,14 . accordingly , more evidence and data that can be used as a reference to provide fitness programs for school children with cp are needed in the future . this study aimed to determine the cardiorespiratory endurance of children with cp using a case series study in order to provide the reference data required for interventions appropriate for south korean cp sufferers , since aerobic ability evaluation and interventions for it for children with cp are not well recognized in south korea . despite success in the study aims , the results should be interpreted with care , owing to the small number of participating subjects . in conclusion , this case report reports cardiorespiratory parameter values that are similar to those that have been reported previously . fitness training for aerobic fitness is an important intervention method for children with cp . accordingly , it is necessary to provide evidence of cardiorespiratory fitness levels for children with cp when such fitness programs are conducted . furthermore , it is necessary to provide precise methods and measurements for base data in every assessment to obtain accurate reference values . in this regard , our study will provide information about vo2 peak values for an aerobic - fitness - related study conducted with children with cp in the future .

[ purpose ] this preliminary study aimed to determine the cardiorespiratory endurance of children with cerebral palsy ( cp ) using a case series study in order to provide the reference data required for interventions appropriate for south korean cp sufferers , since aerobic ability evaluation and interventions for children with cp are not well recognized in south korea . [ subjects and methods ] four children and adolescents with cp gmfcs ( gross motor function classification system ) level i and ii and two normally developing children ( nd ) ( age : 715 years ) were studied . cycle ergometer testing was performed to determine their vo2 peak and rer peak concentrations as well as ve peak and 6mwt distance . [ results ] the vo2 peak was lower in subject e ( cp ) at 44.5 than in subject b ( nd ) , and it was lower in subject a ( nd ) at 22.9 than in subject c ( cp ) . the 6mwt distance was longer in subjects a and b ( nd ) than in age - matched cp subjects . [ conclusion ] this case report demonstrates that the cardiorespiratory parameters values of cp children were similar to those reported previously . further research is required to evaluate the normative values of cp and the optimal cardiorespiratory parameters .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC1524905

the identification of genomic regulatory elements is an important but unsolved problem in genome annotation ( 1 ) . of the 5% of mammalian genome that is estimated to be under evolutionary selection pressure , less than a third is coding ( 2,3 ) . the remaining portion is believed to be composed of untranslated regions , non - coding genes , chromosomal structural elements and regulatory elements that control a variety of biological processes including gene expression , translation , chromosomal replication and condensation . whereas the number of coding genes in many of the sequenced organisms can now be reasonably estimated , there is no clear estimate of the number of functional regulatory elements in these genomes , especially in the metazoa . in eukaryotes ranging from the nematodes to flies to the mammals , the number of coding genes is similar ( 2,46 ) , and it is now thought that organismal complexity may be attributed to phenomena such as alternative splicing , dna rearrangement and increased number of transcriptional regulatory elements as well as transcription factors ( tfs ) which regulate gene expression ( 7 ) . the identification of cis - regulatory elements controlling gene expression , and characterization of their interaction with the respective tfs , thus lie not only at the very heart of understanding of the network of gene interactions but also of explaining the origins of organismal complexity and development . as such these representations are general and can be used to describe any class of short dna sequence elements . however the theories for weight matrix model , which is a common way to represent a collection of dna elements , are based on the biophysical considerations of protein dna interactions ( 811 ) ( as described in the following section ) . therefore , here we primarily discuss the transcriptional regulatory elements , more specifically the dna sites that are bound by the tfs . it is estimated that there are 2000 tfs in the mammalian genomes ( 2,5 ) and 1000 in the flies and worms ( 7 ) . however , only for a minority of the tfs ( 900 in human , 700 in mouse , 200 in drosophila and 100 in caenorhabditis elegans ) is there currently any known information on binding sites or interacting protein partners ( 12 ) . dna - binding site models are available for 500 vertebrate tfs , and < 5000 genomic sites are known in all vertebrates in fewer than 3000 genes ( 12 ) . based on the information available from a few of the well - studied genes ( 13 ) , it appears that the total number of such sites in the multicellular genomes could be at least an order of magnitude higher than the number of coding genes , i.e. in the order of hundreds of thousands or more . thus , our knowledge of the tfs , and especially their binding sites and regulated genes , is severely limited at this present time . computational methods for modeling and identification of dna regulatory elements have been developed over the past two and a half decades ( 1420 ) . orthogonal information from comparative genomics or co - regulation at the transcriptional level have also been integrated into these methods to identify cis - regulatory sites ( 2125 ) . more recently , methods have been developed ( 2632 ) to analyze composite regulatory elements , i.e. modules consisting of multiple dna sites bound by the regulatory factors ( 33 ) . all of these methods have been valuable in expanding our limited knowledge of regulatory elements in the genome . here we discuss the progress made in the computational identification of genomic regulatory elements , recent advances , the utility of orthogonal data , existing challenges in the field and some selected examples where these methods have been successfully applied to discover novel functional elements . rather than exhaustively covering the literature , we focus on the key concepts . significant progress has also been made in the experimental characterization of regulatory sequences , a discussion of which is beyond the scope of this article , and the reader is referred elsewhere for further information ( 3443 ) . recurrent motifs in a collection of dna sites are most commonly modeled by sequence patterns ( also called regular expressions ) , or by position weight matrices ( pwms , also called profiles and position - specific scoring matrices , pssms ) . the sequence patterns are simply strings over the 4-letter alphabets [ a , c , g , t ] that form the dna . in order to capture variation in a specific position , the degenerate iupac nucleic acid codes ( 44 ) ( , nomenclature for incompletely specified bases in nucleic acid sequences ) are used ( figure 1 ) . an l - long sequence motif can also be represented by a 4 l matrix with weights giving the frequency of the four dna bases ( or the logarithm , see below ) in each of the l positions ( 18,45,46 ) ( figure 2 ) . it is worth noting here that some dna - binding site motifs are bipartite , i.e. have two halves that are sometimes separated by a spacer element in between . such bipartite motifs can often be palindromic , e.g. 5-cggnnnnnnnnnnnccg-3 , the binding site for yeast tf , gal4 ( 47 ) . the pattern and pwm representations serve as complementary approaches and have been used widely since the 1980s . the dna sequence patterns are simpler in representation , and advantageous in terms of exact enumeration of their significance in the genome using statistical methods ( explained in detail later ) . the pwms are able to capture information on the variability of a collection of dna sites in a quantitative manner , which is not possible with the dna patterns . however when detecting significant pwms in dna sequences , heuristic methods have to be used instead of exhaustive enumeration . perhaps the most important practical utility of these models is their application in scanning dna sequences for new regulatory element candidates . for this , one needs the appropriate motif models representing the regulatory elements , a statistical framework to score sites and determine their significance , and suitable thresholds to minimize false positives and false negatives . currently there are two comprehensive and curated databases containing information on tfs binding site profiles ( 12,48 ) . jaspar ( 48 ) contains a smaller set that is non - redundant ( i.e. each tf has only one profile ) , while transfac ( 12 ) contains multiple profile models for some tfs . in addition to the above generic databases , other organism - specific databases exist that host transcriptional regulation data ( 47,4951 ) ( ) . therefore if a large enough sampling of sites is not available , they may not capture all possible variations of the functional sites . this tends to produce false negative calls on new sites that do not match well with the previously characterized ones . the binding sites for structurally related tfs are often similar , and in such cases building familial binding profiles instead of profiles for each tf may be useful ( 52 ) . in both patterns as well as pwm representations , the significance of a particular motif is given by a measure of statistical surprise ( or likelihood ) for the motif given the data . in the case of patterns , significance can be calculated given the distribution of all occurrences of patterns using standard statistical procedures ( 20,53 ) . this has been used in several software packages that discover over - represented patterns from input sequences ( 5458 ) . in the methods using weight matrix models , the measure of significance is commonly given by the information content ( ic , also called relative entropy ) ( 8,9,11 ) : 1i(p)=j=1li = atfi , j logfi , jpi , where i(p ) is the ic for the pwm representing a pattern p , l is the pattern length , i is the index of a base { range a through t } at position j of the pwm , fi , j is the frequency of base i at position j of the pwm , and pi is the probability of observing that base in the data . based on biophysical models of protein dna binding , it has been shown that the contributions of the individual positions of a site to the total binding free energy of the tf may be given by log(fi , j / pi ) ( 8,11,46 ) , which are often used as the weights ( or log - odds scores ) in a pwm ( figure 2c ) . the ic is therefore the weighted average of the binding energies from each of the sites represented in the matrix , and lower ic indicates higher variability ( consequently lower specificity ) in the sites . statistical methods for computing the p - value of ic have been defined ( 59,60 ) . several algorithms use the ic measure to identify optimal motifs from input sequences ( 59,6164 ) . given a dna motif , searching sequences for candidate sites is straightforward ; but distinguishing real sites from artifactual ones is difficult . the problem of distinguishing true versus false sites arises from the fact that tf dna - binding sites are usually degenerate and many subsequences may match a given motif . the situation is illustrated with the collection of eight known sites for a saccharomyces cerevisiae tf , rox1 [ taken from the scpd database ( 47 ) ] ( figure 2 ) . even though there is a preferred base at 11 of the 12 positions , at 8 of those more than one base is tolerated ( in a larger collection of sites , even these 4 conserved positions may show variations ) . only two of the known binding sites correspond to the consensus pattern , ( t / c)ccattgttctc ( conserved positions are shown in boldface ) , so a search of the genome with this consensus pattern would miss many functional sites . on the other hand , the iupac representation of the sites , yyhmtkgttbdb , captures the variation of all the sites but is also likely to find a large number of false positives and non - functional sites in a genome - wide search . given a sequence and a pwm , one can score any subsequence in that input . suppose the length of the pwm is l and the weight of base i at position j is wi , j , the score of a subsequence ( s ) , when aligned to the pwm , is then given by the following equation : 2scores=j=1li = atwi , jsi , j , where xi , j = 1 if base i occurs at position j of subsequence s and 0 otherwise . this simple scheme is commonly used ( 59,65,66 ) , and assumes an additive contribution from each position towards the score . methods have been described to calculate the p - values of these scores based on their distribution ( 59,65,67 ) . similar to dna patterns , site predictions with the pwms can suffer from high false positive rates if motifs are degenerate . if base composition of a genome is assumed to be random ( which is not an accurate assumption , but helps us to discuss the following point in an approximate but simplified way ) , the rox1 pwm ( figure 2c ) , which has an ic of 11.2 bits , would be expected to have a site every 2500 nt [ for details see ( 9 ) ] , or 4700 total sites in the yeast genome . there are motifs for which the ic is even lower , leading to a much higher number of possible sites . for example , a collection of 13 known binding sites for uash ( 47 ) has an ic of only 8.5 bits , giving 32 000 potential sites in the yeast genome ( or 1 every 370 nt ) . thus , the problem of distinguishing real from false ( biologically non - functional ) sites is an important but challenging one . in addition to the motif degeneracy , there are other biological issues relating to the predictions of functional tf - binding sites in the genome , which are discussed below . although there are limitations , the pwm models work fairly well in representing the specificity of dna - binding sites and predicting tf - binding probability to a given site ( 11,18 ) . some tfs are by nature moderately or poorly specific in their dna binding and achieve higher specificity only in the context of other binding partners . one also has to remember that the chromatin structure and dna methylation play important roles in gene regulation ( 6870 ) . large portions of the chromosomal dna are sequestered by histones forming part of the nucleuosomal structure , and are therefore not accessible for binding by the tfs . dna methylation can inhibit interaction of the regulatory proteins with cognate dna sites and also influence the chromatin structure . while doing genome - wide searches for putative binding sites using a motif model , one typically does not know the chromosomal regions that are open for the regulatory proteins to bind , or ( with a few exceptions ) the binding partners for a given tf . a blind search of the entire genome without such information usually returns a large number of sites , many of which would probably bind to the tf ( strongly or weakly , depending on the match of the sites to the model and the specificity of the model ) if the dna sequences were open for binding , but are biologically non - functional in vivo . genomic sequences that play an active role in transcriptional regulation , such as the promoters , may often be outside of the nucleosome structure , or at least available a part of the time due to chromatin remodeling ( 71 ) . so the rate of non - functional site predictions in these sequences is likely to be lower than other parts of the genome . however , without the information on dna availability , methylation status or other binding partners , the binding site predictions with individual models are unlikely to achieve the same level of specificity that are achieved in vivo by the tfs . despite the fact that for individual pwm models determining thresholds for distinguishing real versus non - functional sites is difficult a sample - size adjusted ic may be defined as the true ic ( equation 1 ) minus the ic expected from an arbitrary alignment of an equal number of random sites . the patser program ( 59 ) ( ) uses a default cutoff score for which the loge(probability ) ( i.e. the probability of observing a score greater or equal to the cutoff ) equals the sample - size adjusted ic . another approach is based on prediction rates on sequences that contain known sites and on sequences that are likely not to contain sites ( 66,72 ) . false negative rates are computed from predictions made on the experimentally characterized tf - binding sites , and false positive rates may be computed , for example , from predictions in exons , where the number of regulatory elements is expected to be low ( 66 ) . given these prediction rates , the selection of the appropriate cut - off score depends largely on the user 's objectives . a third approach has been to use a distribution of scores for a pwm and use one or two standard deviations below the mean score as a threshold for filtering out low - scoring sites ( 73 ) , but retaining most of the true positive ones . recently , djordjevic et al . ( 10 ) have described a support vector machine ( svm ) method for representation of dna motifs based on thermodynamic principles of protein dna binding . the biophysical treatment and optimization of the svm automatically provides a threshold to distinguish the binding energies of the real sites versus those that are likely to be false . this natural and objective thresholding is one of the strengths of their approach and it provides a way to avoid the use of the more subjective or user - defined thresholds that are frequently employed . since many of the tfs bind dna in the context of other tfs , where information is available about the organization of multiple dna - binding sites or the binding partners for a given tf , it can be utilized to reduce the rate of false positive predictions . even when individual pwms tend to be fairly non - specific , searching for co - occurrence of binding sites that form regulatory modules has been shown to be an effective approach to increasing prediction specificity without losing sensitivity ( 74 ) . finally , we discuss one limitation of the additive pwm models in representing dna sites , and recent developments which address this issue . as seen from equations 1 and 2 , the simple pwm approach assumes independent contributions from each position within a dna site towards the binding free energy of the tf ( mononucleotide model ) . this has been shown not to hold true in several situations ( 37,75,76 ) where sufficient binding site data are available for detailed analysis . in such cases , higher - order models , i.e. those that consider interdependence between positions within a site , are better representations and give more accurate predictions when searching for candidate sites ( 10,7679 ) . most of the higher - order models ( 10,7581 ) use di - nucleotide interactions , which is a reasonable compromise between the mononucleotide pwms and the more complete models with interactions between multiple ( more than two ) positions . this is because ( i ) the large number of characterized binding sites that is needed to build complex models ( with increased number of parameters ) is rarely available for any particular tf ( 12 ) and ( ii ) in a few cases where sufficient experimental data are available ( 37,75 ) , interdependencies between adjacent nucleotide positions appear to be the most significant of the within - site interactions . from the dna - binding site data available for a small number of tfs , it has been estimated that 25% of sites may show significant within - site positional correlations ( 76 ) . even in cases where intra - site interactions exist , the simpler additive model has been suggested to be a good approximation ( 80 ) . as more experimental data become available with the application of high - throughput methods for characterizing tf - binding sites , it remains to be seen if significant interdependence between positions of dna - binding sites is prevalent in tf dna interactions , and whether the more complex sequence models provide a considerable enhancement in modeling accuracy over the additive pwms in many cases . since the structural contexts of both dna and protein can contribute towards specific dna binding by tfs ( 82 ) , some studies have investigated the use of structural information of protein the structure - based approaches are promising as they can predict binding sites for tfs where no previous sites have been characterized before ( 85 ) , and can provide improved predictive power over the simple sequence profile models ( 83 ) . however , they have been limited in their general use so far , since derivation of the quantitative structural parameters is dependent on the small number of solved protein dna complex structures . a more thorough discussion of the structural aspects of protein dna recognition is beyond the scope of this review and the readers are referred to the articles above ( and references therein ) for further information on this topic . with larger datasets , the issues with modeling complex dna protein interactions can be investigated more thoroughly than what has been done so far , and the building of models ( with or without structural parameters ) with higher accuracy is likely to become more feasible . discovery of motifs in sequence data was an early problem to be addressed in computational biology . the dna motif discovery algorithms that have been developed can be divided into two categories , namely pattern driven methods ( those that identify dna patterns ) and sequence driven or alignment driven methods ( those that identify profile models ) ( 16,20 ) . the concepts behind these algorithms , their advantages and limitations , and a few example methods are discussed below . in the pattern driven methods , given a set of dna sequences and a length l of pattern that we wish to find , the challenge is to identify the most significant patterns of that length . the solution to the problem can be obtained by generating all possible patterns of length l , searching for the number of occurrences of each pattern , and then reporting the ones with highest frequency as being the most significant in the given data ( 5258,8991 ) . this enumerative approach is exact and guaranteed to find optimal solutions in the restricted search space . approximate sequence patterns , or patterns that contain degeneracy at one or more positions , can also be identified from the sequence . the similarity between any two patterns may be given by the hamming distance ( the number of positions in which they differ ) or the levinstein distance ( the number of substitutions , insertions or deletions needed to transform one string into another ) ( 20 ) . when multiple patterns are close in terms of their distance , they can be merged into one approximate pattern ( 89,92 ) . although the exact enumeration is an advantage of these methods , one limitation is that searching for long patterns is computationally expensive , and an exhaustive search through the sequence space of 4 words often becomes impractical for l > 10 ( 93 ) . two general strategies have been taken to address this limitation : ( i ) the use of efficient methods [ e.g. pattern graphs ( 93 ) or projections ( 94 ) ] for pre - processing the data so that the search space is reduced and actual pattern search becomes less expensive , and ( ii ) combining the shorter overlapping patterns found from the data to yield longer or more complex patterns ( 89,92,95,96 ) . in addition to the exact enumerative methods , efficient data structures like the suffix trees ( 97 ) have also been applied to the dna pattern discovery problem ( 98100 ) . although not exact algorithms , the advantage of the suffix trees is that they allow one to search for patterns of longer lengths since the search time is not exponential in the length of the patterns , but exponential in the number of mutations to be tolerated in the sites ( 98,100 ) . in the sequence driven methods the challenge is to find the location of the sites and the representative pwm using only the sequence data , without any assumptions on the statistical distributions of patterns in the sequences . if the locations of sites are known , building a dna profile for them is trivial . however in the ab initio motif discovery problems , this information is not known ( missing information ) and has to be learned from the input data . such problems with missing information can be solved by employing machine learning algorithms . several machine - learning approaches have been applied to the problem of motif finding ( 59,6264,76,101107 ) . unlike some pattern driven methods where the most significant motifs can be identified by exact enumeration , obtaining the globally optimal results motifs of arbitrary lengths can be searched , since the search time does not depend significantly on the length of sites . the first amongst the sequence driven methods was the greedy algorithm ( 59,61,62 ) . given a set of n sequences , and a motif length l to be searched , this algorithm progressively builds matrices by including the sites which maximize the ic ( equation 1 ) . the algorithm first builds a set of significant matrices by comparing all pairs of sequences . in subsequent iterations , sites that increase the ic of the alignment another method that has been commonly used is the expectation maximization ( em ) algorithm ( 63,102,103 ) . the em algorithm simplifies the analysis of problems with missing information by iteratively substituting the locations of sites by expected locations . the algorithm starts with a guess pwm , which can be random or based on some prior knowledge about the binding sites [ see e.g. ( 52,94 ) ] . using the pwm , the probability of each subsequence being a binding site is estimated , and the pwm is updated based on those probabilities . a stochastic variant of the em algorithm that is now very widely used in sequence motif recognition is the gibbs sampling method ( 64,106 ) . gibbs sampling , which is a type of markov chain monte carlo ( mcmc ) algorithm , tends to provide a more robust optimization of the pwms as the probabilistic sampling of sites helps the avoidance of local minima . variations of the gibbs sampling technique have been implemented in many motif finding software that are used in the bioinformatics community ( 76,104,105,108,109 ) . for stochastic algorithms like the gibbs sampling , multiple searches have to be performed with the input dataset in order to confirm that the same motifs are discovered starting from different random points in the sequence space . controlling for background sequence is an important issue in dna motif discovery that has been effectively addressed in recent years . variation in local base composition can adversely affect sequence alignment and discovery of relevant motifs . because such variations can be complex , and since binding motifs are often at- or gc - rich , these adverse effects can be difficult to control using existing masking algorithms . in addition , some low complexity sequence motifs [ like ploy(a ) or poly(gc ) ] are widespread in genomes of certain organisms . as a consequence , often the strongest motifs that are discovered from any input data are these common motifs that are prevalent in the genome but do not represent the specific regulatory elements that are being sought . it is therefore important to detect those motifs that are significantly more frequent in the positive sequence set ( the input set in which we want to discover motifs ) relative to the background ( 91,92 ) . the available programs which identify such motifs ( often called discriminative motifs ) do so by modeling the background with a markov chain ( 105,109,110 ) , or by accounting for the motifs that are frequent in a given background set ( 26,92,108 ) through sampling or enumeration . recently , tompa et al . ( 111 ) reported the most comprehensive comparative study yet performed for different ab initio motif - finding algorithms . assessment was done for 13 commonly used dna motif discovery tools that do not use any auxiliary information , such as comparative sequence analysis , mrna expression levels or chromatin immunoprecipitation ( chip chip ) data . predictions were compared with known binding sites , using various statistics to assess their accuracy . one important , but not unexpected , observation from this work was that a few different tools tend to complement each other 's performance . for example , motifsampler 's ( 109 ) predictions complement well the predictions of meme ( 101,102 ) , oligo / dyad - analysis ( 56,90 ) , ann - spec ( 108 ) and ymf ( 110 ) . the authors rightly suggest that biologists would be well advised to use a few complementary tools in combination rather than relying on a single one , and to pursue the top few predicted motifs of each rather than the single most significant motif ( 111 ) . there are currently some examples where ab initio motif finding algorithms have been employed to identify novel regulatory elements that have been validated by follow - up experimental studies . a few of these are described below with the objective of illustrating the types of input data that can be used and the approaches that were taken in the studies . using the program alignace on the upstream promoter region of 248 distinct groups of genes in yeast s.cerevisiae , hughes et al . ( 104 ) , generated a list of 3311 putative regulatory motifs . by applying stringent thresholds and selecting motifs which had highest specificity for certain groups of genes , this large set was reduced to a small set of 54 motifs that were then grouped to 25 motif clusters based on the similarity of multiple motifs . of the 25 motif clusters , one of the previously unidentified motifs , representing the binding sites for the tf rpn4 , was verified using mrna expression analysis of the rpn4 protein knockout and overexpressing strains . ( 112 ) have described the identification of one novel regulatory element in the nematode c.elegans heat - shock response starting from a set of microarray experiments in which genes were robustly upregulated on heat - shock treatments at both early and late time points . using a set of 28 heat - shock upregulated genes they used the ab initio motif discovery algorithms , ann - spec ( 108 ) and consensus ( 59,108 ) , to identify two strong motifs that are over - represented in the promoters of these genes . one of those motifs was the previously characterized heat - shock element that is broadly conserved in eukaryotes and known to bind to the tf called heat - shock factor . the second motif , which was novel , was shown to be biologically functional in vivo through transgenic and mutational studies . in fact , the two elements were shown to function in a cooperative manner ; the contribution to heat - shock mediated expression by either one was weak , but when placed together in close proximity they strongly regulated expression . studies similar to the one described above was done to identify several new muscle regulatory elements in c.elegans , which were shown to contribute to muscle expression in a cooperative fashion ( 113 ) . the identified elements were also highly predictive of additional muscle - specific genes in that organism ( 114 ) . these studies show that ab initio prediction methods can be valuable in elucidating unknown regulatory elements not only in unicellular organisms , but also the more complex metazoan genomes . although much progress has been made in the methods for ab initio detection of dna regulatory elements , the problem still remains a difficult one , especially where the input sequences are long and motifs are weak . therefore , the incorporation of auxiliary information into such methods can be of significant benefit . since the availability of many complete genomes , the application of comparative genomics in the identification of dna regulatory elements has become an important area of study and it is covered in detail in the next section . here , as an example , we discuss a different approach which leverages mrna expression data in dna motif discovery . ( 24 ) described recently a method to discover regulatory elements that uses correlation of dna patterns with the expression levels of genes in a single profiling experiment . they use a simple linear regression model to fit the logarithm of the expression of each gene to the sum of contributions from a set of dna patterns in the upstream promoter region . all the genes are simultaneously fit , and statistically significant patterns that best fit the expression data are selected . using yeast expression datasets , they reconfirm most of the motifs originally found by clustering of expression data and then running motif finding algorithms on clustered gene sets ( 115,116 ) . since there are frequently cooperative ( non - additive ) interactions between tfs regulating a gene , the modeling is simplistic . however , the advantage of this method is the fact that limited expression data are required for analysis and discovery of the dna motifs . in addition to the above methods which integrate motif discovery with expression data , several tools and studies have been described that identify dna - binding site motifs for tfs from a set of sequences defined by mrna profiling ( 112,118,119 ) or chip chip data ( 120,121 ) . in multicellular organisms the sequence space in which regulatory elements can be present in the genome is vast . in addition to the core promoter region , auxiliary transcription regulatory elements like enhancers , silencers and insulators can be present in the distant 5 upstream region , 3 downstream region and the introns ( 122 ) . in drosophila such dna elements can be spread over a region of 10 kb around the genes whereas the average transcribed dna is 23 kb , and in mammals these elements can be scattered over distances of hundreds of kb ( 123 ) . approaches that help to limit this search space are hence of significant value to the analysis of regulatory elements . also , it is intriguing to study those regulatory mechanisms and elements that are likely to be of fundamental importance in maintaining certain cellular functions and therefore conserved in evolution . consequently , phylogenetic footprinting ( 124,125 ) , which is based on the premise that functional elements are likely to be under selection pressure and thereby evolve at a rate that is slower than the surrounding non - functional sequence , has been widely applied in recent years to the identification of regulatory sequences ( 21,123,126129 ) . purely for the purpose of illustration , a simple example of the application of phylogenetic footprinting for the identification of putatively conserved tf - binding sites is given in figure 3 . enrichment of regulatory elements has been clearly demonstrated in non - coding dna in the human mouse conserved regions ( 21,127,130 ) . for example , wasserman et al . ( 21 ) found that 98% of the known muscle regulatory elements are located within 19% of the sequence that is most conserved between human and mouse . non - coding sequences that can be aligned across two or more organisms have been shown to have functional regulatory roles ( 123,129 ) . analysis of motif frequencies and correspondence in conserved regions across multiple species has been used to identify known and novel regulatory elements in organisms ranging from yeast ( 25,131133 ) to the mammals ( 134,135 ) . rodent sequence alignments , and known sets of regulatory sequences and ancient repeats in those genomes , methods have been developed to distinguish conserved regulatory regions from neutral sequences ( 136,137 ) . in addition to its application in eukaryotic genomes , comparative genomic approaches have helped in elucidation of regulatory elements in prokaryotes and archaea ( 22,138141 ) . therefore , significant developments have been made over the past several years in utilizing the sequences from multiple species to identify functional regulatory elements in all phyla . there are now numerous methods that are available for alignment of genomic sequences from two or more species ( 142151 ) . some of these have been integrated with motif finding and visualization programs to provide practical tools for analysis of regulatory elements within cross - species conserved regions ( 152154 ) . multiple sequence alignment methods can be advantageous in comparative genomics since they utilize more information relative to pairwise sequence alignments ; they can also be more powered to identify regulatory motifs ( 135,140,155 ) . prakash and tompa ( 135 ) recently compared the performance of six global and local multiple alignment tools with respect to their potential of identifying highly conserved short ( 10mers ) dna patterns from the immediate upstream promoter sequences of orthologous genes from multiple vertebrate species ( human , chimp , mouse , rat , chicken ) . two of the methods tested , namely mlagan ( 147 ) and tba ( 156 ) , appeared to perform better than several others for this purpose ( 135 ) . more advanced methods have now been developed that directly take into consideration the phylogenetic distances between the organisms that are being aligned in order to identify conserved dna motifs ( 107,126,157159 ) . in addition to phylogenetic footprinting , comparative genomics is now being utilized in other sophisticated and intriguing ways to identify regulatory elements of potentially conserved function . some methods have not only utilized sequence conservation but also gene network conservation ( based on the hypothesis that multiple sets orthogonal genes may be regulated by common tfs ) to identify sets of regulatory motifs ( 133,160 ) . although developed in yeast , one of these studies show that such methods can be sufficiently powered to detect regulatory elements in much longer sequences in the multicellular organisms , including mammalian genomes ( 133 ) . there are now many examples where comparative genome sequence analysis has been used successfully to elucidate novel regulatory elements which would have been difficult to identify without that information ; three are illustrated below . mccue et al . ( 140 ) used an extended gibbs sampling algorithm to identify probable transcription regulatory sites upstream of escherichia coli genes by cross - species comparison . a set of 184 genes with orthologs from two or more other gamma proteobacterial organisms were analyzed . of their predictions 81% corresponded with the documented sites known to regulate these genes , whereas 67% corresponded when data from only one other species were available , suggesting that addition of one more species aids in sensitivity of the binding site detection . one of the novel predictions , a dna site bound by the tf yijc , was verified by experiments . in an elegant study , loots et al . ( 123 ) demonstrated the utility of phylogenetic footprinting by identifying a regulatory region conserved between human and mouse for il-5 that is located 120 kb away from the gene itself . in another study comparing genomic sequences from multiple primate species , boffelli et al . ( 129 ) , identified regulatory elements for apoa , a recently evolved primate gene . a region of high conservation adjacent to the transcription start site was shown to interact with one or more dna - binding proteins using electrophoretic mobility shift assays with nuclear extracts from liver cells . identification of such primate - specific functional elements would be unattainable through the comparison of species that are evolutionarily more distant . as evident from the discussions above , approaches that utilize conservation across species are very useful in elucidating functional dna regulatory elements . significant advances have been made in this area over the past 6 years . despite its utility and widespread use , there are limitations in phylogenetic footprinting approaches with respect to their use in the identification of regulatory elements . the short regulatory elements may be missed if the genomic sequences that are being aligned come from distant organisms ( 112,161,162 ) . if the organisms are too close , however , the alignments are extensive and therefore unable to distinguish the conserved functional elements from the non - functional ones ( 161,162 ) . it is unclear at this time whether cross - species alignments would be equally useful in finding regulatory elements in all phylogenetic clades ( 163,164 ) . in a recent comparison of two drosophila species , the known regulatory elements were found to be only modestly enriched in the conserved regions ( 164 ) , although the amount of conservation in the non - coding regions of these drosophila species was roughly the same as in human mouse . in another study , individual binding sites appear to be conserved across two nematode species , but they were not located in sequences that were aligned by the software for phylogenetic footprinting ( 112 ) . therefore , whether cross - species sequence alignment is likely to be an effective approach in all organisms in elucidating most of their functional regulatory elements , as well as the issues such as optimal phylogenetic distances , and the number of organisms needed to detect these elements , are still matters of debate and investigation ( 163 ) . in eukaryotes , tfs rarely act alone in regulating the expression of a given gene . in most cases multiple factors bind dna , often in close proximity with each other , forming regulatory modules ( 13,33,165,166 ) . by utilizing combinatorial interactions between multiple factors these cis - regulatory modules ( crms ) confer specific spatial and temporal patterns of transcription . therefore identification of composite modules and higher order regulatory structures is currently an active area of research in computational analysis of regulatory sequences . the problem is difficult however since the combinatorial interactions between the regulating factors can be very complex ( e.g. see the array of regulatory interactions in the immediate upstream region of endo16 in sea urchin ( 13 ) ] . there have been some developments in the past 6 years in addressing this problem in dna sequence analysis . the current approaches can be classified as follows : methods that identify modules given a set of dna motifs representing sites for tfs that are known to act together in regulating transcription ( 27,2931,74,167171).methods for ab initio identification of multiple dna motifs representing the sites for a crm ( 26,93,98,105,172174 ) . methods that identify modules given a set of dna motifs representing sites for tfs that are known to act together in regulating transcription ( 27,2931,74,167171 ) . methods for ab initio identification of multiple dna motifs representing the sites for a crm ( 26,93,98,105,172174 ) . softwares that identify crms given a set of known dna motifs fall into two categories , those that use hidden markov models to represent the crms ( 27,169 ) , and those that rely on observation of frequent joint occurrence of sites within a certain window , modeling the frequency of multiple sites with an appropriate statistical ( e.g. poisson ) distribution ( 30,31,74,167,169,170,175,176 ) . because the dna - binding site models for the majority of tfs are currently unknown and the information on tfs that bind dna together in crms is very limited ( 166 ) , several ab initio methods have been developed to identify composite dna elements given just a set of input sequences ( 26,28,98,105,172174 ) . a few of these methods use efficient suffix - tree structures to identify multiple or dyad patterns ( 28,98 ) , and others employ gibbs sampling or monte carlo strategy to identify multiple pwms that may represent binding sites in regulatory modules ( 26,105,172174 ) . by combining information from multiple sites , these methods have the potential to identify motifs that are too weak ( i.e. information poor ) to be identified individually ( 26 ) . two examples of the application of computational methods for identification of novel crms are discussed . the first describes the utility of an ab initio motif finder in identifying a novel composite motif regulating cell - cycle genes in yeast , and the second describes how the information on tfs that are known to bind dna jointly can be leveraged to make genome - wide predictions of regulatory modules involving sites for those factors . with the application of an ab initio composite motif discovery program , ( 177 ) identified a crm involving the binding sites for yox1 and mcm1 from the promoters of a set of 28 genes upregulated in the yeast yox1 knockout strain . yox1 , which represses the expression of genes in the m / g1 interval of yeast cell - cycle , binds dna in conjunction with the generic mads family tf , mcm1 . the binding sites for both tfs were jointly identified using the software and subsequently validated through mutational and gel mobility shift studies . the second example is of the transcriptional program in drosophila embryo . by using known dna binding specificity data for five tfs , berman et al . ( 170 ) identified genomic regions containing unusually high concentrations of predicted binding sites for these factors . a significant fraction of these binding site clusters overlap known crms . in addition , many of the remaining clusters were adjacent to genes that were expressed in a pattern characteristic of those regulated by these factors . the authors tested one of the newly identified clusters , mapping upstream of the gap gene giant ( gt ) , and showed that it acted as an enhancer that recapitulates the posterior expression pattern of gt . although the above approaches show potential , the developments in computational identification of crms are recent and there is significant room for further investigations and improvement , since the arrangements of sites in the modules are complex . the number of datasets containing collections of known composite regulatory elements that exist today is very limited [ a few examples are ( 74,164,167 ) ] , which poses an obstacle in the training and testing of general computational methods in this realm . understanding the mechanisms of transcriptional regulation has been an object of extended and difficult quest in biological disciplines . clearly , significant advances have been made over the past two and half decades not only in the representation and modeling of the dna regulatory elements , but also methods for their identification in genomic dna . however , our knowledge of the transcriptional regulatory elements in the genome and their contribution to gene expression in different spatial and temporal contexts is still limited . given the complex pattern of regulatory interactions , the challenges involved in the complete elucidation of these elements in the genome are substantial . one of the major challenges is to associate the computationally identified regulatory elements with their cognate tfs . genome - wide analyses often identify a host of putative regulatory elements ( 25,132135 ) . in order to get an understanding of the regulatory processes it is essential to associate the regulatory proteins with these elements . there have been some investigations into this problem ( 119,178,179 ) in prokaryotes and yeast , but further developments are required . an important utility of characterizing the cis - regulatory elements is their use in the computational reconstruction of gene regulatory networks . several studies have applied the information on cis - regulatory elements , either in isolation or in combination with other orthogonal sources of information ( e.g. microarray expression data ) , to construct regulatory networks ( 180183 ) . these studies demonstrate how the information on transcriptional regulatory elements can be integrated to create gene networks . however , there are significant opportunities for investigations in this area . the ab initio motif discovery tools and comparative genomics approaches have made it possible to detect regulatory elements in many genomes . in addition , information that can guide the search for regulatory elements to the most relevant regions of the genome is becoming available , e.g. the accurate location of transcriptional start sites ( 184 ) , dna - ase hypersensitive sequences within nuclear chromatin that represent regulatory regions ( including promoters , enhancers , silencers , locus - control regions ) ( 43 ) , and tf binding locations from the chip chip experiments ( 38,40,41 ) . individually , there are methodological or practical limitations in the computational and experimental approaches in elucidating the location and function of the full set of regulatory elements . for example , the computational dna motif finding algorithms have limitations in terms of the sensitivity and specificity of signals they can detect , whereas the high - throughput tf - binding site location technologies ( e.g. chip chip ) are currently limited in the extent of intergenic sequences they can explore ( 185,186 ) . therefore it appears that the most efficient path to elucidating the novel regulatory elements and mechanisms will lie in the judicious integration of the various methods and data ( 182 ) . despite inherent challenges in the field , rapid progress has been made over the past few years in the computational identification of regulatory elements . successes of the computational methods have been demonstrated through experimental validations , and efficient methods for comparative genomics and analysis of crms are being fruitfully utilized to elucidate complex regulatory elements in organisms ranging from the unicellular bacteria to the mammals . the iupac ( international union of pure and applied chemistry ) code for representing degenerate nucleotide sequence patterns . ( a ) the collection of eight known rox1-binding sites taken from scpd ( 47 ) . the cells represent the number of times a base i is observed at position j in the alignment of sites . the frequencies , fi , j , of base i at position j of the binding sites can be obtained by dividing the values in the cells of the alignment matrix by the total number of sites , e.g. fc,1 = ft,1 = 4/8 = 0.5 . each weight is given by log2(fi , j / pi ) ( see text ) , where pi is the probability of observing the base i in the data ; here we have taken pa = pt = 0.32 , and pc = pg = 0.18 ( corresponding to the s.cerevisiae genome ) . this matrix was used to score the sites in a. as an example , the score of the site in red ( sequence ccaattgttttg , score 13.87 ) is given by the summation of the scores that are circled in red . note that the scores of the two consensus sequences , cccattgttctc and tccattgttctc are different because pc pt . ( d ) sequence logo representation ( 187 ) of the alignments , visually showing the ic and conservation at each of the alignment positions . the ic of this matrix is 11.3 bits or 7.83 nats ( equation 1 ) . the genomic regions , along with 5 kb upstream and 2 kb downstream , of the ckm gene were extracted from the human and mouse genomes and aligned using the blastz software ( 151 ) . the blastz alignments were then fed into the rvista program ( 153 ) through the website . binding sites for several tfs that are known to regulate gene expression in the muscle tissue were then predicted on the human sequence using the pwm models available from the transfac database ( 12 ) . the predicted sites can be dynamically viewed and clustered through the above website . for the purpose of this current figure , we required that at least two binding sites belonging to different tfs be present within a window of 100 nt . a cluster of sites was observed in the immediate 5 upstream region of this gene ( boxed ) . percent conservation between the two sequences is shown ; regions with 75% conservation are colored .

identification and annotation of all the functional elements in the genome , including genes and the regulatory sequences , is a fundamental challenge in genomics and computational biology . since regulatory elements are frequently short and variable , their identification and discovery using computational algorithms is difficult . however , significant advances have been made in the computational methods for modeling and detection of dna regulatory elements . the availability of complete genome sequence from multiple organisms , as well as mrna profiling and high - throughput experimental methods for mapping protein - binding sites in dna , have contributed to the development of methods that utilize these auxiliary data to inform the detection of transcriptional regulatory elements . progress is also being made in the identification of cis - regulatory modules and higher order structures of the regulatory sequences , which is essential to the understanding of transcription regulation in the metazoan genomes . this article reviews the computational approaches for modeling and identification of genomic regulatory elements , with an emphasis on the recent developments , and current challenges .

INTRODUCTION REPRESENTATION AND SEARCH OF DNA REGULATORY ELEMENTS DISCOVERY OF DNA MOTIFS COMPARATIVE GENOMICS IN THE SEARCH FOR REGULATORY ELEMENTS COMPOSITE MOTIFS AND CONCLUDING REMARKS Figures and Tables

PMC3707231

about 80% of strokes are ischaemic , resulting from an obstruction of blood flow , while about 15% are due to a primary intracerebral hemorrhage . stroke is one of the leading causes of chronic adult disability and death in western industrialized countries . neurological deficits reflect the location of the tissue damage and , in particular , the extent of the neuronal loss . neurons deprived of their normal metabolic substrates cease to function in seconds and show signs of structural damage after only 2 minutes . as energy - dependent processes fail , neurons are unable to maintain their normal transmembrane ionic gradients , resulting in ion and water imbalance that triggers apoptotic and necrotic cell death cascades and , ultimately , leads to focal neurological signs and symptoms . according to the who 's international classification of function , disability , and health ( icf , who 2001 ) , the impairment of brain functions may originate different activity limitations ( disability ) and participation restriction ( handicap ) . motor impairments , including hemiparesis , incoordination , and spasticity , are the most common deficits after stroke . however , functional recovery frequently occurs following stroke , although its extent is highly variable . some patients with initial severe hemiparesis may eventually achieve full recovery , while others have little or no improvement and remain permanently disabled . there are many reasons for the variable degrees of recovery , including the age of the patient , the location and extent of the lesion , and individual variations in anatomical and functional connections . the neural bases for poststroke recovery rely on the concept of plasticity , namely , the ability of central nervous system ( cns ) cells to modify their structure and function in response to a variety of external stimuli ( experience ) . the plastic / reparative properties of the brain are determined by the balance between cell - intrinsic mechanisms and extrinsic regulatory molecules , which is regulated by activity - dependent processes and different kinds of interaction with the external world [ 4 , 5 ] . molecules in the adult cns milieu , such as myelin - associated proteins ( e.g. , nogo , mag , and omgp ) , factors secreted by astrocytes near the stroke site ( e.g. , chondroitin sulfate proteoglycans ) , and repulsive axonal guidance cues ( e.g. , semaphorins , netrins , and members of the ephrin family ) , constrain axonal sprouting and hamper the formation of new connections . in preclinical stroke models it has been shown that pharmacological blockade of nogo , nogo receptor antagonism , or digestion of chondroitin sulfate proteoglycans by chondroitinase induce axonal sprouting and promote functional recovery [ 79 ] . blocking the semaphorin pathway other growth inhibitors , such as epha4 and ephrin - a5 , have also recently been identified , which limit functional recovery and are promising targets for repair after stroke [ 11 , 12 ] . interestingly , inhibition of rock , a downstream target of several growth inhibitors , greatly improves outcome after ischemic stroke . several studies have also uncovered pharmacological targets that promote a neuronal growth state in the adult cns . for example , inosine triggers a serine / threonine kinase ( mst3b ) , enhancing axonal sprouting [ 13 , 14 ] . the therapeutic potential of replacement strategies in laboratory models of stroke is also under investigation . transplantation of neural progenitor cells , bone marrow - derived mesenchymal stem cells or human - induced pluripotent stem cells into the ischemically lesioned brain have been proved to be a safe and efficient approach to promote significant functional recovery in experimental animals [ 1517 ] . nonetheless , the mechanisms underlying the beneficial effects of cell transplantation in the ischemic cns remain uncertain , and , most importantly , to date there is no clear evidence that donor cells may directly contribute to the structural repair of neuronal circuits . in addition to pharmacological or replacement therapies , clinical and preclinical studies are currently focusing on noninvasive strategies for post - stroke rehabilitation . motor rehabilitation after hemiparetic stroke typically involves combinatory approaches , including neurofacilitation techniques , task - specific training , and task - oriented training [ 19 , 20 ] . furthermore , stroke units , in which patients have access to daily skill training therapies in highly stimulating environments , such as during physical , occupational , or language therapy , result in decreased deficits , increased performance on self - care tasks , lower 1-year mortality , and lower probability to be in a nursing home at followup . finally , in recent years brain stimulation , mirror therapy , action observation , or mental practice with motor imagery is emerging as interesting options as add - on interventions to standard physical therapies . here , we will provide an overview of recent noninvasive strategies employed to enhance functional recovery in patients after stroke and discuss the current knowledge of rehabilitative strategies and the associated neural plastic events in preclinical models of stroke . stroke rehabilitation aims to guarantee that stroke survivors reach the maximum physical , functional , and psychosocial recovery possible within the limits of their impairment . in order to help stroke patients to fully participate in life , the final goal of rehabilitation should be to maximize performance of activities of daily living and independence . through learning - dependent processes recovery of stroke patients is extremely heterogeneous and determined by a combination of processes including functional restoring of damaged nervous tissue , relearning of lost skills through reorganization of spared pathways ( plasticity ) , adaptation , and compensation for deficits . compensation reflects the use of alternate behavioral strategies in order to solve a specific task . most recovery of specific neurological focal deficits occurs during the first 3 to 6 months after stroke , but it is largely accepted that improvements can continue for years after stroke . general principles of stroke rehabilitation include the start of intensive rehabilitation programs carried out in a stroke unit within the first few days after stroke [ 24 , 25 ] . evidence demonstrates that comprehensive intensive rehabilitation , as well as the presence of a structured multidisciplinary team , may be more effective than less intense programs . in agreement with the learning nature of the rehabilitative process , involvement , engagement , and motivation of patients , families and caregivers most recent neurorehabilitative approaches are based on a task - oriented model of motor learning , whose main feature is an intensive training with specific tasks in an environmental context ( task - specific and context - specific trainings ; [ 2730 ] ) . in this context , a number of new rehabilitative techniques potentially capable of stimulating cerebral plasticity have been proposed and tested in the last years . among these techniques , large interest is devoted to treatment approaches aimed to improve motor functions , including constraint - induced movement therapy , mental practice , mirror therapy , virtual reality , robotics , and brain stimulation techniques . constraint - induced movement therapy ( cimt ) involves the restriction of usage of the unaffected limb , forcing the use of the paretic one , and aiming to contrast the maladaptive learned nonuse of the paretic limb ( the subject learns to ignore the damaged limb because of its lack of functionality and learns to use exclusively the healthy limb ) . a number of studies including randomized controlled trials and a cochrane review have shown that cimt is effective in improving motor performance in human patients after stroke [ 31 , 32 ] with a large effect size and robust effects especially on arm function . in particular , the ecxite trial demonstrated that daily intensive cimt training for upper limb paresis was superior to the control treatment 3 to 9 months after stroke , and that a modest improvement in motor function persisted in the cimt group after 2 years . important limitations to the routine use of cimt training derive from the fact that it is labor intensive and suitable only for patients with some conservation of motor functions ( in particular wrist and finger ) , thus its use is recommended only for selected patients . mental practice with motor imagery is considered a promising additional treatment to improve motor functions of severely affected upper limb , although its clinical effectiveness is not yet clearly proven . this approach grounds on the statement that imaging a movement requires activation of brain circuits involved in the preparation and execution of the same movement and consists in a repetitive cognitive training during which the patient imagines performing a task or body movement without actually physically performing it . it has been demonstrated that mental practice may modulate cerebral perfusion and neural activity in brain regions similar to those activated during actual movements [ 34 , 35 ] . following few systematic reviews [ 36 , 37 ] suggesting that mental practice may be beneficial for post - stroke disabilities in addition to conventional treatments , a recent cochrane review concluded that there is only limited evidence that mental practice may increase the effectiveness of usual physiotherapy and occupational therapy . another approach based on multisensory stimulation is represented by the mirror therapy . in this technique a mirror is placed at 90 in the patient midsagittal plane , so that the paretic limb is hidden behind the mirror and the patient watches the image on the mirror of the unaffected arm as if it was the affected arm . in a certain sense , the patient receives the impression that the affected limb is functioning . it has been demonstrated that viewing the image of one 's moving hand reflected by the mirror increases the excitability of neurons in the ipsilateral primary motor cortex more than directly viewing the inactive hand . mirror therapy effects ( as well as those related to mental practice ) may be related to the activity of the so - called mirror neurons , which discharge both following performance of motor acts and simply observing the same action done by another individual [ 40 , 41 ] . in fact , by means of fmri it has been demonstrated that prolonged and repetitive observation of an action may enhance the activity in the ventral premotor cortex , the supplementary motor area , and the superior temporal gyrus . a recent systematic review including 14 studies and a total of 567 patients treated with mirror therapy concluded that , when compared to other rehabilitative approaches , this treatment has a significant effect on motor function even though this result is strongly influenced by the type of intervention used as control . thus , it remains unclear if mirror therapy should replace other treatments for motor rehabilitation after stroke , while its role as additional intervention is confirmed . moreover , mirror therapy improves activities of daily living , but this statement is limited by the small number of studies ( four ) examining this effect . the virtual reality idea is based on the possibility that a computer can generate a three - dimensional graphical environment from numerical data , so that , by using visual , aural , or haptic devices , the operator can experience the environment as if it were a part of the world . a key feature of all virtual reality applications is interaction : virtual environments are created to allow the user to interact also with virtual objects within the environment . in some systems , the interaction may be achieved via a mouse or a joystick button , while in others , a representation of the user 's hand may be generated within the environment with movement of the virtual hand reflecting the user 's hand , thus allowing a more natural interaction with objects . therefore , virtual reality represents a unique instrument to achieve several requirements for effective rehabilitation , such as repetitive practice , feedback about performance , and motivation to endure practice [ 45 , 46 ] . specifically , by using virtual reality it is possible to drive and control exercises for patient rehabilitation within a functional , purposeful , and motivating context . different virtual reality approaches have been used , in particular , for upper limb motor rehabilitation . a cochrane review published two years ago , analyzing 19 randomised and quasi - randomised trials of virtual reality that involved 565 participants , concluded that there is a limited evidence that virtual reality and interactive video games may be beneficial in improving arm function and activity of daily living function when compared with the conventional treatments . another , contemporary meta - analysis , including 12 studies ( 5 randomized controlled trials and 7 observational studies ) for a total of 195 patients , showed that in the large majority ( 11 over 12 ) of these studies virtual reality added a significant benefit on arm motor recovery after stroke . however , to gain convincing evidence of virtual reality effectiveness in poststroke rehabilitation , further research is needed based on good randomized controlled trials . in the last years a growing interest has been addressed to robot - assisted rehabilitative treatments after stroke . in theory several studies have demonstrated a significant result in motor recovery of the upper limb of patients who trained with robotic devices but no significant effect on functional ability . however , the conclusion of a randomized controlled trial ( ul - robot ) and a cochrane meta - analysis limited the significance of these results . in the ul - robot trial two groups of patients receiving 36 therapy sessions over 12 weeks of robot - assisted therapy or intensive conventional physical therapy , respectively , were compared with patients receiving usual ( not intensive ) care . the study failed to demonstrate a superiority of the intensive robot therapy when compared to intensive conventional physical therapy , but both techniques were superior to usual care , suggesting that intensity of training may be a crucial factor for motor recovery . the cochrane review , including 19 trials and 666 patients , concluded that electromechanical and robot - assisted arm training after stroke may improve generic activities of daily living as well as paretic arm function , but not arm muscle strength . a phase iii randomized and controlled trial ( leaps- ) , designed to test the efficacy of a popular technique that utilizes partial body - weight support with treadmill training , was concluded in 2011 . the leaps trial included 408 patients randomly assigned to three groups : two groups were subjected to a locomotor training with treadmill and body - weight support ( one group initiating treatment 2 months after stroke and the second 6 months after stroke ) , the third group received a home exercise program . the results were someway surprising : no significant difference was found between the three groups concerning the improvement in walk speed , motor recovery , balance , functional status and quality of life . thus , locomotor training with body - weight support and treadmill can not be considered superior to a structured , progressive , and intensive at home treatment . also , in this trial all intensive interventions were more effective when compared to non - intensive and structured care . a promising robotic interface has been recently developed by courtine 's group to evaluate , enable , and train pattern generation and balance during walking in rats . the devise continuously and independently assists or perturbs propulsion and balance along four degrees of freedom , while rats are progressing overground within a large workspace . in a model of stroke the use of noninvasive techniques of brain stimulation to stimulate adaptive plasticity is very appealing , and the results obtained are exciting . two main techniques are available to obtain both cortical enhancement and inhibition : repetitive transcranial magnetic stimulation ( rtms ) and transcranial direct current stimulation ( tdcs ) . rtms , using a coil placed on the scalp , generates a focal magnetic field , which induces ( transiently , focally , and reversibly ) an electric current in the underlying cortex . low frequency stimulation ( in the range of 1 hz ) reduces cortical excitability , while higher stimulation frequencies increase the cortical excitability . in tdcs , weak direct currents are delivered to the cortex through two electrodes that polarize the underlying tissue . electrode position is crucial to modulate the distribution and direction of the current flow : anodal stimulation has an excitatory effect by cortical neuron depolarization , while cathodal tdcs hyperpolarizes neurons by decreasing cortical excitability . in general , two different approaches can be described using noninvasive brain stimulation : one addressed to increase excitability of ipsilateral damaged hemisphere ( e.g. , by stimulating primary motor cortex ) , and the other one directed to reduce the activity of intact surrounding or contralateral area that can produce intra- or interhemispheric inhibition . the purpose of these applications is to restore the unbalance between intact and lesioned hemisphere according to the interhemispheric competition model . moreover , bestmann and coworkers suggested an unexpected role of the contralesional dorsal premotor cortex , with an elegant demonstration by means of rtms which showed the supporting activity of contralesional dorsal premotor cortex to ipsilesional sensorimotor regions in particular for greater clinical and neurophysiological impaired patients . the application of these approaches have produced very promising results , in both acute and chronic stroke patients , recently reviewed by corti et al . . that review suggests that rtms applied to the affected hemisphere is safe and could be considered effective for modulating brain function and contributing to motor recovery after stroke . however , the authors stressed the need of double - blinded , sham - controlled phase ii and phase iii clinical trials involving larger sample sizes to validate this treatment . in a meta - analysis of 18 randomized controlled trials dedicated to the effects of rtms on upper limb motor impairment , hsu et al . found a significant effect size ( 0.5595% ci , 0.370.72 ) for motor outcome function , with more clear effects for subcortical stroke and low - frequency rtms applied to the unaffected hemisphere . questioned about the real duration and anticipated size of the treatment effects in chronic stroke patients . in such patients they showed with a small semirandomized clinical trial that rtms application does not augment the gains from a late rehabilitation program . the need for randomized controlled trial is even more evident to validate efficacy of tdcs , considering that its use in stroke patients is quite new [ 59 , 60 ] . recently , khedr et al . provide an interesting evidence that both anodal and cathodal tdcs are superior to sham stimulation in enhancing the effect of rehabilitation training to improve motor recovery after subacute stroke in a pilot randomized controlled trial . however , it must be stressed that our knowledge about mechanisms underlying brain stimulation are largely incomplete . rehabilitative conditions in stroke units , such as physical therapy and various kinds of stimulating activities , can be partially mimicked in animal studies by housing the animals in an enriched environment ( ee ) . ee is a widely employed paradigm to study the influence of external stimuli on brain plasticity in animal models both in physiological conditions and after damage . environmental enrichment refers to housing conditions that facilitate enhanced sensory , social , cognitive stimulation , and motor activity . home cages used for enrichment are larger than standard cages to allow room for several objects , which generally vary in composition , shape , size , texture , smell , and colour . enrichment may also involve access to running wheels for enhanced voluntary exercise ( figure 1 ) . key aspects appear to be the provision of environmental complexity , with objects that offer a range of opportunities for visual , somatosensory and olfactory stimulation , and environmental novelty , obtained by changing the objects and their position in the cage , which might provide additional cognitive stimulation . increased complexity and novelty social interactions are also favored by housing rather large groups of animals of both sexes together ( see for review ) . several studies show that in experimental models of stroke , ee strongly promotes recovery of motor functions , such as skilled limb function [ 6468 ] and gait . compensatory mechanisms have been shown to substantially contribute to functional improvement after stroke [ 70 , 71 ] . compensation reflects the use of alternate behavioral strategies in order to solve a specific task [ 70 , 72 ] . to what extent ee enhances functional outcome after stroke due to compensation for lost functions rather than their restoration is not entirely clear . witte and coworkers addressed this question by focusing on the time course of functional recovery versus motor compensation in skilled forelimb movements after cerebral ischemia in rats . the skilled reaching task allows the distinction between recovery and compensation by quantitative ( reaching success ) and qualitative ( movement pattern ) analysis . it has been shown that ee facilitates effective compensation in skilled reaching , while it does not promote restitution of function . namely , rotating movements of the forelimb during reaching are permanently impaired and require functional compensation through intensified use of the upper body . interestingly , in one of the first studies on the effects of ee on stroke animals , ohlsson and johansson addressed whether preoperative and postoperative environments can differently influence functional outcome after focal brain ischemia . rats were subjected to ligation of the right middle cerebral artery ( mca ) then transferred from a non enriched to an enriched environment or reared in an ee already before the operation . rats kept in an ee before and after the mca ligation improved sooner and to a slightly higher degree than those placed in the ee only after the ischemia . the beneficial effects of ee in the animals enriched before mca ligation suggest that complex experiences during healthy conditions may provide a brain reserve against late brain damage , according to previous findings [ 7378 ] . among the ee - induced changes in physiological conditions , the development of new synapses [ 79 , 80 ] and dendritic spines [ 8183 ] has been demonstrated . in addition , there is evidence that exposure to ee reduces the expression of growth - inhibitory molecules in the intact cns tissue [ 84 , 85 ] . therefore , it is conceivable that reduced inhibitory mechanisms together with a reserve of synapses in enriched animals may provide neuroprotection and facilitate functional compensation after stroke . a bulk of evidence highlights the functional benefits induced by motor training after focal ischemic injury in humans . a useful method of training for chronic and acute individuals after a stroke is treadmill training ( figure 1 ) . when applied to ischemic rats starting 24 h after ischemia , it leads to a significant reduction of infarct volume and improves neurological function . interestingly , functional recovery after stroke ( such as forelimb foot placing , parallel bar crossing , and rope or ladder climbing ) can be further improved by complex motor training ( which can be obtained by using rotarod ) rather than simple repetitive exercise , such as treadmill training . this suggests that repeated complex movements involving motor balance and coordination are more effective for functional recovery after stroke than either simple activity or inactivity . in line with this view , specific behavioral experience , such as skilled - reaching training ( figure 1 ) , after focal experimental infarct , provides substantial behavioral recovery of skilled hand function in monkeys . in experimental animals , skilled reaching training consists of daily practice of the impaired forelimb to retrieve food pellets . this kind of rehabilitation provides positive reinforcement ( i.e. , food reward ) associated with use of the impaired limb , thereby encouraging animals to practice spared motor function or promoting development of compensatory motor strategies , resulting in lessened functional deficiency . interestingly , by combining both enriched living conditions and daily skilled - reaching training , biernaskie and corbett obtained dramatic long - term improvement both in skilled use of the impaired forelimb and digits and in limb placement in stroke rats . these findings reinforced the idea that skilled learning therapy coupled with enriched surroundings may facilitate neurologic recovery in humans . it should be noted , however , that the effect of forced exercise on functional recovery after stroke is controversial . forced exercise , such as treadmill running or constraint - induced movement therapy , has been shown to enhance the functional recovery of motor skills after experimental ischemic stroke [ 90 , 91 ] . other studies , however , demonstrate that treadmill running produced negative physiological adaptations induced by stress , and a constraint - induced movement study did not show improved functional outcome after brain ischemia . patients with high levels of social support or large social networks exhibit more rapid and extensive functional recovery after stroke than socially isolated individuals [ 94 , 95 ] . the importance of social influences on stroke outcome have been also highlighted in experimental animals by johansson and ohlsson ( figure 1 ) . these authors assessed the relative importance of postoperation physical activity and social interaction for functional outcome . rats were housed together in a large cage with no equipment or housed individually in cages with free access to a running wheel and compared to rats kept in an ee . interestingly , rats housed together in a large cage with no activity - stimulating facilities improve more than rats housed in individual cages with access to a running wheel . however , rats housed in an ee improve significantly more than the other two groups , suggesting that , although increased physical or social activity alone might result in some of the beneficial effects observed with enrichment , they do not fully account for the broader behavioural improvements observed following exposure to complex stimuli . to study social influences on experimental stroke outcome , devries ' group addressed the effects of social isolation versus pair housing on stroke - induced infarct size and functional recovery in mice . they observed that pair housing decreased infarct size and improved functional outcome of stroke mice when compared to socially isolated mice . social interaction influences locomotor activity and introduces auditory , olfactory , and visual stimuli , which in turn may influence pathophysiological mechanisms and recovery . further , the same authors asked whether one aspect of social interaction , namely , physical contact , may mediate the effects of social interaction . to control for the element of physical contact during pair housing , the experiment included the use of standard cages fitted with a grid partition that allowed the experimental mouse to see , hear , and smell its partner but not engage in physical contact . interestingly , only paired animals that were in unobstructed physical contact showed smaller infarct volumes and exhibited recovery of locomotor activity following mca occlusion , indicating that physical contact during social interactions influences stroke outcome . further clinical research is , therefore , needed to determine the influence of physical contact on patient recovery . another potential noninvasive treatment that might have a significant impact upon recovery of skilled motor behaviors after stroke is tactile stimulation ( figure 1 ) . when stroke rats are given tactile stimulation , which involves petting animals individually with a baby hairbrush or a paintbrush , they show dramatic improvement in the single pellet reaching task relative to untreated lesioned animals . these data suggest that massage therapy might be beneficial in resolving motor deficits in human stroke patients . interestingly , intermittent single whisker stimulation , if initiated within 2 h of permanent mca occlusion in the rat , induces complete protection from ischemic stroke by 24 h after injury , preventing the expected damage and deficits . an initial absent or severely disrupted whisker functional representation is followed by gradual recovery to baseline responses over the treatment period . evoked subthreshold activity and spiking and blood flow levels , which are severely decreased immediately after occlusion , return gradually to preocclusion levels . blood flow data suggest that the protection induced by early stimulation is due to reorganized blood flow via collateral vessels ( interarterial connections ) . in contrast , animals that do not receive treatment until 3 h post - mca occlusion show compromised function and large infarcts [ 101 , 102 ] . these studies raise hope for the development of stimulation - based strategies to mitigate stroke pathology in humans . recent studies employed animal models to investigate the positive effects of tdcs and define the optimal time window of its application after stroke ( figure 1 ) . both early ( 1 day after ischemia ) and late ( 1 week after ischemia ) anodal tdcs treatments exert beneficial effects on cognition , behavioral function ( i.e. , improved barnes maze performance and motor behavioral index scores ) , and neural plasticity , without exacerbating ischemic volume and metabolic alteration . however , only the rats receiving late tdcs treatment showed improvement in the beam balance test . accordingly , in the study by jiang et al . anodal and cathodal tdcs applications from day 1 to day 3 after cerebral infarction do not improve the beam walking test scores of rats on day 3 , but significant amelioration of motor function is observed if the animals receive continuous application of tdcs till day 7 or 14 . these findings suggest that late application of tdcs may result in stronger motor function improvement than earlier intervention after stroke . accordingly , one study , in which anodal tdcs was applied during five daily sessions to the ipsilesional primary motor cortex in acute stroke patients starting on the 2nd day , did not reveal any significant difference in motor function between the tdcs and sham groups , indicating that tdcs application from day 2 to day 5 after stroke does not promote functional recovery . ltp and ltd may be candidates processes to explain the cellular correlates for tdcs - induced effects [ 106 , 107 ] . despite the observed beneficial effects in humans ( see for review ) , the cellular / molecular mechanisms underlying rtms action are far from clear . it is likely that rtms induces ltp or ltd , which , in turn , produce enduring changes on neocortical excitability and synaptic connections [ 109111 ] . in humans , an increase in motor - evoked potential amplitude [ 110 , 112 ] , regional cerebral blood flow , glucose metabolism , and eeg response amplitude has been reported . studies in animal models ( figure 1 ) have shown that rtms effects depend on changes in nmda receptor activity . provided the first evidence that rtms induces changes in bdnf - trkb signaling in the rat brain , which are reflected in lymphocytes . transcription of glial fibrillary acidic protein ( gfap ) is increased in astrocytes of the mouse dentate gyrus ( the magnitude of this response depends on the number of stimulus trains ) , suggesting that rtms induces the first stage of a reactive response that is similar to what occurs following nervous tissue injury . however , the consequences of rtms on experimental animals after stroke have been poorly investigated . report a significant recovery of neurological severity score in stroke rats treated with tms , which is accompanied by increased expression of c - fos and bdnf in the cerebral cortex surrounding the infarction areas . after clinical stroke , the initiation of physical rehabilitation programs varies from days to several weeks after the insult . determining whether there is a period during which the poststroke brain is most sensitive to physical rehabilitation is essential to maximize the functional gains from such therapy . hypothesized that implementing rehabilitative treatment early after the stroke would enhance functional outcome . to characterize a potential critical period for successful rehabilitation after stroke , animals received enriched rehabilitative training at 5 d , 14 d , or 30 d after mca occlusion . early initiation of enriched rehabilitation ( 5 d after stroke ) provides enhanced functional outcome relative to ischemic animals receiving delayed rehabilitation , suggesting that the poststroke brain is in a state of heightened sensitivity to behavioral experience . in line with those findings , barbay et al . demonstrate a time - dependent , rehabilitation - induced map reorganization after ischemic injury in primates . similarly , early treadmill training ( started 24 h post - mca occlusion ) was found to have significant effects in reducing brain infarct volume and in improving neurologic function , when compared with late training ( started 1 week post - mca occlusion , ) . nevertheless , some evidences suggest that early training after focal brain ischemia in rats exacerbates brain damage and worsens the general outcome after excessive use of the impaired limb . namely , when the intact forelimb is constrained immediately after the surgical procedure , thus forcing the animal to overuse the impaired forelimb for postural support and movements , functional improvement is reduced [ 120 , 121 ] . the intensity of training may contribute to early exclusive use - dependent exaggeration of injury . for example , in the study by yang et al . , the intensity of treadmill training for 30 min / day seems to be mild compared to forced use by casting procedures . excessive sensorimotor activation too early after the insult may exacerbate injury through a use dependent , nmda - mediated process , possibly stimulating an excitotoxic cascade . this process may dissipate over days , explaining why rehabilitative experience beginning 35 d after insult does not worsen injury size or behavioral outcome [ 89 , 123 ] . in addition , during the first week after injury , the tissue surrounding the infarct is reported to show decreased phasic inhibition and thus become hyperexcitable . however , carmicheal 's group show that while phasic gaba signaling is reduced in the first weeks after stroke , tonic gaba signaling is potentiated in peri - infarct motor neurons . behavioral and electrophysiological studies in mice suggest that the overall effect in terms of motor cortex circuitry is a diminished neuronal excitability , which when reversed leads to recovery . therefore , the precise signaling systems in brain excitability that are deleterious in the early phases , become beneficial in later phases of recovery ( see for a comprehensive review on brain excitability in stroke ) . rehabilitation may act by affecting this delicate balance between hypo- and hyperexcitability of neuronal circuits in peri - infarct cortex . interestingly , immediate exposure to ee improves functional outcome , despite exacerbation of ischemic injury [ 67 , 126 ] , perhaps as a consequence of removal of functionally abnormal neurons . nonetheless , early ee combined with training enhances recovery when compared with conditions in which rehabilitation is started later and is not accompanied by any exacerbation of injury . in addition , a window of opportunity extends also to neurovascular changes , which can facilitate full protection . in summary , the efficacy of rehabilitative therapy after stroke is influenced by the time of its initiation , with mild intensity physical training provided early after brain injury being beneficial for functional improvement . delaying the beginning of rehabilitation may instead reduce the efficacy of treatment and , as a consequence , more intense or longer duration therapies are required to achieve the same functional gains . much of the recovery after stroke is likely due to brain plasticity , with some areas of the brain taking over the functions previously performed by the damaged regions . proposed mechanisms include : ( i ) redundancy of brain circuitry with alternative pathways taking over when another one has been damaged ; ( ii ) unmasking of previously existing but functionally inactive networks ; ( iii ) sprouting of fibers from surviving neurons with formation of new synapses [ 127 , 128 ] . when damage to a functional system is partial , within - system recovery is possible , whereas after complete destruction , substitution by a functionally related system may be the only alternative . in stroke patients , improved arm and hand movement and clinical scores have been found in correlation with an enlargement of the hand region in the ipsilesional cortex [ 130135 ] . activity changes in specific cortical areas may result from a reduction in inhibition from horizontal or callosal connections . alternatively , new connections may form due to lesion - induced sprouting at the cortical or subcortical level [ 137 , 138 ] . reorganization of neuronal connectivity around the lesion site and also in the undamaged contralateral cortex has been detected [ 139141 ] . interestingly , following an ischemic subtotal lesion of the rat forelimb motor cortex , spontaneous recovery of forelimb function is correlated with hindlimb corticospinal neurons forming new connections with cervical , forelimb - related , spinal cord neurons . for example , while an ischemic lesion confined to a small portion of the representation of one hand results in a further loss of hand territory in the adjacent , undamaged cortex , early rehabilitative training prevents the loss of hand territory adjacent to the infarct . in some instances , the hand representation expands into regions formerly occupied by representations of the elbow and shoulder . functional reorganization in the undamaged motor cortex is accompanied by behavioral recovery of skilled hand function . moreover , stroke rats housed in an ee or receiving tactile stimulation have significantly increased dendritic branching and spine density on pyramidal cortical neurons than control stroke rats , suggestive of increased sprouting of intracortical connections in the enriched / stimulated group . indeed , ee can influence a number of factors , such as functional enforcement of existing neuronal circuits , sprouting , formation of new connections , and angiogenesis [ 63 , 144 ] . ee may also modulate ischemia - induced glutamate excitotoxicity , thus leading to attenuated oxidative damage and neurodegeneration . one candidate mechanism underlying the beneficial effects of ee on functional recovery after stroke involves upregulation of neurotrophic factors [ 146 , 147 ] , which may stimulate neuritic remodeling and synaptogenesis . cortical neurons that sprout a new connection after stroke activate a neuronal growth program that consists of transcription factors , cell adhesion , axonal guidance , and cytoskeletal modifying molecules . it is known that ee modulates the expression of several genes in the infarcted cortex . namely , postischemic ee or social interaction modulate the expression of substances associated with neuronal plasticity , such as nerve growth factor - induced gene a ( ngfi - a ) and ngfi - b . ngfi - a ( also known as egr1 , krox24 , zif/268 , and tis8 ) , a transcription factor belonging to the early growth response family , is associated with stabilisation of ltp and learning [ 151 , 152 ] . ngfi - a target genes are synapsin - i and -ii , which are involved in synaptic vesicle trafficking and release as well as synaptogenesis [ 153155 ] . synapsin - i and ii are increased in the ipsilateral cortex of stroke rats following skilled training . in addition , ngfi - a is a master switch for the initiation of inflammatory gene expression under ischemic stress . ngfi - b ( also known as nur77 , n10 , tis1 , or tr3 ) , a member of the steroid / thyroid receptor family without any known ligand , has also been associated with ltp . at one month following mca occlusion the mrna expression of ngfi - a and ngfi - b is increased after ee in the cerebral cortex and the hippocampus . however , other reports show a decreased expression of ngfi - a in both cortices of ee rats [ 161163 ] , likely reflecting the suppression of postischemic inflammation in the brain . differences in the intensity and the duration of exercise administered to the rats may account for the different results obtained . postlesional plasticity in the adult brain is not restricted to structural modifications at the level of axons , dendrites , and synapses but also comprises the generation , differentiation , and maturation of new neurons in circumscribed brain regions ( reviewed by ) . numerous studies utilizing different experimental models have shown that an ischemic cns lesion leads to a substantial increase in proliferation of neural stem cells and subsequently increased generation of new neurons in the subgranular zone of the dentate gyrus and in the subventricular zone ( svz ) ( see for review ) . dentate neurogenesis is stimulated by focal ischemic infarcts even when the site of the injury is located in remote cortical brain areas [ 165 , 166 ] . newborn neurons in the svz are recruited to infarcted areas and may start to express region - specific mature neuronal markers [ 167172 ] . however , newborn cells expressing mature and region - appropriate neuronal markers have only been observed in the ischemic striatum but not in the cerebral cortex , with low fractions of newly generated cells surviving into maturity [ 167 , 168 , 173 ] . possible reasons for the reduced incidence of neuronal replacement in the ischemic striatum and cortex could be low cell survival or hampered neuronal phenotypic maturation due to detrimental factors in the perilesional environment , lack of neurotrophic support and of necessary developmental cues . notably , ablation of doublecortin - positive neuronal precursors from the rostral svz and dentate gyrus abolishes neurogenesis and associated neuronal migration induced by focal cerebral ischemia . this results in increased infarct size and worsened neurologic deficits , indicating that neurogenesis contributes to neuroprotection and short - term functional outcome after experimental stroke in mice . those beneficial effects may depend on the release of chemical mediators ( e.g. , growth factors ) by immature neurons . studies on the effects of ee and exercise on the adult germinal niches in intact animals have shown that both these paradigms lead to increased neurogenesis in the hippocampus and the svz . for example , postischemic ee enhances cell proliferation in the svz , with stronger effects in the chronic poststroke phase [ 171 , 176 ] , while wheel - running exercise after neocortical infarction attenuates the early poststroke activation of the svz germinal niche . interestingly , no effect of ee or exercise on hippocampal progenitor cell proliferation is reported after transient global ischemia in rats , suggesting that common pathways of regulation by lesion and environmental interventions may exist [ 178180 ] . in contrast , specific rehabilitative training of the impaired forelimb ( skilled reaching training ) is able to increase dentate neurogenesis relative to nontrained stroke rats , although at lower levels when compared with sham - operated animals . moreover , increased levels of newborn granule cells generated in the dentate gyrus correlate with better functional outcomes [ 181 , 182 ] . interestingly , postischemic ee combined with spatial learning ( which simulates occupational therapy in human rehabilitation and activate hippocampus and prefrontal cortex ) restores the perturbed dentate gyrus neuroblast production resulting from focal ischemic insult and increases neuroprotection in the ischemic penumbra . the lack or inadequacy of endogenous neuronal replacement after brain lesions encouraged investigations on the role of glial cells in poststroke recovery process . increasing evidence indicate that glial cells crucially contribute to the degenerative and regenerative processes following ischemic brain lesions [ 184 , 185 ] . also , some of the beneficial effects of ee on the postischemic brain might be mediated by a dynamic modulation of different glial populations . it is well known that astrocytes are essential for optimal neuronal function and take an active part in synaptic generation and plasticity as well as in maintenance of neuronal and synaptic homeostasis [ 186188 ] . recently , it has been revealed that astroglia may represent neural stem cells in the adult brain and may also direct neuronal differentiation of adult neural stem cells [ 189191 ] . they immediately proliferate in response to the lesion , increase their expression of gfap , and contribute to the formation of the glial scar [ 192 , 193 ] . reactive astroglia might provide a protective environment in the perilesional zone by shielding neurons from oxidative stress [ 194 , 195 ] or producing antiapoptotic and trophic factors . accordingly , they might promote neuronal survival , synaptic remodelling , and neurite outgrowth [ 184 , 196199 ] . postischemic ee or daily training of the impaired forelimb enhances astrogliosis in the perilesional area [ 171 , 176 , 193 ] . reactive astroglia , although representing an impediment for axon growth , may fulfill important protective and reparative functions after ischemic injuries and rehabilitation [ 199203 ] . immediately after the ischemic insult , resting microglia change their morphology from a ramified to an activated hyperramified phenotype and express the cd68 antigen . the activated microglia migrate towards the lesion , remove the necrotic tissue by phagocytosis , and thereby become macrophages [ 205 , 206 ] . some macrophages derive from monocytes that cross the blood - brain barrier after the ischemic lesion [ 207 , 208 ] . besides the degradation of necrotic cells , activated microglia and macrophages release growth factors and scavenge - free radicals [ 209 , 210 ] . however , activated microglia could also harm the injured brain with the synthesis of potentially toxic substances like nitric oxide and reactive oxygen radicals or the release of glutamate and proinflammatory cytokines [ 209 , 211216 ] . indeed , recent studies show that suppression of activated microglia and macrophages significantly improve functional recovery after focal ischemic infarcts [ 217 , 218 ] . in stroke animals exposed to ee or training a reduction of proliferating microglia and macrophages finally , proliferation and survival of immature and mature oligodendrocytes are only slightly influenced by ee . it has been shown that ee increases the number of ng2-positive glia , in intact ipsi - and contralateral cortical regions remote from the infarct . ng2-positive cells possess some characteristics of multipotent progenitor cells , may support neuronal function , and can turn into myelin - forming oligodendrocytes [ 220223 ] . however , the role of this cell population in the injured brain is still obscure . novel noninvasive interventions for stroke patients , such as mental practice , mirror therapy , virtual reality , robotics , and brain stimulation techniques , are emerging as potentially efficient strategies to promote functional recovery , but in most cases only when provided in combination with physical rehabilitation [ 43 , 224 ] . the expansion of rehabilitative programs with a wide range of possible interventions is more likely the key to obtain optimal results , by stimulating different reparative and adaptive brain processes . particularly , the use of noninvasive techniques of brain stimulation to promote adaptive plasticity , such as tdcs and rtms , is very appealing , and the results obtained in preclinical and clinical models of stroke are exciting . in this context , however , randomized controlled trials are needed to validate the efficacy of these techniques . this knowledge may allow the identification of biological markers suitable to monitor plastic processes in human patients undergoing specific rehabilitative programs , predict the outcome of the treatments , and optimise existing procedures . in conclusion , in the last few years there has been an enormous progress in the field of rehabilitative trials after stroke , for example , in terms of standardized interventions and tools for assessment of function and patient selection ( e.g. , recruitment of homogeneous groups of patients ) . crucial issues , however , remain to be addressed in future studies , including the sample wideness , repeatability of the results , and effective outcome measurements .

stroke is a common and disabling global health - care problem , which is the third most common cause of death and one of the main causes of acquired adult disability in many countries . rehabilitation interventions are a major component of patient care . in the last few years , brain stimulation , mirror therapy , action observation , or mental practice with motor imagery has emerged as interesting options as add - on interventions to standard physical therapies . the neural bases for poststroke recovery rely on the concept of plasticity , namely , the ability of central nervous system cells to modify their structure and function in response to external stimuli . in this review , we will discuss recent noninvasive strategies employed to enhance functional recovery in stroke patients and we will provide an overview of neural plastic events associated with rehabilitation in preclinical models of stroke .

1. Introduction 2. Novel Noninvasive Strategies for Patients Rehabilitation 3. Noninvasive Therapies in Animal Models of Stroke 4. Is There a Critical Period for Successful Rehabilitation? 5. Cellular and Molecular Correlates of Rehabilitation-Induced Plasticity 6. Conclusions

PMC3881779

the periodontium is a topographically complex organ consisting of epithelial tissue and soft and mineralized connective tissue . the structures comprising the periodontium include gingiva , cementum , bone and periodontal ligament . several diseases affect the composition and integrity of periodontal structures causing the destruction of the connective tissue matrix and cells , the loss of fibrous attachment and the resorption of alveolar bone following an intense inflammatory response to bacteria . such diseases are highly prevalent among different world populations , being an important impact factor in health oral programs . the main bacterial pathogens of periodontal diseases are gram - negative anaerobic species that express a number of potential virulence factors that stimulate an unbalanced production of several molecules and pro - inflammatory factors , being an important determinant in the disease outcome . innumerous studies have shown that , although largely nonspecific , basic treatment consisting of oral hygiene instruction and subgingival instrumentation is able to keep the microbial load at low levels and , if followed by daily self - performed oral hygiene and regular professional periodontal maintenance care , leads to periodontal health in most cases . however , due to its unique patient and site - specific characteristics , a small proportion of the population suffers from ' ' refractory '' types of periodontitis , showing inadequate resolution of inflammation and failure to return tissue to homeostasis . since knowledge of the pathways and processes underlying resolution of periodontal tissue inflammation have grown significantly , an increased interest in substances and/or drugs that may contribute to the restoration of tissue homeostasis has occurred as a more sophisticated biological treatment modality for recurrent types of periodontal disease . several biological systems are candidates to modulate the inflammatory process involved in periodontal disease . evidence from animal experiments and short - term clinical trials demonstrate that anti - inflammatory drugs are able to decrease the rate of periodontal bone destruction by local inhibition of pro - inflammatory molecules . in brazil , the leaves of species of mikania laevigata schultz bip . ex baker , popularly known as " guaco " , have been widely used as infusions or plasters , while the crude extract of this species is commonly commercialized as phytomedicine . guaco is also popular in brazil as an anti - inflammatory , antispasmodic and pain - reliever for rheumatism , arthritis , intestinal inflammation and ulcers . among the few pharmacological and phytochemical studies published , preparations obtained from aerial parts of mikania laevigata have been described as anti - ulcerogenic , antimicrobial , bronchodilatory and anti - inflammatory , possibly accounted for by the presence of several chemical constituents . our research group has recently assessed the pharmacological properties and the underlying molecular mechanisms of mikania laevigata , demonstrating that , in animals , its hydroalcoholic extract was able to reduce neutrophil migration and vascular permeability , and prevent the release of tnf- and il-1. since such findings could promote a protective effect in periodontal tissues against damage , the aim of this study was to evaluate the immunomodulatory action of systemic injection of a " guaco " extract in a model of experimental periodontitis in rats . the extract used in this study was previously obtained and tested as described elsewhere . briefly , the leaves of mikania laevigata were collected at the farm school of the university of uberaba ( tringulo mineiro , mg , brazil ) . a voucher specimen ( hufu 54.748 ) has been deposited at the herbarium of the federal university of uberlndia , brazil . , between days 15 and 17 of november and allowed to dry at 30c in an oven with air renovation for 15 days . the dry plant , triturated by knife mill , was extracted by maceration with 70% ethanol : water under continuous agitation ( shaker ) for three times during 7 days each totalizing 21 days [ the end ratio between plant and solvent was 1:8 ( w / v ) ] , obtaining a crude extract . the crude extract was dried and filtered using filter paper , concentrated in air - forced chamber at 30c until dry crude extract was obtained . forty male wistar rats ( 250 - 350 g ) were used in the study . the animals were kept in plastic cages with access to food and water ad libitum . prior to the surgical procedures , all animals were allowed to acclimatize to the laboratory environment for a period of 5 days . all experiments were conducted in accordance with national health guidelines for the welfare of experimental animals and were approved by the ethics committee of the university of uberaba ( protocol # 001/2008 ) . more specifically , under general anesthesia obtained by intramuscular administration of ketamine ( 1.0 ml / kg ) , ligature was placed and immobilized around both mandible first molars of each animal . the ligature was left in position for the whole experimental period so that inflammation could be constantly induced by the colonization of bacteria inside of it . one day following ligature placement , the animals were randomly assigned to one of the following groups ( n=10 ) : 1 ) animals without ligature placement receiving administration of empty vehicle ( control ) ; 2 ) animals without ligature placement receiving administration of mle ; 3 ) animals with ligature receiving administration of saline ; 4 ) animals with ligature receiving administration of subcutaneous mle ( 10 mg / kg / day ; 200 l . mle or vehicle was administered daily for 30 days . twenty - four hours after the last injection the current dose was chosen based on a previous study showing that it was effective to diminish the carrageenan - induced peritonitis . neutrophil infiltration to the gingival tissues of rats was evaluated by mpo kinetic - colorimetric assay as previously described . samples of gingival tissue were collected in 50 mm k2hpo4 buffer ( ph 6.0 ) containing 0.5% hexadecyl trimethylammonium bromide ( htab ) and kept at -80c until use . samples were homogenized using a polytron ( pt3100 ) and centrifuged at 13,000 g for 4 min . the resulting supernatant devoid of debris was subjected to mpo activity assay determined by spectrophotometer at 450 nm ( spectra max ; molecular devices inc . , briefly , 10 l of sample was mixed with 200 l of 50 mm phosphate buffer ph 6.0 , containing 0.167 mg / ml o - dianisidine dihydrochloride and 0.0005% hydrogen peroxide . the results were presented as mpo activity ( number of neutrophils x mg gingival tissue ) . the right and left jaws were dissected , fixed in 10% buffered neutral formalin for 48 h and decalcified in a decalcifying solution of ethylenediaminetetraacetic acid ( edta ) 10% for 3 months . after that , briefly washed in running tap water , dehydrated and embedded in paraffin wax . sections were mounted on glass slides and stained with hematoxylin and eosin ( he ) for the evaluation of bone resorption . using an image analysis system ( image j - national institute of health ) , the area of bone loss in the furcation region was histometrically determined as previously described . additional sections were mounted on glass slides pre - treated with 3-aminopropyltriethoxy - silane ( sigma chemical co. , st . sections were treated with 3% hydrogen peroxide ( h2o2 ) for 30 min to block endogenous peroxidase . to block the nonspecific binding of antibody , the sections were treated with phosphate buffered saline ( pbs)-1% bovine serum albumin ( vector laboratories , burlingame , ca , usa ) for 30 min at room temperature before incubation with the primary antibody ( polyclonal antibodies against rankl ( 1:50 ) ( santa cruz biotechnology , santa cruz , ca , usa ) for 24 h at 4c . biotinylated secondary antibody was used ( sigma;1:100 ) for 60 min at room temperature . the slides were treated with vectastain abc - ap kit ( diluted at 1:100 ; vector laboratories ) for 60 min at room temperature , and the specific reaction by each antibody was visualized using 3 , 3'diaminobenzidine ( dab ) . the slides were then counterstained with mayer 's hematoxylin , dehydrated through graded ethanol , cleared in xylene , and mounted in slides with the help of permount mounting media ( fisher scientific ) . at least 10 representative sections of each specimen were analyzed under a light microscope ( olympus bx50 , tokyo , japan ) . immunohistochemical analysis was performed individually by two previous calibrated examiners ( jcc and vjs - f ; kappa index=0.97 ) that were blind to the treatment conditions . the number of stained cells was counted and normalized in the area where bone resorption was taking place . statistical comparisons between groups were made using anova analysis of variance followed by bonferroni test . the extract used in this study was previously obtained and tested as described elsewhere . briefly , the leaves of mikania laevigata were collected at the farm school of the university of uberaba ( tringulo mineiro , mg , brazil ) . a voucher specimen ( hufu 54.748 ) has been deposited at the herbarium of the federal university of uberlndia , brazil . , between days 15 and 17 of november and allowed to dry at 30c in an oven with air renovation for 15 days . the dry plant , triturated by knife mill , was extracted by maceration with 70% ethanol : water under continuous agitation ( shaker ) for three times during 7 days each totalizing 21 days [ the end ratio between plant and solvent was 1:8 ( w / v ) ] , obtaining a crude extract . the crude extract was dried and filtered using filter paper , concentrated in air - forced chamber at 30c until dry crude extract was obtained . forty male wistar rats ( 250 - 350 g ) were used in the study . the animals were kept in plastic cages with access to food and water ad libitum . prior to the surgical procedures , all animals were allowed to acclimatize to the laboratory environment for a period of 5 days . all experiments were conducted in accordance with national health guidelines for the welfare of experimental animals and were approved by the ethics committee of the university of uberaba ( protocol # 001/2008 ) . more specifically , under general anesthesia obtained by intramuscular administration of ketamine ( 1.0 ml / kg ) , ligature was placed and immobilized around both mandible first molars of each animal . the ligature was left in position for the whole experimental period so that inflammation could be constantly induced by the colonization of bacteria inside of it . one day following ligature placement , the animals were randomly assigned to one of the following groups ( n=10 ) : 1 ) animals without ligature placement receiving administration of empty vehicle ( control ) ; 2 ) animals without ligature placement receiving administration of mle ; 3 ) animals with ligature receiving administration of saline ; 4 ) animals with ligature receiving administration of subcutaneous mle ( 10 mg / kg / day ; 200 l . mle or vehicle was administered daily for 30 days . twenty - four hours after the last injection , the current dose was chosen based on a previous study showing that it was effective to diminish the carrageenan - induced peritonitis . neutrophil infiltration to the gingival tissues of rats was evaluated by mpo kinetic - colorimetric assay as previously described . samples of gingival tissue were collected in 50 mm k2hpo4 buffer ( ph 6.0 ) containing 0.5% hexadecyl trimethylammonium bromide ( htab ) and kept at -80c until use . samples were homogenized using a polytron ( pt3100 ) and centrifuged at 13,000 g for 4 min . the resulting supernatant devoid of debris was subjected to mpo activity assay determined by spectrophotometer at 450 nm ( spectra max ; molecular devices inc . , sunnyvale , ca , usa ) with three readings within 1 min . briefly , 10 l of sample was mixed with 200 l of 50 mm phosphate buffer ph 6.0 , containing 0.167 mg / ml o - dianisidine dihydrochloride and 0.0005% hydrogen peroxide . the results were presented as mpo activity ( number of neutrophils x mg gingival tissue ) . the right and left jaws were dissected , fixed in 10% buffered neutral formalin for 48 h and decalcified in a decalcifying solution of ethylenediaminetetraacetic acid ( edta ) 10% for 3 months . after that , briefly washed in running tap water , dehydrated and embedded in paraffin wax . sections were mounted on glass slides and stained with hematoxylin and eosin ( he ) for the evaluation of bone resorption . using an image analysis system ( image j - national institute of health ) , the area of bone loss in the furcation region was histometrically determined as previously described . additional sections were mounted on glass slides pre - treated with 3-aminopropyltriethoxy - silane ( sigma chemical co. , st . sections were treated with 3% hydrogen peroxide ( h2o2 ) for 30 min to block endogenous peroxidase . to block the nonspecific binding of antibody , the sections were treated with phosphate buffered saline ( pbs)-1% bovine serum albumin ( vector laboratories , burlingame , ca , usa ) for 30 min at room temperature before incubation with the primary antibody ( polyclonal antibodies against rankl ( 1:50 ) ( santa cruz biotechnology , santa cruz , ca , usa ) for 24 h at 4c . biotinylated secondary antibody was used ( sigma;1:100 ) for 60 min at room temperature . the slides were treated with vectastain abc - ap kit ( diluted at 1:100 ; vector laboratories ) for 60 min at room temperature , and the specific reaction by each antibody was visualized using 3 , 3'diaminobenzidine ( dab ) . the slides were then counterstained with mayer 's hematoxylin , dehydrated through graded ethanol , cleared in xylene , and mounted in slides with the help of permount mounting media ( fisher scientific ) . at least 10 representative sections of each specimen were analyzed under a light microscope ( olympus bx50 , tokyo , japan ) . immunohistochemical analysis was performed individually by two previous calibrated examiners ( jcc and vjs - f ; kappa index=0.97 ) that were blind to the treatment conditions . the number of stained cells was counted and normalized in the area where bone resorption was taking place . statistical comparisons between groups were made using anova analysis of variance followed by bonferroni test . to assess whether mle would affect bone loss in the furcation region , he sections were histometrically analyzed . the resorption area measured after the experimental period demonstrated that ligature induced experimental periodontitis significantly increased bone loss ( p<0.05 ) ( figure 1c ) when compared with both vehicle ( 0.310.03 mm ) and mle ( 0.350.06 mm ) non - ligated groups , which were not different when compared with each other ( p>0.05 ) ( figures 1a and 1b ) . additionally , the results demonstrated that mle administration ( 10 mg / kg , daily ) was able to significantly inhibit ( 0.560.13 mm ; p<0.05 ) the volume of bone loss in ligated teeth ( figure 1d ) , however still higher when compared with the non - ligated teeth ( p<0.05 ) . the values of the resorption area of the three groups were represented in figure 1 . histology at the furcation of first molars sampled from the rats euthanized after 30 days of experiments is shown [ hematoxylin and eosin(he ) staining ] ; ( a ) non - ligated animals treated with vehicle , ( b ) non - ligated animals treated with ( mle ) ( 10 mg / kg / day for 30 consecutive days ) , ( c ) ligature - induced periodontitis treated with vehicle for 30 days , ( d ) ligature - induced periodontitis treated with subcutaneous mle ( 10 mg / kg/ day for 30 consecutive days ) . ( e ) results are expressed as mean area ( mm ) standard deviation of 10 animals in each group . different symbols indicate intergroup statistical significance ( anova followed by bonferroni s test ) . magnification at 40 objective ( scale bar=100 m ) next , the possible interference of mle on neutrophil migration was investigated . according to mpo assay , mle administration did not exert any significant effect on neutrophil accumulation on non - ligated teeth ( 55.7425.84 versus 53.1415.21 neutrophils x 10 ; p>0.05 ) by measuring gingival tissue mpo . on the other hand , the increased levels of neutrophil accumulation observed on ligated teeth ( 113.8238.96 neutrophils x 106 ; p<0.05 ) were significantly decreased by mle administration ( 61.2310.12 neutrophils x 10 ; p<0.05 ) ( figure 2 ) . thus , mle was able to reduce neutrophil accumulation and consequently inflammation in the gingival tissue . mikania laevigata extract ( mle ) decreases neutrophil migration to the gingival tissue . in order to estimate the relative numbers of infiltrating neutrophils in the gingival tissue , myeloperoxidase ( mpo ) activity present in the gingival tissue homogenates was measured . # p<0.05 compared with control animals ; * p<0.05 compared with ligature - induced periodontitis treated with vehicle ( anova followed by bonferroni 's test ) on the other hand , since a reduced bone resorption was observed in the mle - treated group , we tested the hypothesis that mle decreases the release of osteoclast regulatory factors . in this way , we used an immunohistochemistry assay to visualize the expression of rankl on periodontal tissues . as shown in figure 3 , animals with ligated - induced periodontitis treated with mle presented significantly less rankl - immunostained cells ( 76.2564.40 immunostained cells ; p<0.05 ) in the periodontal tissue when compared with ligated - animals treated with vehicle ( 242.0046.23 immunostained cells ) . non - ligated animals ( 49.6618.23 immunostained cells ) were not affected by mle administration ( 45.7522.42 immunostained cells ; p>0.05 ) . expression patterns of rankl in the periodontal tissue at the furcation of first molars sampled from the rats euthanized at 30 days are shown . low number of positive staining cells for rankl was observed in the furcation of non - ligated animals treated with vehicle ( a ) as well the non - ligated animals treated with mikania laevigata extract ( mle ) ( b ) . increased number of rankl positive cells were found in the furcation of the ligated - animals treated with vehicle ( c ) . lower number of rankl stained cells were found in the furcation of the ligated - animals treated with mle ( 10 mg / kg / day for 30 consecutive days ) . ( e ) data are presented as median standard deviation of the average rankl positive cells / per area of furcation . negative control staining was carried out by the incubation with secondary antibody alone that showed lack of immunostaining pattern in all experimental groups ( data not shown ) . # p<0.05 compared with control animals ; * p<0.05 compared with ligature - induced periodontitis treated with vehicle ( anova followed by bonferroni s test ) . the figure is representative of at least 4 different sections obtained from each group . scale bar at lower ( 40x ) magnification=100 m , and scale bar at higher ( 400x ) magnification=20 m to assess whether mle would affect bone loss in the furcation region , he sections were histometrically analyzed . the resorption area measured after the experimental period demonstrated that ligature induced experimental periodontitis significantly increased bone loss ( p<0.05 ) ( figure 1c ) when compared with both vehicle ( 0.310.03 mm ) and mle ( 0.350.06 mm ) non - ligated groups , which were not different when compared with each other ( p>0.05 ) ( figures 1a and 1b ) . additionally , the results demonstrated that mle administration ( 10 mg / kg , daily ) was able to significantly inhibit ( 0.560.13 mm ; p<0.05 ) the volume of bone loss in ligated teeth ( figure 1d ) , however still higher when compared with the non - ligated teeth ( p<0.05 ) . the values of the resorption area of the three groups were represented in figure 1 . histology at the furcation of first molars sampled from the rats euthanized after 30 days of experiments is shown [ hematoxylin and eosin(he ) staining ] ; ( a ) non - ligated animals treated with vehicle , ( b ) non - ligated animals treated with ( mle ) ( 10 mg / kg / day for 30 consecutive days ) , ( c ) ligature - induced periodontitis treated with vehicle for 30 days , ( d ) ligature - induced periodontitis treated with subcutaneous mle ( 10 mg / kg/ day for 30 consecutive days ) . ( e ) results are expressed as mean area ( mm ) standard deviation of 10 animals in each group . different symbols indicate intergroup statistical significance ( anova followed by bonferroni s test ) . next , the possible interference of mle on neutrophil migration was investigated . according to mpo assay , mle administration did not exert any significant effect on neutrophil accumulation on non - ligated teeth ( 55.7425.84 versus 53.1415.21 neutrophils x 10 ; p>0.05 ) by measuring gingival tissue mpo . on the other hand , the increased levels of neutrophil accumulation observed on ligated teeth ( 113.8238.96 neutrophils x 106 ; p<0.05 ) were significantly decreased by mle administration ( 61.2310.12 neutrophils x 10 ; p<0.05 ) ( figure 2 ) . thus , mle was able to reduce neutrophil accumulation and consequently inflammation in the gingival tissue . mikania laevigata extract ( mle ) decreases neutrophil migration to the gingival tissue . in order to estimate the relative numbers of infiltrating neutrophils in the gingival tissue , myeloperoxidase ( mpo ) # p<0.05 compared with control animals ; * p<0.05 compared with ligature - induced periodontitis treated with vehicle ( anova followed by bonferroni 's test ) on the other hand , since a reduced bone resorption was observed in the mle - treated group , we tested the hypothesis that mle decreases the release of osteoclast regulatory factors . in this way , we used an immunohistochemistry assay to visualize the expression of rankl on periodontal tissues . as shown in figure 3 , animals with ligated - induced periodontitis treated with mle presented significantly less rankl - immunostained cells ( 76.2564.40 immunostained cells ; p<0.05 ) in the periodontal tissue when compared with ligated - animals treated with vehicle ( 242.0046.23 immunostained cells ) . non - ligated animals ( 49.6618.23 immunostained cells ) were not affected by mle administration ( 45.7522.42 immunostained cells ; p>0.05 ) . expression patterns of rankl in the periodontal tissue at the furcation of first molars sampled from the rats euthanized at 30 days are shown . low number of positive staining cells for rankl was observed in the furcation of non - ligated animals treated with vehicle ( a ) as well the non - ligated animals treated with mikania laevigata extract ( mle ) ( b ) . increased number of rankl positive cells were found in the furcation of the ligated - animals treated with vehicle ( c ) . lower number of rankl stained cells were found in the furcation of the ligated - animals treated with mle ( 10 mg / kg / day for 30 consecutive days ) . ( e ) data are presented as median standard deviation of the average rankl positive cells / per area of furcation . negative control staining was carried out by the incubation with secondary antibody alone that showed lack of immunostaining pattern in all experimental groups ( data not shown ) . # p<0.05 compared with control animals ; * p<0.05 compared with ligature - induced periodontitis treated with vehicle ( anova followed by bonferroni s test ) . the figure is representative of at least 4 different sections obtained from each group . scale bar at lower ( 40x ) magnification=100 m , and scale bar at higher ( 400x ) magnification=20 m tooth loss , caused by poor periodontal health ( which affects up to 20% of the adult population worldwide ) can lead to significant morbidity and premature death . the economic impact of oral diseases is an important consideration with up to 10% of public health expenditure in developed countries related to curative dental care . in cases where traditional and basic treatments are not able to arrest periodontal disease progression , the adjunct use of alternative substances aimed at modulating the host immune response is desirable . however , despite several agents being commercially available , most of these chemicals may present undesirable side effects and limited scientific evidence so far . it is recognized that most of the new drugs discovered in the last few decades have originated from nature . chemical constituents obtained from medicinal plants and other natural products have been increasingly used to treat many inflammatory diseases . in the present study , it was demonstrated that an extract obtained from mikania laevigata , a popular medicinal plant in south american countries , was able to decrease alveolar bone loss , which may be explained , at least in part , by the lower expression of rankl and decreased neutrophil migration observed on mle - treated animals . a growing body of research suggests that chronic periodontal disease involves a failure of inflammation resolution pathways to restore tissue homeostasis . therefore , strategies to slow or arrest periodontal disease progression by the modulation of the host immune response have been investigated . among the commercially available substances earliest evaluated so far to modify resolution pathways , are the nonsteroidal anti - inflammatory drugs ( nsaids ) . these agents antagonize proinflammatory pathways and/or signaling , preventing the production arachidonic acid metabolites that are proinflammatory mediators implicated in a variety of bone resorptive and tissue degrading processes . although in vitro and experimental studies have shown promising results , periodontal clinical trials have not reached consistent clinical benefits when combined with conventional therapy besides having undesirable side effects when used in a long - term basis . hence , the search for alternative products continues and natural phytochemicals isolated from plants used in traditional medicine are considered as good alternatives to synthetic chemicals . more recently , the number of reports of the use of traditional plants and natural products for the treatment of periodontal disease has increased . in vitro studies evaluating the antimicrobial potential of substances in addition , in vivo studies in animals using compounds of natural plants such as baicalin and cannabinoids observed a protective role against periodontal tissue breakdown in ligature - induced periodontitis , which was partially explained by their inhibitory effects on prostaglandins ( cox-2 ) , interleukins ( il-1 and tnf- ) , nitric oxide and the bone related molecules rank and rankl . our results corroborate with such previous findings , with the administration of mle significantly decreasing alveolar bone resorption and promoting an inhibition of neutrophils influx and rankl production . in the progression of inflammatory diseases , the interactions of recruited neutrophils in the site of inflammation stimulate the local production of several inflammatory mediators that may further amplify the inflammatory response and injure the surrounding tissues . in fact , our group recently assessed the pharmacological properties and the underlying molecular mechanisms of the hydroalcoholic mle to confirm the popular wisdom as a putative anti - inflammatory drug . we could observe that the anti - inflammatory effect of mle was able to reduce neutrophil migration and vascular permeability in a peritonitis model . mle additionally prevented the release of both tnf- and il-1 and contributed to a reduction in leukocyte adhesion and transmigration across the endothelium as observed by intravital microscopy . taken together , the results suggest that use of the medicinal guaco extract may be able to suppress the development of inflammatory lesions , which are initiated by neutrophil recruitment including periodontal conditions . the discovery of the rank - rankl - osteoprotegerin ( opg ) system has brought rapid progress in the understanding of the regulatory mechanisms of osteoclast differentiation and activation exerted by the immune system . recent studies suggest the involvement of rankl and opg in the pathogenesis of periodontitis . not only osteoblasts but also other resident cells , including cells of the periodontal ligament , the findings of the present study , demonstrated that the administration of mle inhibited the expression of rankl . to the best of our knowledge , no direct effect of mle on the rank / rankl / opg system has been addressed in the literature . however , several investigations have addressed the close relation between the production of inflammatory mediators and the modulation of rankl / opg balance . during the progression of periodontal disease , high levels of pro - inflammatory cytokines have been positively related to rankl expression , suggesting that such inflammatory molecules play a major role in periodontal bone resorption . interestingly , a recent work demonstrated that lipopolysaccharide ( lps ) , a toll - like receptor 4 ligand , up - regulated the expression of membrane rankl in human blood neutrophils . besides , lps - activated human and murine neutrophils , cocultured with human monocyte - derived osteoclasts and raw 264.7 cells , respectively , stimulated bone resorption . therefore it is possible to suggest that the potent anti - inflammatory properties of mle may have caused the diminished production of rankl observed in mle - treated animals observed in the present study and indirectly participated in the decreased volume of bone loss during ligature - induced periodontal disease progression . there is considerable evidence that plant extracts have the potential to be developed into agents that can be used as preventative or treatment therapies for oral diseases . the results of the present investigation indicate that " guaco " extract may be useful to control bone resorption during progression of experimental periodontitis in rats in animal studies . while our results are encouraging , further controlled trial studies will be important to establish whether guaco is able to offer clinical therapeutic benefits .

objectivesthe mikania laevigata extract ( mle ) ( popularly known in brazil as " guaco " ) possesses anti - inflammatory properties . in the present study we tested the effects of mle in a periodontitis experimental model in rats . we also investigated possible mechanisms underlying such effects . material and methodsperiodontal disease was induced by a ligature placed around the mandibular first molars of each animal . male wistar rats were divided into 4 groups : non - ligated animals treated with vehicle ; non - ligated animals treated with mle ( 10 mg / kg , daily ) ; ligature - induced animals treated with vehicle and ligature - induced animals treated with mle ( 10 mg / kg , daily ) . thirty days after the induction of periodontal disease , the animals were euthanized and mandibles and gingival tissues removed for further analysis . resultsmorphometric analysis of alveolar bone loss demonstrated that mle - treated animals presented a decreased alveolar bone loss and a lower expression of the activator of nuclear factor-b ligand ( rankl ) measured by immunohistochemistry . moreover , gingival tissues from the mle - treated group showed decreased neutrophil migration myeloperoxidase ( mpo ) assay . conclusionsthese results indicate that mle may be useful to control bone resorption during progression of experimental periodontitis in rats .

INTRODUCTION MATERIAL AND METHODS Source of Mikania laevigata and preparation of the "guaco" extract Animals Experimental animal design MPO activity assay Histological and immunohistochemical analyses Statistical analysis RESULTS Effect of MLE on furcation bone loss Effect of MLE on neutrophil migration and RANKL expression DISCUSSION CONCLUSION ACKNOWLEDGEMENTS AND FUNDING

PMC4573065

new , large genomic data sets are providing more in - depth insights into the diagnosis and treatment of disease . in the past decade , new and innovative methods have continued to add value to the underlying data and uncover the secrets of the genome . visual data inspection by experienced researchers is an important quality control element in the analytical process . unfortunately , this part of the process is tedious and time consuming , and the increasing volumes of high - throughput sequencing data of various types and platforms are proving to be a major analytical challenge . here , we report a visualization tool that allows researchers to explore their data at a very rapid speed and significantly reduce the burden of reviewing tens and hundreds of thousands of variant calls . areas with systematic read errors can be quickly identified , and inefficient attempts to verify results in noisy regions can be avoided . alview is a fast and portable visualization tool . the core code interfaces with heng li et al s samtools library1 for parsing bam files . the program is written in platform - independent c. peculiarities specific to an operating system are isolated with if defined ( ifdef ) directives ; so , for instance , when microsoft visual c provides alternate support for a portable operating system interface ( posix ) standard function , a handcrafted , native interface work around is supplied . for graphical user interface ( gui ) frameworks , alview uses win32 interface for windows , the gtk2 interface for linux , and bsd unix - based systems and cocoa for apple mac os x. samtools1 is written to posix standards , but different microsoft visual compilers provide various levels of support for these unix style standards . as a result , the source code for third - party libraries that were modified for windows is provided to facilitate compiling and linking alview on windows . the main code for alview , in the file alviewcore.cpp , is written to be portable between operating systems and emphasizes speed of execution . the code can be compiled as a stand - alone executable and must be linked with the zlib2 and samtools1 libraries . sequence reads are processed via custom samtools callback functions arranged in in - memory data structures and represented by an aesthetic , annotated image . the image is then output to the screen as a native graphics object or to the disk as a standard image format file . alview can also be compiled as a webserver daemon that uses the common gateway interface ( cgi)3 standard . the cgi version produces interactive html output and uses dynamic html54 features , including zoom in by selection via a jquery5 library . the cgi webserver alview version loads a list of permitted - to - access bam files from a user - maintained text file ; so custom lists of bam files of interest are easy to generate and use . the source code is free and open to modification so that users and local system operators can implement their own security . the alview cgi webserver version provides modifiable url access , so that , for instance , cells in a spreadsheet can link to viewable results for any sample or location . stand - alone alview accepts parameters that specify bam file name and genomic coordinates . invoking alview in a script can create a slideshow of interesting regions . for example , fields in a single nucleotide polymorphism ( snp ) detection output file can be used to specify a series of calls to alview to generate images for each purported polymorphism or mutation . the burden of reviewing ten and hundreds of thousands of mutation calls can therefore be significantly reduced . the source code is available at github.6 the readme file there points to links for selected executables and complete download packages that include the associated reference genome data . a live webserver version of alview for examining public human cancer short - read datasets is available at https://cgwb.nci.nih.gov/cgi-bin/alview . alview source code and executables for several operating systems are available at the national cancer institute ( nci)/national cancer informatics program s ( ncip s ) github site : https://github.com/ncip/alview . nci retains the copyrights to national cancer institute and associated images , which may not be used in forked projects . alview provides a solid substructure that allows for various types of access to short - read data across different operating systems . figure 1 . demonstrates the various navigation and information buttons available in the web version of alview and shows how selection via mouse provides zoom in capabilities . alview is a trim , fast , precise tool and complements existing programs such as the integrated genomics viewer ( igv),7 bamview,8 and gbrowse 2.0.9 the benefits of alview are extreme speed and a sharp focus on exploring short reads . different implementation philosophies can influence memory usage and performance but provide useful alternative paths to solving similar problems . igv provides much more functionality than alview by supporting many other input file types other than bam sequence read files . igv s java implementation provides write once , run anywhere portability via implementations of the java virtual machine . alview s implementation relies on low - level operating system and native gui toolkit api calls . igv requires registration for download for running off of disk , whereas alview does not . desktop igv may require internet for full , easy , simple operation , whereas alview does not require network connection ( though it may call user - invoked external webpages ) . 7 intel core i52400 cpu at 3.10 ghz and 8 gb ram , restarts of igv v2.3 took from 12 to 18 seconds . restarts of alview took a small fraction of one second . for a small view of a genomic region , the java platform se binary for igv took up 292 mb , while alview took up 11 mb .

the name alview is a contraction of the term alignment viewer . alview is a compiled to native architecture software tool for visualizing the alignment of sequencing data . inputs are files of short - read sequences aligned to a reference genome in the sam / bam format and files containing reference genome data . outputs are visualizations of these aligned short reads . alview is written in portable c with optional graphical user interface ( gui ) code written in c , c++ , and objective - c . the application can run in three different ways : as a web server , as a command line tool , or as a native , gui program . alview is compatible with microsoft windows , linux , and apple os x. it is available as a web demo at https://cgwb.nci.nih.gov/cgi-bin/alview . the source code and windows / mac / linux executables are available via https://github.com/ncip/alview .

Introduction Features and Methods Results

PMC5021116

the data presented here were obtained from an ongoing prospective multicentre clinical study to search for biomarkers of periodontal disease progression . a consecutive sample of 329 participants was recruited between january 2012 and april 2015 at four centres in the united states : the forsyth institute ( cambridge , ma ) , new york university college of dentistry ( new york , ny ) , southern illinois university school of dental medicine ( alton , il ) , and the university at buffalo , state university of new york ( amherst , ny ) . participants were examined clinically by calibrated examiners every 2 months for 12 months to monitor for periodontal disease progression based on cal measurements . the study was approved by the institutional review board at each centre prior to initiation . the inclusion and exclusion criteria can be found in the online supporting information , further details can be obtained at clinicaltrials.gov ( https://clinicaltrials.gov/ct2/home ) under the identifier nct01489839 . from 29,189 replicate sitespecific cal measurements from the 329 participants baseline data , we computed the average standard deviation ( sd ) of cal within each subject and across subjects . data obtained from 16 clinical examiners based on 318,237 replicate measures across all visits were used to calculate intraexaminer agreement . periodontal assessments performed on each subject included up to 168 sites per subject ( 6 sites per tooth mesiobuccal , buccal , distobuccal , mesiolingual , lingual and distolingual for up to 28 teeth excluding third molars ) and included : presence or absence of gingival redness and plaque ; probing depth ( pd ) ; measurement of distance from the cementoenamel junction to the free gingival margin ( b measure ) ( in case of recession , a negative value was assigned ) ; cal ( calculated by subtracting the b measure from the pd ) ; presence or absence of plaque , gingival redness , bop and suppuration . pd and the b measure were measured using a north carolina manual periodontal probe ( pcpunc 15 hufriedy co , chicago , il ) , rounding down to the nearest millimetre and at premolars and the first and second molars these variables were measured twice . cal was calculated for each pass by the electronic data capturing ( edc ) system . if the difference between the 2 measurements was 2 mm , the examiner was prompted by the edc to obtain pd and the b measure a third time . subjects with 6 sites with cumulative loss of attachment 2 mm from baseline during monitoring phase had their monitoring interrupted , and proceeded to treatment . participants displaying 4 mm of cal increase at a given site received periodontal rescue therapy at such sites and continued with monitoring . after the monitoring phase , periodontally healthy subjects received professional dental prophylaxis and exited the study , whereas participants with periodontal disease received nonsurgical mechanical periodontal therapy . the dataset used in this report consisted of participants who had enrolled in the study up until april 12 , 2015 . if a subject received rescue therapy in some but not all sites , data for such sites were removed from the analysis and the subject was otherwise retained in the analysis for any remaining sites . in addition , sites with extreme variations in cal ( i.e. a difference between the minimum cal and maximum cal > 5 mm ) were also excluded . the analyses proceeded in three stages : we ( i ) calculated the observed proportion of sites with progression and regression according to different thresholds ; ( ii ) performed an alternating logistic regressions ( alr ) implementation of the generalized estimating equations ( gee ) procedure to assess whether the proportion of progressing sites changes over time and ( iii ) applied lmms to predict subjectspecific trends in cal for each site and from which classifications of progression and regression were made . stage 1 the observed proportion of sites with changes in cal from the baseline values greater or equal to 1 , 2 and 3 mm , respectively , were summarized for every visit in each clinical group separately . in addition , we also summarized the proportion of progressed sites with reversals at the subsequent visit ( i.e. being progressed a visit j , and then notprogressed at visit j + 1 ) . for these calculations , missing data points in the observed cal measurements were imputed by locf ( lastobservationcarriedforward ) from the previous visit . in contrast , all lmms and the gee analysis described below were conducted without imputation of missing data points . stage 2 the alr ( carey et al . 1993 ) implementation of gee for populationaveraged modelling was performed using all the sites simultaneously to assess whether the populationaveraged proportions of observed cal increases 1 mm from baseline to 2 , 4 , 6 , 8 , 10 and 12 months changed over time , while adjusting for disease cohort ( healthy , mild , severe ) . for additional details on this model stage 3 for each of the 168 tooth sites , a separate linear mixed effects model ( laird & ware 1982 , holditchdavis et al . 1998 ) with a cubic polynomial for time ( months ) was fitted to quantify the course of progression within individuals . specifically , the model for calit , the value of attachment loss from the i subject at time = t ( for t = 0 , 2 , 4 , 6 , 8 , 10 or 12 months ) iscalit=0+b0i+1agei+2femalei+3bcali+(4+b1i)time+(5+b2i)time2+6time3+7bcali2+8ageitime+9femaleitime+10bcalitime+it the model includes fixed effects for age , gender ( with males as the reference group ) , time , timesquared and timecubed , the twoway interactions age by time and gender by time , mean baseline cal for the subject ( bcali ) and its square , as well as the interaction of bcali and ( linear ) time . this is a fairly rich model for fixed effects with respect to inclusion of polynomial and interaction terms . the rationale was to account for betweensubject variability and thereby reduce error in the prediction of subjectspecific trends , which in turn should improve accuracy of classification of withinsubject change . to this end , the model additionally includes random effects b0i , b1i and b2i for subjects , time and timesquared respectively ; these and the random error term ij , are independent and normally distributed with unknown variances . the random effects component for each subject is the difference between the subject 's regression and the populationaveraged regression ( the latter determined from fixed effects ) . this random effects component is a measure of how progression of periodontal disease at the site for each subject systematically differs from the typical course of progression in the whole population after accounting for age , gender and baseline cal . furthermore , each tooth site of each subject has his or her own regression curve for cal given by the sum of the fixed effects and random effects components . from the subjectspecific curves generated from the regression models ( one model per site ) , predicted values of cal were computed at baseline and at 2 , 4 , 6 , 8 , 10 and 12 months for each site and person . model assessment was based on the predicted residual error sum of squares ( press ) residual e(i)t , which is the difference between observed and predicted cal from the ith subject at the tth visit based on the fit of the model that leaves out that subject . the prediction accuracy of the lmm for each site was calculated using two summaries of press residuals , the press statistic ( liu et al . 1999 ) and the sum of absolute predicted residual errors , each divided by the total number of observations across all subjects and visits for the site . these statistics enable identification of sites where prediction of cal is best and where it is poorest ( for additional details , see online supplementary material ) . we developed a threshold for progression empirically based on the prediction standard errors from a second series of lmms ( again , one per site ) fitted to calit , which is the change in cal value from baseline to time = t ( for t = 2 , 4 , 6 , 8 , 10 or 12 months ) for subject i. these models are identical to the models described above , except that the outcome is calij . the threshold for change was based on the 75 percentile of the distribution of the standard errors for subjectspecific predicted calij . sites were then classified as progressing based on the predictions from the first series of lmms using the threshold established from the second series . in particular , considering that the halfwidth of a 95% prediction interval for predicted cal is 1.96q75 ( mm ) , we grouped sites based on changes in pcal ( pcal ) into : ( i ) regressing sites ( pcal < 2q75 ) ; ( ii ) stable sites ( 2q75 pcal 2q75 mm ) ; ( iii ) intermediate sites ( 2q75 < pcal < 4q75 mm ) and ( iv ) progressing sites ( pcal 4q75 ) ( for additional details on this model , see online supplementary material ) . the high threshold of 4q75 for progression was motivated by the desire for high specificity in the classification of progression of periodontitis potentially at the expense of lower sensitivity . to show the effect of classification of sites based upon lmm prediction , the mean observed and predicted cal values were calculated at each visit across all sites and subjects within each category of disease progression . in addition , the observed proportion of sites with changes in observed cal from the baseline values greater or equal to 1 , 2 and 3 mm , respectively , were summarized for every visit in each site category of progression . we also summarized the proportion of progressed sites with reversals at the subsequent visit . to illustrate further the fluctuation of cal over time for individual sites and the smoothing effect of the lmm on the profile of longitudinal changes in cal measurements , we selected a participant ( subject x ) with a large number of sites ( n = 30 ) with increase in cal 2 mm from baseline to 12 months . the observed and estimated values for cal over time and the data presented here were obtained from an ongoing prospective multicentre clinical study to search for biomarkers of periodontal disease progression . a consecutive sample of 329 participants was recruited between january 2012 and april 2015 at four centres in the united states : the forsyth institute ( cambridge , ma ) , new york university college of dentistry ( new york , ny ) , southern illinois university school of dental medicine ( alton , il ) , and the university at buffalo , state university of new york ( amherst , ny ) . participants were examined clinically by calibrated examiners every 2 months for 12 months to monitor for periodontal disease progression based on cal measurements . the study was approved by the institutional review board at each centre prior to initiation . the inclusion and exclusion criteria can be found in the online supporting information , further details can be obtained at clinicaltrials.gov ( https://clinicaltrials.gov/ct2/home ) under the identifier nct01489839 . from 29,189 replicate sitespecific cal measurements from the 329 participants baseline data , we computed the average standard deviation ( sd ) of cal within each subject and across subjects . data obtained from 16 clinical examiners based on 318,237 replicate measures across all visits were used to calculate intraexaminer agreement . periodontal assessments performed on each subject included up to 168 sites per subject ( 6 sites per tooth mesiobuccal , buccal , distobuccal , mesiolingual , lingual and distolingual for up to 28 teeth excluding third molars ) and included : presence or absence of gingival redness and plaque ; probing depth ( pd ) ; measurement of distance from the cementoenamel junction to the free gingival margin ( b measure ) ( in case of recession , a negative value was assigned ) ; cal ( calculated by subtracting the b measure from the pd ) ; presence or absence of plaque , gingival redness , bop and suppuration . pd and the b measure were measured using a north carolina manual periodontal probe ( pcpunc 15 hufriedy co , chicago , il ) , rounding down to the nearest millimetre and at premolars and the first and second molars these variables were measured twice . cal was calculated for each pass by the electronic data capturing ( edc ) system . if the difference between the 2 measurements was 2 mm , the examiner was prompted by the edc to obtain pd and the b measure a third time . subjects with 6 sites with cumulative loss of attachment 2 mm from baseline during monitoring phase had their monitoring interrupted , and proceeded to treatment . participants displaying 4 mm of cal increase at a given site received periodontal rescue therapy at such sites and continued with monitoring . after the monitoring phase , periodontally healthy subjects received professional dental prophylaxis and exited the study , whereas participants with periodontal disease received nonsurgical mechanical periodontal therapy . the dataset used in this report consisted of participants who had enrolled in the study up until april 12 , 2015 . if a subject received rescue therapy in some but not all sites , data for such sites were removed from the analysis and the subject was otherwise retained in the analysis for any remaining sites . in addition , sites with extreme variations in cal ( i.e. a difference between the minimum cal and maximum cal > 5 mm ) were also excluded . the analyses proceeded in three stages : we ( i ) calculated the observed proportion of sites with progression and regression according to different thresholds ; ( ii ) performed an alternating logistic regressions ( alr ) implementation of the generalized estimating equations ( gee ) procedure to assess whether the proportion of progressing sites changes over time and ( iii ) applied lmms to predict subjectspecific trends in cal for each site and from which classifications of progression and regression were made . statistical significance was defined as p < 0.05 throughout . all statistical analyses were conducted using sas software . stage 1 the observed proportion of sites with changes in cal from the baseline values greater or equal to 1 , 2 and 3 mm , respectively , were summarized for every visit in each clinical group separately . in addition , we also summarized the proportion of progressed sites with reversals at the subsequent visit ( i.e. being progressed a visit j , and then notprogressed at visit j + 1 ) . for these calculations , missing data points in the observed cal measurements were imputed by locf ( lastobservationcarriedforward ) from the previous visit . in contrast , all lmms and the gee analysis described below were conducted without imputation of missing data points . stage 2 the alr ( carey et al . 1993 ) implementation of gee for populationaveraged modelling was performed using all the sites simultaneously to assess whether the populationaveraged proportions of observed cal increases 1 mm from baseline to 2 , 4 , 6 , 8 , 10 and 12 months changed over time , while adjusting for disease cohort ( healthy , mild , severe ) . for additional details on this model stage 3 for each of the 168 tooth sites , a separate linear mixed effects model ( laird & ware 1982 , holditchdavis et al . 1998 ) with a cubic polynomial for time ( months ) was fitted to quantify the course of progression within individuals . specifically , the model for calit , the value of attachment loss from the i subject at time = t ( for t = 0 , 2 , 4 , 6 , 8 , 10 or 12 months ) iscalit=0+b0i+1agei+2femalei+3bcali+(4+b1i)time+(5+b2i)time2+6time3+7bcali2+8ageitime+9femaleitime+10bcalitime+it the model includes fixed effects for age , gender ( with males as the reference group ) , time , timesquared and timecubed , the twoway interactions age by time and gender by time , mean baseline cal for the subject ( bcali ) and its square , as well as the interaction of bcali and ( linear ) time . this is a fairly rich model for fixed effects with respect to inclusion of polynomial and interaction terms . the rationale was to account for betweensubject variability and thereby reduce error in the prediction of subjectspecific trends , which in turn should improve accuracy of classification of withinsubject change . to this end , the model additionally includes random effects b0i , b1i and b2i for subjects , time and timesquared respectively ; these and the random error term ij , are independent and normally distributed with unknown variances . the random effects component for each subject is the difference between the subject 's regression and the populationaveraged regression ( the latter determined from fixed effects ) . this random effects component is a measure of how progression of periodontal disease at the site for each subject systematically differs from the typical course of progression in the whole population after accounting for age , gender and baseline cal . furthermore , each tooth site of each subject has his or her own regression curve for cal given by the sum of the fixed effects and random effects components . from the subjectspecific curves generated from the regression models ( one model per site ) , predicted values of cal were computed at baseline and at 2 , 4 , 6 , 8 , 10 and 12 months for each site and person . model assessment was based on the predicted residual error sum of squares ( press ) residual e(i)t , which is the difference between observed and predicted cal from the ith subject at the tth visit based on the fit of the model that leaves out that subject . the prediction accuracy of the lmm for each site was calculated using two summaries of press residuals , the press statistic ( liu et al . 1999 ) and the sum of absolute predicted residual errors , each divided by the total number of observations across all subjects and visits for the site . these statistics enable identification of sites where prediction of cal is best and where it is poorest ( for additional details , see online supplementary material ) . we developed a threshold for progression empirically based on the prediction standard errors from a second series of lmms ( again , one per site ) fitted to calit , which is the change in cal value from baseline to time = t ( for t = 2 , 4 , 6 , 8 , 10 or 12 months ) for subject i. these models are identical to the models described above , except that the outcome is calij . the threshold for change was based on the 75 percentile of the distribution of the standard errors for subjectspecific predicted calij . sites were then classified as progressing based on the predictions from the first series of lmms using the threshold established from the second series . in particular , considering that the halfwidth of a 95% prediction interval for predicted cal is 1.96q75 ( mm ) , we grouped sites based on changes in pcal ( pcal ) into : ( i ) regressing sites ( pcal < 2q75 ) ; ( ii ) stable sites ( 2q75 pcal 2q75 mm ) ; ( iii ) intermediate sites ( 2q75 < pcal < 4q75 mm ) and ( iv ) progressing sites ( pcal 4q75 ) ( for additional details on this model , see online supplementary material ) . the high threshold of 4q75 for progression was motivated by the desire for high specificity in the classification of progression of periodontitis potentially at the expense of lower sensitivity . to show the effect of classification of sites based upon lmm prediction , the mean observed and predicted cal values were calculated at each visit across all sites and subjects within each category of disease progression . in addition , the observed proportion of sites with changes in observed cal from the baseline values greater or equal to 1 , 2 and 3 mm , respectively , were summarized for every visit in each site category of progression . we also summarized the proportion of progressed sites with reversals at the subsequent visit . to illustrate further the fluctuation of cal over time for individual sites and the smoothing effect of the lmm on the profile of longitudinal changes in cal measurements , we selected a participant ( subject x ) with a large number of sites ( n = 30 ) with increase in cal 2 mm from baseline to 12 months . the observed and estimated values for cal over time and the longitudinal changes in cal were then plotted for every progressing site . out of the 533 participants who attended a baseline visit , 51 subjects had their monitoring interrupted due to rescue therapy , whereas 350 had already completed the 12month monitoring ( fig 21 were excluded because of a change in examiner , resulting in 329 participants in the final analysis . periodontally healthy subjects tended to be younger , more likely to be female , and to have fewer missing teeth than subjects with mild or severe periodontal loss ( table 1 ) . one can also observe that subjects in the healthy category presented less plaque , gingival redness , bop and suppuration than the periodontitis groups . periodontally healthy were not necessarily periodontally intact and had an average cal of 1.2 mm . this was the result of our inclusion criteria , which allowed for the presence of recessions , with the intent of allowing for the recruitment of older subjects in this category . among these participants , 48 sites were excluded due to a fluctuation in cal greater than 5 mm and 107 sites were excluded due to rescue therapy for a final number of 52,441 sites included in analyses . flow chart of subject recruitment for the study : 2,533 subjects were telephone screened for this study ; 1,072 subjects were enrolled ( consented ) in the study ; 549 enrolled subjects were deemed eligible for the study after clinical screening ; and 533 subjects attended a baseline visit . of those , 51 subjects were moved to the treatment phase due to rescue therapy and 350 subjects completed their 12month visit by april 12 , 2015 . twentyone of these individuals were excluded due to change in the examiner during the monitoring phase , resulting in 329 subjects ( 93 periodontally healthy ; 113 with mild periodontal loss and 123 with severe periodontal loss ) . demographic and clinical parameters of study subjects in the three clinical categories : periodontally healthy subjects , subjects with mild periodontal loss and subjects with severe periodontal loss aa , african american ; c , caucasian ; nd , not disclosed . the subjectlevel mean sd of duplicate measures was 0.26 mm ( range 0 to 0.79 mm ) . the intraexaminer agreement for the 16 clinical examiners was : exact agreement ( sd ) a total of 2,192 sites ( 4.2% ) that had missing cal data points had data carried forward from the previous visit in the first ( descriptive ) stage of the analysis . within 12 months , overall , 21.2 , 2.8 and 0.3% of sites progressed according to the thresholds of 1 , 2 and 3 mm respectively . the proportion of sites that progressed according to simple thresholds increased over time , with a high proportion of reversals , irrespective of the diagnostic threshold and the clinical group ( table 2 ) . the data indicate a higher proportion of progressing sites in the periodontal disease groups compared to healthy subjects for all three thresholds . the proportion of sites reversing was relatively independent of the duration of followup , though the proportion of sites classified as reversing within a given time period increased greatly as the progression threshold increased . observed percentage of sites with progression and percentage of progressed sites with reversals at the subsequent visit ( i.e. no longer being in the state of progression ) based on changes in cal from baseline greater than or equal to 1 , 2 and 3 mm at each visit ; data for all subjects and stratified for the three clinical groups all three groups healthy , mild and severe periodontal disease experienced a statistically significant increase in progression of periodontitis over time ( fig . 2 ) . plots of probabilities that clinical attachment loss ( cal ) 1 mm against time for the three disease categories : periodontally healthy , mild periodontal loss and severe periodontal loss , calculated using alternating logistic regression . the distribution of mean values ( i.e. minimum , 25 quartile , median , 75 quartile and maximum ) for the press statistics and absolute value of press residuals for each of the 168 sites measured were : 0.51 ; 0.79 ; 0.95 ; 1.22 ; 2.12 and 0.53 ; 0.64 ; 0.71 ; 0.79 ; 1.06 respectively . the latter set of results indicate that for the site with the best prediction , site 445 , the average absolute press residual was 0.53 mm . for the site with the worst prediction , site 273 , the mean lack of fit was 1.06 mm ( see online supplementary material for additional details ) . from the lmm for changes in cal , the 75 percentile for the standard errors of prediction was 0.238 and the width of the 95% prediction interval for predicted cal 0.47 mm ( see online supplementary material ) . using this threshold to classify sites according to their progressing state , progression occurred in 0.2% , 0.9% and 0.7% of the sites for healthy , mild and severe subjects ( table 3 ) . line plots of mean observed and predicted cal ( sd ) for sites in the four categories of progression illustrate how the classification scheme resulted in distinguishable patterns of changes in cal for both observed and predicted values of cal ( fig . the proportion of progressing and reversing sites based on observed cal measurements , stratified for the 4 categories of progression , revealed that progressing sites had the highest proportions of sites crossing the thresholds for cal change and the lowest percentages of reversal from the progressing state ( table 4 ) . one can also observe that for the 1 and 2 mm thresholds , the rates of reversals decreased over time in progressing sites . number of subjects and number and percentage of sites ( in parenthesis ) for each of the four categories of progression from baseline to month 12 with classifications based on linear mixed model predictions of cal for subjects in the three distinct clinical groups thresholds for categories of progression : ( i ) regressing sites ( pcal < 0.47 mm ) ; ( ii ) stable sites ( 0.47 mm pcal 0.47 mm ) ; ( iii ) intermediate sites ( 0.47 mm < pcal < 0.94 mm ) ; and ( iv ) progressing sites ( pcal 0.94 mm ) . line plots of mean changes in clinical attachment loss ( cal ) for observed and predicted values over time for sites grouped in the four categories of progression based in the linear mixed models : regressing 1,918 sites from 238 subjects ; stable 46,661 sites from 329 subjects ; intermediate observed percentage of sites with progression and percentage of progressed sites with reversals at the subsequent visit ( i.e. no longer being in the state of progression ) based on changes in cal from baseline greater than or equal to 1 , 2 and 3 mm at each visit and stratified for the four site categories of progression there were no progressing sites at 10 months and , therefore , no subsequent reversal . using all sites from a single participant ( subject x ) with changes 2 mm at 12 months to illustrate , plotting the observed cal measurements or their changes from baseline does not distinguish profiles for the different sites ( figs s1a and b respectively ) . in contrast , the lmm smoothed the longitudinal profiles of cal measurements , helping to differentiate profiles from different sites ( fig . for instance , the three curves that had the greatest change in pcal , including the one highlighted in red , were the only three classified as being progressed based on the lmm approach ( fig . the subjectlevel mean sd of duplicate measures was 0.26 mm ( range 0 to 0.79 mm ) . the intraexaminer agreement for the 16 clinical examiners was : exact agreement ( sd ) a total of 2,192 sites ( 4.2% ) that had missing cal data points had data carried forward from the previous visit in the first ( descriptive ) stage of the analysis . within 12 months , overall , 21.2 , 2.8 and 0.3% of sites progressed according to the thresholds of 1 , 2 and 3 mm respectively . the proportion of sites that progressed according to simple thresholds increased over time , with a high proportion of reversals , irrespective of the diagnostic threshold and the clinical group ( table 2 ) . the data indicate a higher proportion of progressing sites in the periodontal disease groups compared to healthy subjects for all three thresholds . the proportion of sites reversing was relatively independent of the duration of followup , though the proportion of sites classified as reversing within a given time period increased greatly as the progression threshold increased . observed percentage of sites with progression and percentage of progressed sites with reversals at the subsequent visit ( i.e. no longer being in the state of progression ) based on changes in cal from baseline greater than or equal to 1 , 2 and 3 mm at each visit ; data for all subjects and stratified for the three clinical groups all three groups healthy , mild and severe periodontal disease experienced a statistically significant increase in progression of periodontitis over time ( fig . 2 ) . plots of probabilities that clinical attachment loss ( cal ) 1 mm against time for the three disease categories : periodontally healthy , mild periodontal loss and severe periodontal loss , calculated using alternating logistic regression . the distribution of mean values ( i.e. minimum , 25 quartile , median , 75 quartile and maximum ) for the press statistics and absolute value of press residuals for each of the 168 sites measured were : 0.51 ; 0.79 ; 0.95 ; 1.22 ; 2.12 and 0.53 ; 0.64 ; 0.71 ; 0.79 ; 1.06 respectively . the latter set of results indicate that for the site with the best prediction , site 445 , the average absolute press residual was 0.53 mm . for the site with the worst prediction , site 273 , the mean lack of fit was 1.06 mm ( see online supplementary material for additional details ) . from the lmm for changes in cal , the 75 percentile for the standard errors of prediction was 0.238 and the width of the 95% prediction interval for predicted cal 0.47 mm ( see online supplementary material ) . using this threshold to classify sites according to their progressing state , progression occurred in 0.2% , 0.9% and 0.7% of the sites for healthy , mild and severe subjects ( table 3 ) . line plots of mean observed and predicted cal ( sd ) for sites in the four categories of progression illustrate how the classification scheme resulted in distinguishable patterns of changes in cal for both observed and predicted values of cal ( fig . 3 ) . the proportion of progressing and reversing sites based on observed cal measurements , stratified for the 4 categories of progression , revealed that progressing sites had the highest proportions of sites crossing the thresholds for cal change and the lowest percentages of reversal from the progressing state ( table 4 ) . one can also observe that for the 1 and 2 mm thresholds , the rates of reversals decreased over time in progressing sites . number of subjects and number and percentage of sites ( in parenthesis ) for each of the four categories of progression from baseline to month 12 with classifications based on linear mixed model predictions of cal for subjects in the three distinct clinical groups thresholds for categories of progression : ( i ) regressing sites ( pcal < 0.47 mm ) ; ( ii ) stable sites ( 0.47 mm pcal 0.47 mm ) ; ( iii ) intermediate sites ( 0.47 mm < pcal < 0.94 mm ) ; and ( iv ) progressing sites ( pcal 0.94 mm ) . line plots of mean changes in clinical attachment loss ( cal ) for observed and predicted values over time for sites grouped in the four categories of progression based in the linear mixed models : regressing 1,918 sites from 238 subjects ; stable 46,661 sites from 329 subjects ; intermediate 3,535 sites from 319 subjects ; and progressing 327 sites from 147 subjects . observed percentage of sites with progression and percentage of progressed sites with reversals at the subsequent visit ( i.e. no longer being in the state of progression ) based on changes in cal from baseline greater than or equal to 1 , 2 and 3 mm at each visit and stratified for the four site categories of progression there were no progressing sites at 10 months and , therefore , no subsequent reversal . using all sites from a single participant ( subject x ) with changes 2 mm at 12 months to illustrate , plotting the observed cal measurements or their changes from baseline does not distinguish profiles for the different sites ( figs s1a and b respectively ) . in contrast , the lmm smoothed the longitudinal profiles of cal measurements , helping to differentiate profiles from different sites ( fig . for instance , the three curves that had the greatest change in pcal , including the one highlighted in red , were the only three classified as being progressed based on the lmm approach ( fig . the use of lmms to predict periodontal disease progression showed several advantages over traditional methods described in the literature . first , the lmms could be applied to all visits from all subjects simultaneously while accounting for variation in cal measurements within subjects over time and between subjects . specifically , by introducing subjectspecific intercepts , slopes and quadratic trends ( i.e. random effects ) , the models accounted for the random fluctuations in cal measurements over time , while providing smoothed predicted profiles of sitespecific change in predicted cal for each site . the predictions were additionally based on fixed effects , which are similar to the more familiar coefficients from standard linear regression models . in particular , the model adjusted for potential influences on cal changes from age , gender and baseline mean cal ( subject level ) differences . using a cutoff based on the standard errors of predicted change in cal , we classified a small percentage of sites as progressing . although we can not verify the biological or clinical accuracy of this classification , this proportion agrees with literature suggesting that periodontal disease progression affects a very small percentage of examined sites ( lindhe et al . 1983 , 1989 , 2003 ) . also in accord with the literature , we classified as progressing a higher percentage of sites in subjects with versus without periodontitis ( le et al . different approaches in the literature have been used to overcome the influence of error in measurement of cal in classifying periodontal disease progression ( haffajee et al . most strategies focus on identifying thresholds of change in cal that would compensate for variance in cal assessments , as determined by pairs of crosssectional measurements . however , given the high level of errors in cal measurements in periodontitis subjects , detection of changes in cal are subjected to high rates of falsepositives ( corraini et al . another weakness of this approach is its lack of accounting for longitudinal sources of variation in cal measurements ( espeland et al . an additional challenge is that apparent longitudinal fluctuation in cal that is partly due to measurement error may be indistinguishable from actual reversal in cal . as illustrated by the high proportion of sites demonstrating reversal after crossing the threshold of 3 mm of cal increase in our dataset , raising the threshold for progression does not avoid issues associated with reversal of changes in cal and in fact was associated with a high overall proportion ( 7080% ) classified as reversing within any time interval . indeed , one of the first questions raised after we detected relatively high rates of reversal was if disease progression had occurred at all in this population . to globally address this issue we tested if the proportion of sites crossing certain thresholds of increases in cal would accumulate over time the use of alr provided strong evidence that the percentage of sites with increases in cal accumulated over time in all clinical groups . that the apparent high rate of reversal did not nullify increases in cal noteworthy , when the proportions of sites crossing the preset thresholds of cal increase and the rates of reversal were computed for sites stratified in the four categories of progression , progressing sites had the highest proportions of sites crossing the three thresholds of increases in cal and the lowest rates of reversal . these results indicate that our classification of progression was able to identify sites with a high likelihood of having undergone irreversible progression . because the diagnosis of periodontal disease progression relies on changes in cal , which are known to have many sources of error ( corraini et al . 2013 ) , we made every effort to minimize the error in the cal measurements . values for the average sd of duplicate sitespecific cal measurements reported in the literature have varied from 0.63 mm ( zappa et al . our results compared favourably with these figures with an overall mean sd of replicate cal measurements of only 0.28 mm . this could be partly explained by the inclusion of periodontally healthy subjects in this study population ( most studies examined only subjects with periodontitis ) and a lower level of disease in the subjects with periodontitis . some investigators have reported intraexaminer cal or relative attachment level agreement 1.0 mm for replicate measurements of cal varying from 93% ( baelum et al . perhaps the main limitation of the analysis was that it was impossible to determine the diagnostic reliability of the proposed method , as the true diagnosis of disease progression remains unknown . in fact , because we can not separate biologically or clinically meaningful fluctuation from measurement error , it is possible that we smoothed relevant changes in cal along with noise . this problem is not unique to the proposed approach , and we anticipate that as the field accrues more wellcollected longitudinal data , we can begin to develop gold standard measures of progression . further , computational difficulties associated with model complexity , specifically with respect to the number of variance components requiring estimation , prohibited the simultaneous fitting of all sites . nonetheless , the lmm applied to individual sites leveraged repeated measurements data from all participants and should better account for errors in cal measurements than previously proposed approaches . in summary , the lmm accounted for several sources of error in longitudinal cal measurement with the goal of enabling a more accurate identification of progressing sites . the results corroborate previous investigations suggesting that the diagnosis of disease progression based on a pair of visits is prone to a high rate of false positives . figure s1 . ( a ) line plot of clinical attachment loss ( cal ) measurements for 30 sites with changes 2 mm in observed cal measurements from baseline to 12 months from a single participant ( subject x ) . ( c ) predicted cal values from the linear mixed models for the same 30 sites over time . the blue profile highlights an example of a site classified as stable , whereas the red profile illustrates a progressing site based in the lmms . log odds ratio estimates ( standard errors ) from the alr analysis with dichotomous outcome cal 1 mm versus cal < 1 mm with change relative to baseline cal . sites with the highest and the lowest mean absolute press residuals . table s4 .

abstractaimthe goal of this study was to identify progressing periodontal sites by applying linear mixed models ( lmm ) to longitudinal measurements of clinical attachment loss ( cal).methodsninetythree periodontally healthy and 236 periodontitis subjects had their cal measured bimonthly for 12 months . the proportions of sites demonstrating increases in cal from baseline above specified thresholds were calculated for each visit . the proportions of sites reversing from the progressing state were also computed . lmm were fitted for each tooth site and the predicted cal levels used to categorize sites regarding progression or regression . the threshold for progression was established based on the modelestimated error in predictions.resultsover 12 months , 21.2% , 2.8% and 0.3% of sites progressed , according to thresholds of 1 , 2 and 3 mm of cal increase . however , on average , 42.0% , 64.4% and 77.7% of progressing sites for the different thresholds reversed in subsequent visits . conversely , 97.1% , 76.9% and 23.1% of sites classified as progressing using lmm had observed cal increases above 1 , 2 and 3 mm after 12 months , whereas mean rates of reversal were 10.6% , 30.2% and 53.0% respectively.conclusion lmm accounted for several sources of error in longitudinal cal measurement , providing an improved method for classifying progressing sites .

Material and Methods Study design Study population Standard deviations of duplicate measures of CAL and intraexaminer reproducibility Clinical examination Rescue therapy Subjects and sites included in analyses Data analyses Results Standard deviations of duplicate measures of CAL and intraexaminer reproducibility Proportion of sites with progression and reversals Alternating logistic regressions Linear mixed models for CAL measurements Discussion Supporting information

PMC4864530

the open window theory implies that there is altered immunity following an acute bout of exercise , which lasts from three to 72 hours after exercise depending on the parameter measured . according to the theory , there is an increased risk of contracting infectious diseases after physical exercise . the j means that individuals engaged in moderate physical activity are at lower risk compared with sedentary individuals . on the other side , excessive volumes of strenuous endurance exercise it is evident that great numbers of molecules encompass the term proinflammatory cytokines . these cytokines are produced very early in the response to multiple stresses and are important by being involved in both innate and acquired immunity . cytokines are characterised as being inducible and belong to different families , including proinflammatory cytokines ( il-1 , il-2 , il-4 , il-6 , il-8 , and tnf ) , which are cytokines that promote systemic inflammation , and anti - inflammatory cytokines ( il-5 , il-6 , and il-10 ) , which refer to the property of a substance that reduces and protects against inflammation . inflammation in excess is detrimental and excessive production and release of tnf and il-1 may lead to pathology . vascular endothelial growth factor ( vegf ) is a signal protein produced by cells that stimulates vasculogenesis and angiogenesis . proinflammatory cytokines like il-1 , il-6 , il-8 , tgf- , and tnf- variably increase vegf expression , depending upon the dose , cell , or tissue type . surgery , trauma , burn , and sepsis induce a pattern of hormonal and immunological responses with the same response profile as that of exercise . earlier studies regarding exercise and effects on immunological parameters indicate that the profiles of proinflammatory and anti - inflammatory cytokine levels are stimulated based on the intensity and duration of the physical exercise [ 8 , 9 ] . long - distance runners , for example , marathon runners , have shown high postexercise levels of il-1 , il-6 , and il-8 after six hours ' endurance exercise [ 911 ] , while there were no changes observed in plasma cytokine levels after a 5-kilometre run . measurements from cycle ergometer and treadmill running show no response of short - term exercise with high and moderate intensity , an increase in il-6 after one - hour cycling at 75% of maximal oxygen consumption ( vo2max ) and three - hours alternating cycling and treadmill running at 6065% of vo2max showed high postexercise values of il-6 , il-1 , and tnf [ 1315 ] . other studies indicate that long - term sleep and energy deficiency lead to immune depression [ 16 , 17 ] . microsphere - based multiplexing bioassay system has , during the last few years , become an important tool in cytokine detection . the assay is performed with great speed and accuracy by making use of hundreds of specially prepared magnetic beads , or microspheres , and is reporting on the contents of the sample . this is in contrast to the classical analysis of cytokine expression patterns , which has been performed by enzyme - linked immunosorbent assays ( elisa ) for each separate analysis . very few studies within the field of exercise immunology have so far used the multiplex technology in cytokine detection in relation to exercise or physical activity executed by healthy people . according to prior reports , we expect in our study that the marathon / half - marathon race and the ranger - training course will lead to immunological responses in the subjects as seen in surgery and sepsis patients . the aim of this study was to examine the secretion of the cytokines il-1 , il-2 , il-4 , il-5 , il-6 , il-8 , il-10 , il-12 , tnf , ifn , gm - csf , and vegf before and after three different trials of physical exercise : the oslo marathon / half - marathon and a military training course of eight days continuous exercise combined with sleep and energy deprivation . both the spontaneous secretion of the cytokines and the lipopolysaccharide- ( lps- ) induced whole blood stimulation of cytokines were examined . fourteen men participating in oslo marathon race ( mean age 40 ; range 2956 ) , eight women ( mean age 36 ; range 2739 ) and eight men ( mean age 34 ; range 3045 ) participating in oslo half - marathon race , and ten physically well - trained male cadets from the norwegian military academy in the age between 21 and 28 years were recruited to this study . the marathon runners were selected based on the previous year 's result list with the criterion that the expected running time should be 3 h 30 min ; the corresponding criterion for the half - marathon runners was 1 h 30 min for men and 1 h 45 min for women . the ranger - training course took place in the eastern part of norway , in a forest area at 500 m altitude . the temperature during the eight days of the course was between 18 and 30c during daytime and between 5 and 15c at night . the cadets were provided nearly no food during the course and approximately 3 hours/8 d sleep was allowed . in the restitution period ( day 9 through day 11 ) , the cadets followed regular sleeping and eating habits as before the course . the subjects were informed about the study and gave their written informed consent for participation . for the marathon runners and the half - marathon runners , blood samples were collected shortly before the race and immediately after the race . for the military cadets , blood samples were collected at the start of the ranger - training course , after 2 , 4 , and 8 days ( end of the ranger - training course ) , and after one ( day 9 ) and three ( day 11 ) days ' recovery . the blood samples from the cadets were taken between 6 and 8 a.m. for all groups , venous blood was sampled into either edta- , na - citrate- , or heparin - anticoagulated vacuum tubes ( becton dickinson , plymouth , uk ) . the blood samples from the ranger - training course were collected in the field , kept on ice , and transported within 60 minutes to the nearest military training camp , terningmoen , where a temporary laboratory was established . the blood samples from the marathon and the half - marathon runners where collected in the start area of the race , kept on ice , and transported within 30 minutes to ullevl university hospital . plasma was separated at 2500 g for 10 minutes and samples were stored at 80c until analysis . to compare the cytokine production capacities of leukocytes , one set of experiments was performed with heparinized whole blood from the half - marathon runners and the ranger - training cadets . the blood samples were stimulated ex vivo with the potent cytokine inducer lipopolysaccharide ( lps ) , 1 g / ml ( working solution ) ( from escherichia coli 026 : b6 , sigma st . louis , mo , usa ) for 6 h at 37c , before storing plasma samples at 80c until analysis . white blood cell ( wbc ) , platelet and erythrocyte counts , haemoglobin , and haematocrit were assessed in edta blood using the technicon h2 system ( bayer corporation , tarrytown , ny , usa ) at the department of medical biochemistry , oslo university hospital , ullevl . plasma levels of individual cytokines ( il-1 , il-6 , il-8 , il-10 , and tnf ) were measured using the quantikine colorimetric sandwich enzyme - linked immunosorbent assays ( elisa ) from r&d , abingdon , uk , according to the manufacturer 's instructions . we used the multiplex bead - based sandwich immunoassay technology ( luminex , austin , tx , usa ) and a human cytokine 12-plex kit ( r&d ) , strictly following the manufacturer 's instructions , to measure the concentration of the following cytokines before and after the marathon race : il-1 , il-2 , il-4 , il-5 , il-6 , il-8 , il-10 , il-12 , tnf , ifn , gm - csf , and vegf . unfortunately , we only had the multiplex bead - based sandwich immunoassay technology ( luminex ) available for the study with the marathon runners . the levels of cytokine release in the half - marathon runners and the cadets from the ranger - training course were therefore measured using the quantikine colorimetric sandwich enzyme - linked immunosorbent assays ( elisa ) . results are given as means and standard errors of the mean ( mean , sem ) . difference within groups ( marathon and half - marathon ) was analysed using two - tailed paired sample t - test . independent sample t - test was used in order to compare men and women within the half - marathon group . since we did not observe any statistically significant differences between the women and men , we have chosen to present the results from the half - marathon runners as one group . paired sample t - test was used to compare results from day 0 to results after 2 , 4 , and 8 days during the ranger - training course , and after day 1 and 3 days recovery . in the ranger - training course the repeated and longitudinal values obtained before , during , and after the period of exercise which made the subjects being their own controls . the total leukocyte counts ( wbc ) ( 10/l ) increased by 3.2-fold ( values before : 5.1 , values after : 16.4 , p < 0.01 ) after the marathon race , 2.4-fold ( values before : 6.3 , values after : 15.2 , p < 0.01 ) after the half - marathon race , and 1.5-fold ( values before : 6.3 , values after : 9.5 , p < 0.01 ) at the end of the ranger - training course ( day 8) . after the ranger - training course , wbc were back to normal values after one - day ( day 9 ) recovery . the increase in wbc was mainly due to a significant increase in neutrophils after both of the marathon races and after the ranger - training course , but monocyte counts were also consistently increased in all three experimental settings . the platelet counts ( 10/l ) increased significantly after both the marathon race ( values before : 194 , values after : 243 , p < 0.01 ) and the half - marathon race ( values before : 255 , values after : 313 , p < 0.05 ) and after eight days at the ranger - training course ( values before : 214 , values after : 265 , p < 0.05 ) . we observed no changes in haemoglobin values after the marathon and the half - marathon races . a slight decrease in haemoglobin values was observed after eight days in the military training course ( p < 0.05 ) , maintained during the whole recovery period . the haematocrit values remained unchanged after the running races , whereas the values were reduced during days 48 of the ranger - training course as well as during the recovery phase . we performed a plain analysis of plasma levels of 12 cytokines in 14 individuals before and after a marathon race . three of these cytokines were heavily increased when comparing pre / post plasma levels : il-6 ( 26-fold increase , p < 0.001 ) , il-10 ( 28-fold increase , p < 0.000 ) , and il-8 ( 11-fold increase , p < 0.001 ) . the other measured cytokines , il-1 , il-2 , il-4 , il-5 , il-12 , tnf , ifn , gm - csf , and vegf , did not change significantly after the marathon race . half - marathon ( table 1 ) . in the half - marathon race we demonstrated significantly increased plasma levels of il-6 ( 40-fold increase , p < 0.001 ) , il-10 ( 10-fold increase , p < 0.001 ) , and il-8 ( 14-fold increase , p < 0.001 ) , whereas il-1 and tnf were unchanged after the race . when examining lps - stimulated cytokine levels , il-6 , il-1 , and tnf were reduced after the race ( il-6 45% reduced , p < 0.001 ; il-1 24% reduced , p < 0.05 ; and tnf 43% reduced , p < 0.001 ) . on the contrary , il-10 and il-8 were further increased when lps stimulation was superposed ; il-10 225% increased , p < 0.01 , and il-8 242% increased , p < 0.001 . ranger - training course ( tables 2 and 3 ) . the subjects of the ranger - training course showed smaller increase than in the half - marathon race with surprisingly highest levels in the postrace recovery period ( table 2 ) . il-1 , il-6 , il-8 , il-10 , and tnf were all significantly increased at some stage during the course . additional investigations including lps stimulation of whole blood showed reduced production capacity for il-1 , il-6 , and tnf , whereas the il-8 and il-10 levels were further increased in a similar manner as after the half - marathon ( table 3 ) . several studies have observed leukocytosis after physical exercise [ 2 , 18 , 19 ] . physical exercise leads to an excessive increase in total number of neutrophils immediately and is followed by a slightly smaller increase a few hours after the exercise . in our study , the blood samples were taken immediately after the exercise and only the initial increase in leukocytes was measured . we observed a 3.2-fold and a 2.4-fold increase in leukocytes after the marathon race and the half - marathon race , respectively . at the end of the ranger - training course ( day 8) , the levels of leukocytes had increased by 1.5-fold . in agreement with former studies we found that the increase in leukocyte counts is primarily due to increase in neutrophils , although monocytes and lymphocytes were increased as well . the first increase in neutrophils is mainly due to release of marginated cells caused by mechanical shear stress and catecholamines . the reason for the different levels of leukocytes in the three exercise trials is probably due to the type and the duration of the activity , and this corresponds to earlier studies [ 23 , 24 ] . acute exercise in healthy individuals has an effect on cytokines and the inflammatory response as well . the intensity , duration , and type of physical exercise can all influence proinflammatory and anti - inflammatory cytokines and thereby influence the susceptibility to inflammatory diseases . [ 4 , 25 ] reported a 2-fold increase in tnf and il-1 after a marathon race , in addition to a nearly 100-fold increase in il-6 values . exercise studies of three hours ' duration performed in laboratories have shown increase in the same parameters . we observed after the marathon and the half - marathon races a 26-fold and a 40-fold increase in il-6 . the magnitude of increase in il-6 is closely related to the duration of the activity , the age of the athlete , the body mass index ( bmi ) , and the physical condition of the person [ 13 , 14 ] . the participants in our trials were moderate to well - trained individuals executing physical exercise of long duration . the half - marathon runners were on average 5 years younger compared to the marathon runners , they run half the distance of a marathon race , and they probably ran the half - marathon race with higher heart rate compared to the level of intensity of the marathon runners . our study did not separately measure the effect of running intensity and running duration on the plasma cytokine levels after marathon and half - marathon running . for the marathon runners we observed 11-fold and 28-fold increase in il-8 and il-10 , respectively , but no change in tnf and il-1. in the half - marathon runners , we observed a 10-fold increase in il-10 and 14-fold increase in il-8 , but as for the marathon race , no changes in il-1 and tnf values were recorded . it seems that tnf and il-1 do not increase exponentially during exercise , which is different when compared to infections . it could be hypothesized that the high levels of the anti - inflammatory cytokines il-6 and il-10 in our study might prevent the production of tnf- and il-1 after the races . il-6 is called an inflammatory responding cytokine described as a proinflammatory cytokine but can also stimulate anti - inflammatory cytokines . il-6 is highly increased during strenuous physical exercise and thus takes part in the control of the inflammatory response to strenuous physical exercise [ 25 , 28 ] . the proinflammatory tnf is known to be inhibited by il-10 and induced by different stressors [ 30 , 31 ] . the values obtained after physical exercise are inconstant related to the variations in duration and intensity of the exercise tasks . in the ranger - training course , there was no increase in il-6 or il-10 during the period of activity , but , surprisingly , a small increase in the recovery phase ( days 9 and 11 ) . il-1 and il-8 showed a small increase during the course and in the recovery period . the boosting of cytokine responses in peripheral blood leukocytes by lps in many ways mimics the initial innate immune response to bacterial infection , lps stimulation showed that the plasma levels of both il-6 , il-1 , and tnf were significantly reduced after the half - marathon race as well as during and after the ranger - training course . who have observed reduced il-6 , il-1 , and tnf- levels in lps - stimulated samples 30 min and 3 h after exercise in half - marathon runners . the observed reduction after the half - marathon race and the ranger - training course may be due to either actually reduced production capacity as a result of exercise or at least a temporary change in the regulatory pathways leading to altered cytokine synthesis and release . in contrast , il-8 and il-10 were boosted both by the exercise itself and after additional lps stimulation . it seems that the anti - inflammatory il-10 production is more triggered in the long - lasting marathon race ( 28-fold increase ) compared with the shorter workload of the half - marathon race ( 10-fold increase ) . furthermore , the il-6 release was more pronounced in the more work - intensive half - marathon than the longer - lasting but less intensive marathon race ( 40-fold versus 26-fold increase ) , possibly being mainly due to a larger release from the muscle mass of the lower extremities . although a similar cytokine profile was registered in the ranger - training course , the magnitude of cytokine increases was not as impressive as for the corresponding marathon races , again possible due to a lesser work load per time unit as well as the lower work intensity . regardless , we have in all three experimental settings demonstrated an increase of proinflammatory and anti - inflammatory cytokines , which in many ways are similar to the cytokine profile , which characterises inflammatory and infectious diseases [ 8 , 27 ] . the balance between proinflammatory and anti - inflammatory cytokines during exercise may lay the ground for potentiation of the inflammatory status by introducing additional coincidental pathogens of both gram - negative and gram - positive origin , which thereby can tilt the balance into overt disease . both lps of gram - negative bacteria and lipoteichoic acid ( lta ) , peptidoglycan , and purified protein derivative ( ppd ) of gram - positive bacteria all have a number of biological activities , including also being potent cytokine inducers [ 35 , 36 ] . most of the studies do only look at the direct immunological responses occurring immediately after different types of physical activity . the very new part in our study is the three days follow - up after the end of the ranger - training course . it may , however , be noted that the training course was performed at a season of the year where the general burden of common cold or influenza - like disease is at a minimum . in conclusion , our study of long - term physical exercise performed in three different trials showed a significant increase in plasma levels of the cytokines il-6 , il-8 , and il-10 , whereas tnf and il-1 levels were unchanged or reduced . open window of altered immunity after long - term physical exercise and which lasts for at least 72 hours after the exercise . the results support the j - curve theory ; athletes are more sensitive to pathogens in the immediate period after intensive physical exercise , probably due to the exercise - induced inflammatory state that can exaggerate the response to coincidentally occurring pathogens .

the open window theory indicates altered immunity 3 to 72 hours after exercise . the j - curve describes the risk of illness in response to exercise . the aim of this study was to examine the secretion of proinflammatory and anti - inflammatory cytokines before and after long - term strenuous exercise . fourteen marathon and 16 half - marathon runners and 10 military cadets participating in a military ranger - training course were recruited to this study . within - subject design was used measuring levels of plasma cytokines before , during , and after exercise . plasma cytokines were measured using luminex multiplex technology and elisa . comparing pre / post plasma levels both the marathon- and the half - marathon runners showed heavily increased levels of il-6 , il-10 , and il-8 ( p < 0.001 ) . lps stimulation among the half - marathon runners decreased the postrace levels of il-6 , il-1b , and tnf by 45% , 24% , and 43% , respectively ( p < 0.01 ) . during the ranger training course the spontaneous and lps - stimulated levels of il-6 , il-8 , il-10 , il-1b , and tnf changed in a similar fashion as in the half - marathon runners although the fluctuations were smaller . our study supports the open window and the j - curve theory ; the immune system is more activated and the subjects are more threatened to infectious pathogens after intensive physical activity and in the period after exercise .

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

PMC4376303

depression is a common mental health problem in older adults , not only affecting mood but also leading to functional decline in those affected . it reduces quality of life and well - being , and increases the risk of disability , suicide , and mortality . moreover , it has a significant impact on families and health care organizations.1 the world health organization projects that depression could become the single highest contributor to the global disease burden by 2030.2 management of depression in older adults is critical , and strategies to prevent and reduce depressive symptoms are urgently needed . physical activity , being a noninvasive and low - risk behavior , is an alternative or complementary intervention that can be used to prevent and treat depressive symptoms.3,4 physical activity ( pa ) is defined as any bodily movement produced by skeletal muscles that results in energy expenditure , and can be categorized as occupational , sporting , conditioning , household , or other activity.5 it is generally considered an effective intervention for decreasing the prevalence of depressive symptoms and promote their remission.613 however , this conclusion is based mainly on results from studies in the general population6,7 or in special populations such as adolescents,8 postnatal women,9 menopausal women,10 immigrants,11 and patients with certain conditions such as type 2 diabetes or obesity undergoing bariatric surgery,12,13 all of whom are likely to derive a benefit . in older adults , whose ability and practice for pa is age - related decline,14 it is controversial about whether changed patterns of pa ( eg , intensity weakens , duration shortens , or frequency reduces ) are still effective to maintain mental health.4 there are some evidence in support of , as follows . firstly , cross - sectional studies have found an inverse correlation between pa and symptoms of depression in older adults,1517 and agree that engaging in more pa is associated with a lower risk of such symptoms . a nonclinical study by salguero et al in 436 older spanish people aged 6098 years concluded that pa was related to both the physical and mental components of health - related quality of life and the risk of depressive symptoms ( odds ratio [ or ] 0.155 , p<0.01).15 another nonclinical study in the usa , conducted as part of the national social life , health and aging project in 1,349 older people aged 6585 years , showed that pa in later life was correlated inversely with symptoms of depression ( r=0.30 , p<0.001).16 a nonclinical study in 2,727 older subjects ( 65 years ) from the taiwan national health interview survey by chen et al established that leisure time pa was associated with fewer depressive symptoms . compared with participants expending 2,000 + kcal / week on pa during leisure time , the risk of experiencing depressive symptoms was significantly higher in those expending 1999 kcal / week ( or 2.06 , p<0.01 ) and 0 kcal / week ( or 3.72 , p<0.001).17 secondly , many longitudinal studies have confirmed that engaging in pa can reduce the incidence of depressive symptoms in older adults , as well as ameliorate these symptoms or increase the likelihood of their remission.1820 lucas et al conducted a prospective , 10-year clinical and nonclinical study in 49,821 older american women aged 5075 years , and found that higher levels of pa were associated with a lower incidence of depression , and the multivariate relative risk comparing the highest level ( 90 minutes / day ) with the lowest level ( < 10 minutes / day ) of pa was 0.80 ( p<0.001).18 ku et al conducted an 11-year , nonclinical follow - up study in 1,160 older taiwanese adults aged 67 years from the taiwan s health and living status of the elderly survey , and found that initial levels of pa were negatively associated with changes in depressive symptoms ( =0.34 , p<0.05).19 however , the conclusions of longitudinal studies are not always consistent . a 2-year nonclinical follow - up study by walker et al in 909 older australians aged 6074 years reported that pa did not decrease depressive symptoms at any point in time ( f=1.65 , p=0.177).20 thirdly , interventional trials have confirmed that pa is effective in reducing depressive symptoms or enhancing the effect of antidepressant medication in older adults.2125 pereira et al conducted a randomized controlled nonclinical trial in 451 older brazilian women aged 6589 years , and found a significant difference in pre - intervention and post - intervention geriatric depression scale scores in both muscle strength exercise and aerobic exercise ( f=38.18 , p=0.001 ) , indicating that the effects of both exercise protocols were comparable with regard to decreasing depressive symptoms ( f=1.76 , p=0.185).21 other research , such as a nonclinical study by lincoln et al in older american patients with diabetes ( n=58 , 65 years , f=20.38 , p<0.0001)22 and another by williams and tappen in older american patients with alzheimer s disease ( n=45 , 71101 years , f=3.26 , p=0.0492)23 also demonstrated a positive effect of pa training on depressive symptoms . in addition , a nonclinical study of qigong by tsang et al ( n=82 , 65 years , f=2.619 , p=0.041)24 in chinese hong kong and a clinical study of tai chi by lavretsky et al ( n=112 , 60 years , f=2.26 , p<0.05)25 in american patients with major depression were demonstrated to have the same effects . with advances in research on the antidepressant effects of pa , the potential for its use as a treatment for depression is being increasingly recognized . however , there were still challenging results of studies questioning the effects of pa . an important nonclinical randomized controlled trial in the uk published recently in the lancet showed no statistically significant benefit of pa on depression in 891 elderly care home residents aged 65 years ( or 0.76 , p>0.05 ) , and concluded that moderately intense exercise did not reduce depressive symptoms.26 lastly , there have been some positive systematic review findings and guideline recommendations made regarding pa for health purposes in the aged population.2731 a systematic review by bridle et al of the effect of exercise on depressive symptoms in older adults concluded that : for older people who present with clinically meaningful symptoms of depression , prescribing structured exercise tailored to individual ability will reduce depression severity.27 guidelines from the american college of sports medicine and the american heart association on pa for older adults recommend : a minimum of 30 minutes on 5 days each week for moderate - intensity aerobic pa , or a minimum of 20 minutes on 3 days each week for vigorous - intensity aerobic pa.28 in korea , the recommendation from the national council of sports for pa is at least 30 minutes of at least moderate activity on three or more days a week.29 most recommendations reached a consensus that at least moderate - intensity pa has a positive effect on general health . however , a nonclinical study by loprinzi in 708 american older adults ( aged 65 years ) found that both light ( or 0.80 , p=0.01 ) and moderate to vigorous ( or 0.78 , p=0.01 ) intensity pa were inversely associated with depression , and confirmed that even light - intensity pa can have a positive effect on depressive symptoms.30 a systematic review of pa and prevention of depression by mammen highlighted that there is promising evidence that any level of pa , including low levels ( eg , walking up to 150 minutes / weeks ) , can prevent future depression , with study population samples ranging in age from 11 to 100 years.31 to summarize , in spite of the challenge of some studies showing a moderate at best or no statistically significant effect of pa on depression in older adults,26 pa is expected to becoming accepted as a low - risk and highly cost - effective intervention for depressive symptoms . pa could be promoted as a public health strategy to modify depressive symptoms in later life . the usa and europe have undertaken a number of epidemiological and experimental investigations of pa and depressive symptoms in the elderly.15,16,18,22,23,25,26,28,30 korea,29,32 japan,33,34 and taiwan17,19 have also increased their research efforts in this area . however , such studies in the people s republic of china are lacking . to address this gap in the literature , we performed a study in a large cohort of older chinese veterans to investigate the relationship between pa and depressive symptoms in a nationally representative epidemiological project with adjustment for a comprehensive range of confounders . a cross - sectional study was conducted from 2009 to 2011 in 277 military communities located in 18 cities nationwide across the people s republic of china , as a part of the chinese veteran clinical research project investigating chronic noncommunicable diseases in older chinese veterans.35 the study was approved by the ethics committee of the chinese pla general hospital ( 07bjz04 ) , and by the ethical review boards in each of the participating districts . veterans aged 60 years who had lived continuously in the community for at least 1 month and agreed to participate in the survey were enrolled . the cvcr protocol is described in detail elsewhere.35 this paper only describes the methods used to investigate pa and depressive symptoms . a two - stage , stratified , cluster sampling approach was applied . a structured questionnaire was used during a routine , door - to - door census . in the first stage , information on sociodemographics ( age , sex , ethnicity , education , marital status , region of residence , living status ) , lifestyle ( pa , hobbies and interests , smoking , alcohol consumption , daily care , participation in social activity ) , adverse experiences ( negative life events , electromagnetic field exposure ) , and medical conditions ( self - rated health , chronic disease , cognitive function ) was collected . in the second stage , depression status was evaluated using the center for epidemiological studies depression scale ( ces - d ) . the data were obtained directly from subjects if they were cognitively intact or from an informant , who was usually a closely related caregiver , with respect to the subject s condition when the subject was cognitively impaired . the level of self - reported depressive symptoms was measured using the chinese version of the ces - d , which has been evaluated for reliability and validity36 and extensively utilized in epidemiological surveys in the chinese population.3739 the ces - d is a 20-item self - reported instrument , comprising 16 negative affect and four positive affect items , such as i felt depressed , participants were asked about the number of days on which they experienced every effect item during the previous week . respondents reported the frequency of occurrence of each negative affect item on a four - point scale : 0 ( rarely or none of the time ; less than 1 day ) , 1 ( some of the time ; 12 days ) , 2 ( much or a moderate amount of the time ; 34 days ) , or 3 ( most or all of the time ; 57 days ) . higher scores on the ces - d indicate more depressive symptoms . a standardized ces - d score of 15 is considered normal , a score 20 indicates definitive depressive symptoms , and a score > 15 and < 20 indicates possible depressive symptoms . in our study , cases with depressive symptoms included both definitive and possible ones . questions about participation in leisure activities , including pa and participation in social activities , as well as hobbies and interests , were introduced into the structured questionnaire . information on pa was self - reported using a one - year pa questionnaire , which was self - administered and convenient to use,34,40,41 and generally eliminated the influence of season and weather as well as temporary inpatient stays or accident - related events . subjects or their caregivers were asked to respond to whether or not the subject regularly participated in any sport or exercise activities in the last one year with a yes if they partook in pa lasting 30 minutes a day on 3 days a week ( active ) , otherwise as no ( inactive ) . the intensity of pa was identified with self - sensations during pa , both of which were accordant . these included a self - detected increase in heart rate , respiration rate , sweating , and muscle fatigue . 60% to 80% of maximal heart rate ( 220 actual age ) , indicates moderate - intensity pa . according to the above , each type of pa was rated as light intensity ( self - reported slightly increased heart rate and respiration rate , insignificant sweating , no perceived muscle fatigue , and actual heart rate < 60% of maximal heart rate during pa ) or moderate - vigorous intensity ( marked increase in heart rate and respiration rate , significant sweating , perceived muscle fatigue , and actual heart rate 60% of maximal heart rate during pa ) . if a participant only engaged in light - intensity pa , they were categorized as light intensity . if a participant engaged in both light and moderate vigorous intensity pa , or only engaged in moderate - vigorous intensity pa , they were categorized as moderate - vigorous intensity . hobbies and interests were selected from a list , including calligraphy , painting , photography , collectibles , rearing animals , handcrafts , reading , writing a diary , creative writing , watching television / listening to the radio , and playing cards / mahjong . smoking was defined as continuous or cumulative smoking of at least one cigarette a day for more than 6 months during life . negative life events that may affect mental state were elicited , including combat experience , persecution in the chinese cultural revolution , bereavement , and economic hardship . occupational electromagnetic field exposure was sought by questioning for a history of working with radar , communications , missiles , or electricity . self - rated health was evaluated by a standardized multichoice question , how would you rate your health on a three - point scale ( good , moderate , or poor ) . chronic diseases were identified by trained researchers , using a combination of clinical assessment , self - reporting , and medical chart review and were recorded as yes or no . cognitive function was assessed using a comprehensive test battery , and classified as cognitively normal , mild cognitive impairment , or dementia , according to neuropsychological test scores , such as mini - mental state examination , montreal cognitive assessment , and ability to perform activity of daily living . all data were double - entered and verified in epidata ( version 3.1 , the epidata association , odense , denmark ) . descriptive statistics were used to characterize the sample . in univariate analyses ( unadjusted model ) , pearson s chi - squared test , correction for continuity , or fisher s exact probability test were performed as appropriate to calculate the associations for categorical variables , including that between pa and depressive symptoms , to identify underlying correlates . multivariate logistic regression was used to adjust for any of the factors that reached a p - value 0.20 in univariate analyses , to control for possible confounding effects . a univariate general linear model was used , with ces - d score as the dependent variable , age as the covariate , and the aforementioned categorized factors as independent variables , while adjusting for potential confounders . all statistical analyses were performed using statistical package for the social sciences version 18.0 ( spss inc . , a cross - sectional study was conducted from 2009 to 2011 in 277 military communities located in 18 cities nationwide across the people s republic of china , as a part of the chinese veteran clinical research project investigating chronic noncommunicable diseases in older chinese veterans.35 the study was approved by the ethics committee of the chinese pla general hospital ( 07bjz04 ) , and by the ethical review boards in each of the participating districts . veterans aged 60 years who had lived continuously in the community for at least 1 month and agreed to participate in the survey were enrolled . the cvcr protocol is described in detail elsewhere.35 this paper only describes the methods used to investigate pa and depressive symptoms . a two - stage , stratified , cluster sampling approach was applied . a structured questionnaire was used during a routine , door - to - door census . in the first stage , information on sociodemographics ( age , sex , ethnicity , education , marital status , region of residence , living status ) , lifestyle ( pa , hobbies and interests , smoking , alcohol consumption , daily care , participation in social activity ) , adverse experiences ( negative life events , electromagnetic field exposure ) , and medical conditions ( self - rated health , chronic disease , cognitive function ) was collected . in the second stage , depression status was evaluated using the center for epidemiological studies depression scale ( ces - d ) . the data were obtained directly from subjects if they were cognitively intact or from an informant , who was usually a closely related caregiver , with respect to the subject s condition when the subject was cognitively impaired . the level of self - reported depressive symptoms was measured using the chinese version of the ces - d , which has been evaluated for reliability and validity36 and extensively utilized in epidemiological surveys in the chinese population.3739 the ces - d is a 20-item self - reported instrument , comprising 16 negative affect and four positive affect items , such as i felt depressed , participants were asked about the number of days on which they experienced every effect item during the previous week . respondents reported the frequency of occurrence of each negative affect item on a four - point scale : 0 ( rarely or none of the time ; less than 1 day ) , 1 ( some of the time ; 12 days ) , 2 ( much or a moderate amount of the time ; 34 days ) , or 3 ( most or all of the time ; 57 days ) . higher scores on the ces - d indicate more depressive symptoms . a standardized ces - d score of 15 is considered normal , a score 20 indicates definitive depressive symptoms , and a score > 15 and < 20 indicates possible depressive symptoms . in our study , cases with depressive symptoms included both definitive and possible ones . questions about participation in leisure activities , including pa and participation in social activities , as well as hobbies and interests , were introduced into the structured questionnaire . information on pa was self - reported using a one - year pa questionnaire , which was self - administered and convenient to use,34,40,41 and generally eliminated the influence of season and weather as well as temporary inpatient stays or accident - related events . subjects or their caregivers were asked to respond to whether or not the subject regularly participated in any sport or exercise activities in the last one year with a yes if they partook in pa lasting 30 minutes a day on 3 days a week ( active ) , otherwise as no ( inactive ) . the intensity of pa was identified with self - sensations during pa , both of which were accordant . these included a self - detected increase in heart rate , respiration rate , sweating , and muscle fatigue . 60% to 80% of maximal heart rate ( 220 actual age ) , indicates moderate - intensity pa . according to the above , each type of pa was rated as light intensity ( self - reported slightly increased heart rate and respiration rate , insignificant sweating , no perceived muscle fatigue , and actual heart rate < 60% of maximal heart rate during pa ) or moderate - vigorous intensity ( marked increase in heart rate and respiration rate , significant sweating , perceived muscle fatigue , and actual heart rate 60% of maximal heart rate during pa ) . if a participant only engaged in light - intensity pa , they were categorized as light intensity . if a participant engaged in both light and moderate vigorous intensity pa , or only engaged in moderate - vigorous intensity pa , they were categorized as moderate - vigorous intensity . hobbies and interests were selected from a list , including calligraphy , painting , photography , collectibles , rearing animals , handcrafts , reading , writing a diary , creative writing , watching television / listening to the radio , and playing cards / mahjong . smoking was defined as continuous or cumulative smoking of at least one cigarette a day for more than 6 months during life . daily care requirement and social participation were recorded as yes or no . negative life events that may affect mental state were elicited , including combat experience , persecution in the chinese cultural revolution , bereavement , and economic hardship . occupational electromagnetic field exposure was sought by questioning for a history of working with radar , communications , missiles , or electricity . self - rated health was evaluated by a standardized multichoice question , how would you rate your health on a three - point scale ( good , moderate , or poor ) . chronic diseases were identified by trained researchers , using a combination of clinical assessment , self - reporting , and medical chart review and were recorded as yes or no . cognitive function was assessed using a comprehensive test battery , and classified as cognitively normal , mild cognitive impairment , or dementia , according to neuropsychological test scores , such as mini - mental state examination , montreal cognitive assessment , and ability to perform activity of daily living . the level of self - reported depressive symptoms was measured using the chinese version of the ces - d , which has been evaluated for reliability and validity36 and extensively utilized in epidemiological surveys in the chinese population.3739 the ces - d is a 20-item self - reported instrument , comprising 16 negative affect and four positive affect items , such as i felt depressed , participants were asked about the number of days on which they experienced every effect item during the previous week . respondents reported the frequency of occurrence of each negative affect item on a four - point scale : 0 ( rarely or none of the time ; less than 1 day ) , 1 ( some of the time ; 12 days ) , 2 ( much or a moderate amount of the time ; 34 days ) , or 3 ( most or all of the time ; 57 days ) . the four positive affect items were reversed when using the four - point scale . higher scores on the ces - d indicate more depressive symptoms . a standardized ces - d score of 15 is considered normal , a score 20 indicates definitive depressive symptoms , and a score > 15 and < 20 indicates possible depressive symptoms . in our study , cases with depressive symptoms included both definitive and possible ones . questions about participation in leisure activities , including pa and participation in social activities , as well as hobbies and interests , were introduced into the structured questionnaire . information on pa was self - reported using a one - year pa questionnaire , which was self - administered and convenient to use,34,40,41 and generally eliminated the influence of season and weather as well as temporary inpatient stays or accident - related events . subjects or their caregivers were asked to respond to whether or not the subject regularly participated in any sport or exercise activities in the last one year with a yes if they partook in pa lasting 30 minutes a day on 3 days a week ( active ) , otherwise as no ( inactive ) . the intensity of pa was identified with self - sensations during pa , both of which were accordant . these included a self - detected increase in heart rate , respiration rate , sweating , and muscle fatigue . 60% to 80% of maximal heart rate ( 220 actual age ) , indicates moderate - intensity pa . according to the above , each type of pa was rated as light intensity ( self - reported slightly increased heart rate and respiration rate , insignificant sweating , no perceived muscle fatigue , and actual heart rate < 60% of maximal heart rate during pa ) or moderate - vigorous intensity ( marked increase in heart rate and respiration rate , significant sweating , perceived muscle fatigue , and actual heart rate 60% of maximal heart rate during pa ) . if a participant only engaged in light - intensity pa , they were categorized as light intensity . if a participant engaged in both light and moderate vigorous intensity pa , or only engaged in moderate - vigorous intensity pa , they were categorized as moderate - vigorous intensity . subjects were canvassed directly for their sociodemographic data . hobbies and interests were selected from a list , including calligraphy , painting , photography , collectibles , rearing animals , handcrafts , reading , writing a diary , creative writing , watching television / listening to the radio , and playing cards / mahjong . smoking was defined as continuous or cumulative smoking of at least one cigarette a day for more than 6 months during life . daily care requirement and social participation were recorded as yes or no . negative life events that may affect mental state were elicited , including combat experience , persecution in the chinese cultural revolution , bereavement , and economic hardship . occupational electromagnetic field exposure was sought by questioning for a history of working with radar , communications , missiles , or electricity . self - rated health was evaluated by a standardized multichoice question , how would you rate your health on a three - point scale ( good , moderate , or poor ) . chronic diseases were identified by trained researchers , using a combination of clinical assessment , self - reporting , and medical chart review and were recorded as yes or no . cognitive function was assessed using a comprehensive test battery , and classified as cognitively normal , mild cognitive impairment , or dementia , according to neuropsychological test scores , such as mini - mental state examination , montreal cognitive assessment , and ability to perform activity of daily living . all data were double - entered and verified in epidata ( version 3.1 , the epidata association , odense , denmark ) . descriptive statistics were used to characterize the sample . in univariate analyses ( unadjusted model ) , pearson s chi - squared test , correction for continuity , or fisher s exact probability test were performed as appropriate to calculate the associations for categorical variables , including that between pa and depressive symptoms , to identify underlying correlates . multivariate logistic regression was used to adjust for any of the factors that reached a p - value 0.20 in univariate analyses , to control for possible confounding effects . a univariate general linear model was used , with ces - d score as the dependent variable , age as the covariate , and the aforementioned categorized factors as independent variables , while adjusting for potential confounders . all statistical analyses were performed using statistical package for the social sciences version 18.0 ( spss inc . , 8,742 had data available for analysis of depressive symptoms , giving a response rate of 90.35% . the median age was 82.29 ( interquartile range 80.2584.60 ) years , and ranged from 60 to 105 years . the sample comprised predominantly the oldest old ( 80 years , 70.7% ) , males ( 95.7% ) , people of han ethnicity ( 97.9% ) , the more higher educated ( secondary or above education , 60.6% ) , married and remarried ( 86.2% ) , those living in the eastern part of the people s republic of china ( 70.7% ) , and those living with family / other ( 97.1% ) . a greater proportion of the sample participated in pa ( 85.3% ) , with hobbies and interests of reading ( 92.0% ) and watching television / listening to radio ( 94.7% ) , were never smokers ( 57.5% ) , never drinkers ( 52.6% ) , did not require assistance with self - care ( 85.9% ) , did not actively participate in social activities ( 60.4% ) , had good self - rated health ( 55.2% ) , and had not experienced negative life events ( 84.6% ) or electromagnetic field exposure ( 90.9% ) . the most common chronic diseases were prostatic hyperplasia ( 77.3% ) , coronary heart disease ( 68.8% ) , hypertension ( 67.3% ) , and cataract ( 56.3% ) . almost half the sample ( 49.1% ) had some degree of cognitive decline . the overall prevalence of depressive symptoms in the sample was 6.74% ( 95% confidence interval 6.247.24 ) , which is notably lower than the 40.3% ( 95% confidence interval 38.741.8 ) reported previously for the general older chinese population.39 sensitivity analyses were performed to examine the influence of missing data , and little s missing completely at random test indicated that the missing pattern was random ( p>0.05 ) and would be negligible . with regard to leisure time pa , the participants engaged predominantly in light - intensity pa ( 81.84% ) , such as walking , biking , dancing , gardening , gate ball , billiards , qigong , and tai chi . a few participants engaged in moderate - vigorous - intensity pa ( 18.16% ) , such as running , jogging , swimming , tennis , ping pong ball , and working out at the gym . many variables were found as to be potentially associated with depressive symptoms in older veterans , and ten were demonstrated to be independently associated with depressive symptoms in adjusted analyses with multivariate logistic regression , as shown in table 2 . with regard to the main focus of this study , in an unadjusted model with univariate analyses , pa ( defined as 90 minutes a week ) was significantly associated with a decreased likelihood of depressive symptoms ( 5.43% versus 18.83% , p<0.0001 ) . after adjusted analyses with multivariate logistic regression for controlling confounders , pa was still inversely associated with depressive symptoms , and was the only independent protective factor identified in this study ( or 0.57 , 95% confidence interval 0.440.72 , p<0.0001 ) . the risk of prevalent depressive symptoms among subjects who were physically active ( pa 90 minutes a week ) was decreased by 43% when compared with those who were physically inactive ( pa < 90 minutes a week ) . moreover , there was a significant difference in ces - d scores between the physically active and inactive groups of pa in the univariate general linear model ( f=59.07 , p<0.0001 ) , as shown in table 3 . in contrast , hobbies and interests as calligraphy , painting , photography , collectibles , rearing animals , handcrafts , reading , writing a diary , creative writing , watching television / listening to the radio , and playing cards / mahjong , all categorized as relatively sedentary behavior , were not associated with a decrease in depressive symptoms in unadjusted or adjusted analyses ( see table 2 ) . there were no significant difference in ces - d scores between groups with these hobbies and interests and those without in the univariate general linear model ( see table 3 ) . 8,742 had data available for analysis of depressive symptoms , giving a response rate of 90.35% . the median age was 82.29 ( interquartile range 80.2584.60 ) years , and ranged from 60 to 105 years . the sample comprised predominantly the oldest old ( 80 years , 70.7% ) , males ( 95.7% ) , people of han ethnicity ( 97.9% ) , the more higher educated ( secondary or above education , 60.6% ) , married and remarried ( 86.2% ) , those living in the eastern part of the people s republic of china ( 70.7% ) , and those living with family / other ( 97.1% ) . a greater proportion of the sample participated in pa ( 85.3% ) , with hobbies and interests of reading ( 92.0% ) and watching television / listening to radio ( 94.7% ) , were never smokers ( 57.5% ) , never drinkers ( 52.6% ) , did not require assistance with self - care ( 85.9% ) , did not actively participate in social activities ( 60.4% ) , had good self - rated health ( 55.2% ) , and had not experienced negative life events ( 84.6% ) or electromagnetic field exposure ( 90.9% ) . the most common chronic diseases were prostatic hyperplasia ( 77.3% ) , coronary heart disease ( 68.8% ) , hypertension ( 67.3% ) , and cataract ( 56.3% ) . almost half the sample ( 49.1% ) had some degree of cognitive decline . the overall prevalence of depressive symptoms in the sample was 6.74% ( 95% confidence interval 6.247.24 ) , which is notably lower than the 40.3% ( 95% confidence interval 38.741.8 ) reported previously for the general older chinese population.39 sensitivity analyses were performed to examine the influence of missing data , and little s missing completely at random test indicated that the missing pattern was random ( p>0.05 ) and would be negligible . with regard to leisure time pa , the participants engaged predominantly in light - intensity pa ( 81.84% ) , such as walking , biking , dancing , gardening , gate ball , billiards , qigong , and tai chi . a few participants engaged in moderate - vigorous - intensity pa ( 18.16% ) , such as running , jogging , swimming , tennis , ping pong ball , and working out at the gym . many variables were found as to be potentially associated with depressive symptoms in older veterans , and ten were demonstrated to be independently associated with depressive symptoms in adjusted analyses with multivariate logistic regression , as shown in table 2 . with regard to the main focus of this study , in an unadjusted model with univariate analyses , pa ( defined as 90 minutes a week ) was significantly associated with a decreased likelihood of depressive symptoms ( 5.43% versus 18.83% , p<0.0001 ) . after adjusted analyses with multivariate logistic regression for controlling confounders , pa was still inversely associated with depressive symptoms , and was the only independent protective factor identified in this study ( or 0.57 , 95% confidence interval 0.440.72 , p<0.0001 ) . the risk of prevalent depressive symptoms among subjects who were physically active ( pa 90 minutes a week ) was decreased by 43% when compared with those who were physically inactive ( pa < 90 minutes a week ) . moreover , there was a significant difference in ces - d scores between the physically active and inactive groups of pa in the univariate general linear model ( f=59.07 , p<0.0001 ) , as shown in table 3 . in contrast , hobbies and interests as calligraphy , painting , photography , collectibles , rearing animals , handcrafts , reading , writing a diary , creative writing , watching television / listening to the radio , and playing cards / mahjong , all categorized as relatively sedentary behavior , were not associated with a decrease in depressive symptoms in unadjusted or adjusted analyses ( see table 2 ) . there were no significant difference in ces - d scores between groups with these hobbies and interests and those without in the univariate general linear model ( see table 3 ) . the results of this study confirm and extend previous research by demonstrating an inverse relationship between pa and depressive symptoms in a large cohort of older chinese veterans . various correlations with depressive symptoms were sought , and pa was found to the only factor protecting against symptoms of depression . even after controlling for a range of confounders , such as age , sex , variations in lifestyle , negative life events , the most common chronic diseases , and level of cognitive function , the relationship between pa and depressive symptoms remained significant ( or 0.57 , 95% confidence interval 0.440.72 , p<0.0001 ) . the independent protective effect of pa on depressive symptoms was far stronger than any of the other healthy lifestyle components , such as the above - mentioned hobbies and interests , social participation , not smoking , and abstaining from alcohol , which were not found to be associated with decreased depressive symptoms . the median ( interquartile range ) age of the sample was 82.29 ( 80.2584.60 ) , making it highly representative of older - old people , and even the oldest - old . there was an indication that the antidepressant effect of pa also extended to the oldest - old , although many researchers thought it difficult to get positive result of pa antidepressant effect in the oldest - old.4 the majority of study participants engaged in light pa ; active pa was defined as 90 minutes a week , so pa in this study would have been mainly light intensity . it was shown that light - intensity pa was probably also protective against depressive symptoms , even though the guidelines for pa in older adults mainly recommend pa of at least moderate intensity . the mechanisms that may account for the inverse association between pa and depressive symptoms are not completely understood , but there are several plausible hypotheses . the neurobiological hypothesis suggests that pa may stimulate synthesis , release , and metabolism of serotonin and -endorphins , increase neurotransmission of norepinephrine , attenuate the response of hypothalamic - pituitary - adrenal axis to reduce levels of cortisol , a stress hormone , stimulate growth of new nerve cells in the hippocampus , and regulate cerebral blood flow , to modify the biochemical and neuroanatomical changes that occur with depression.4244 the psychological hypothesis suggests that pa may provide a sense of enjoyment and achievement , provide more opportunities for social interaction , provide a distraction from worries and negative thoughts , enhance self - confidence , self - esteem , and self - determination , and improve self - efficacy.42,45,46 further , it should also be considered that pa for older adults yields benefits that extend beyond depression , such as maintenance of functional independence , which might in turn contribute to less likelihood of depression . factors such as functional independence are generally considered to be a mediator between pa and depression , and could help us to investigate potential mechanisms that may account for the effect of pa on depression . a majority of the sample participated in active pa ( 85.3% ) , which was encouraging . this may reflect the predominance of men in the sample , who usually do less housework and spend more time outdoors than women . another important driver of pa in this age group is the social support received from veterans communities , such as health education , and provision of accessible places and convenient facilities for pa , which spurs people into action . as demonstrated by previous studies , although the trend of pa is usually downward with advancing age in older adults,14 pa has been generally considered to have an inverse correlation with depressive symptoms and to be effective in reduce the risk of such symptoms.15,20 our study found similarly encouraging evidence . both with and without adjustment , there was a decreased risk of prevalent depressive symptoms in physically active subjects compared with their physically inactive counterparts . moreover , of all the factors independently associated with depressive symptoms , we found that pa was the only protective one , even in the oldest - old and when of light intensity . thus , differences in the ability to engage in pa ( eg , very old age , requiring assistance with self - care , multiple comorbidities , and cognitive dysfunction are likely to hamper the ability to perform pa ) did not appear to explain the difference in depressive symptoms between physically active elderly and physically inactive elderly . social support has been demonstrated to be advantageous in improving mental health in the older population , and may be a confounder.16,47 all our older veterans enjoy chinese military welfare and are generally provided with optimal health care and social support , including free medicine , regular physical examinations , health management , better social insurance , a good socioeconomic level , good housing , and convenient facilities . there was almost no difference in social support between our physically active and inactive subjects , so the influence of social support on the relationship between pa and depressive symptoms is likely to be negligible . it is suggested that any difference may result from physiological and psychological differences derived from pa , or from other pleasurable pursuits available to physically active subjects . the level of social support was not measured , and some pleasurable activities were not considered , and these need to be studied further . it is often questioned whether pa has a beneficial effect on depressive symptoms in the oldest - old . a paper by steffens published in the lancet pointed out that the lack of consistency of results across studies on this topic results at least in part from the difference in mean age between samples.4 a study sample with a mean age < 75 years is more representative of younger old people , and is likely to yield a significant result ; however , a study sample with a mean age of > 80 years often fails to do so . our sample had a median age of 82.29 years , so mostly represented the oldest - old . however , we obtained a surprising result , in that pa was found to protect against depressive symptoms even in the oldest - old . the distinctive condition was inferred to result from the large sample size of the oldest - old ( 6,069 , 70.7% ) making relatively weak statistical effect more powerful . most recommendations have reached a consensus that moderate - vigorous - intensity pa has a generally beneficial effect on health.28,29 in this study , light - intensity pa , such as walking , was more popular among subjects , and was also shown to be achievable for depressive symptoms . the effect of light - intensity pa in older adults was a distinctive finding and has not been fully studied , except for one study conducted by loprinzi that found light - intensity pa was inversely associated with depressive symptoms in older american adults.30 another finding that supervised low - intensity exercise was more effective in reducing depressive symptoms than moderate - intensity or high - intensity exercise in a meta - analysis of outcomes of pa interventions among healthy adults48 also does not support the common contention that insufficient pa intensity was the reason for poor outcomes.28 it has been hypothesized that moderate - intensity to vigorous - intensity pa is required for general health ( as per the recommendations ) , whereas light - intensity pa is probably enough for mental health . further , light - intensity pa may elicit immediate positive feelings that may not be as apparent with more vigorous pa.3 this deserves more investigation , and if confirmed , promotion of light - intensity pa may be more practical for older adults . it is stressed that hobbies and interests such as calligraphy , painting , photography , collectibles , rearing animals , handcrafts , reading , writing a diary , creating writing , listening to the radio , and playing cards were not found to have an association with depressive symptoms in this study , although they were thought to be good for maintaining mental health . it is speculated these pastimes are sedentary behaviors , so have poorer physiological effects compared with pa . multi - tasking ( eg , listening to the radio while walking , performing stretches while watching television , going outdoors for painting or photography , visiting exhibitions of calligraphy or collectibles , going to the library or reading room to read , going to a senior center or community center to play cards ) is suggested for the elderly to decrease their sedentary behavior and increase pa at the same time , which should be feasible for older adults . here we report the first and largest national epidemiological study in the older chinese population that explores the relationship between pa and depressive symptoms , and its results are encouraging . the major strengths of this study are its large sample size and high response rate , resulting in good statistical power . another of its unique characteristics of significantly predominant proportion and absolute number of the oldest - old and the light - intensity pa patterns provided stronger evidence to pa protective effect on depressive symptoms in the two special conditions . first , pa was not measured with a high - quality assessment , so that it may be relatively less reliable . this should be done using either an objective measure ( eg , monitor , accelerometer , pedometer ) or a more specific questionnaire ( eg , the community healthy activities model program for seniors questionnaire,49 the international physical activity questionnaire,50 the global physical activity questionnaire51 ) , all of which result in a quantified level of pa . in this study , pa was categorized as being of light intensity in general , instead of accurate calculation of its energy expenditure . some other studies have reported the level of pa as estimation of energy expenditure in metabolic equivalents of task,52,53 or classified it as the low , moderate , or high intensity according to the total pa score shown as continuous variable . the second limitation of this study concerns its design ; as with other self - reported questionnaire surveys , there is the possibility of recall and reporting bias . given the cross - sectional nature of our study , causality between pa or other factors and depressive symptoms could not be inferred . other variables that may be possible confounders need to be further explored in a future study . because of the widely differing sociodemographic backgrounds between older veterans and the general population of older adults , the generalizability of our study findings to the aged population in general is uncertain . the results of this study confirm and extend previous research by demonstrating an inverse relationship between pa and depressive symptoms in a large cohort of older chinese veterans . various correlations with depressive symptoms were sought , and pa was found to the only factor protecting against symptoms of depression . even after controlling for a range of confounders , such as age , sex , variations in lifestyle , negative life events , the most common chronic diseases , and level of cognitive function , the relationship between pa and depressive symptoms remained significant ( or 0.57 , 95% confidence interval 0.440.72 , p<0.0001 ) . the independent protective effect of pa on depressive symptoms was far stronger than any of the other healthy lifestyle components , such as the above - mentioned hobbies and interests , social participation , not smoking , and abstaining from alcohol , which were not found to be associated with decreased depressive symptoms . the median ( interquartile range ) age of the sample was 82.29 ( 80.2584.60 ) , making it highly representative of older - old people , and even the oldest - old . there was an indication that the antidepressant effect of pa also extended to the oldest - old , although many researchers thought it difficult to get positive result of pa antidepressant effect in the oldest - old.4 the majority of study participants engaged in light pa ; active pa was defined as 90 minutes a week , so pa in this study would have been mainly light intensity . it was shown that light - intensity pa was probably also protective against depressive symptoms , even though the guidelines for pa in older adults mainly recommend pa of at least moderate intensity . the mechanisms that may account for the inverse association between pa and depressive symptoms are not completely understood , but there are several plausible hypotheses . the neurobiological hypothesis suggests that pa may stimulate synthesis , release , and metabolism of serotonin and -endorphins , increase neurotransmission of norepinephrine , attenuate the response of hypothalamic - pituitary - adrenal axis to reduce levels of cortisol , a stress hormone , stimulate growth of new nerve cells in the hippocampus , and regulate cerebral blood flow , to modify the biochemical and neuroanatomical changes that occur with depression.4244 the psychological hypothesis suggests that pa may provide a sense of enjoyment and achievement , provide more opportunities for social interaction , provide a distraction from worries and negative thoughts , enhance self - confidence , self - esteem , and self - determination , and improve self - efficacy.42,45,46 further , it should also be considered that pa for older adults yields benefits that extend beyond depression , such as maintenance of functional independence , which might in turn contribute to less likelihood of depression . factors such as functional independence are generally considered to be a mediator between pa and depression , and could help us to investigate potential mechanisms that may account for the effect of pa on depression . a majority of the sample participated in active pa ( 85.3% ) , which was encouraging . this may reflect the predominance of men in the sample , who usually do less housework and spend more time outdoors than women . another important driver of pa in this age group is the social support received from veterans communities , such as health education , and provision of accessible places and convenient facilities for pa , which spurs people into action . as demonstrated by previous studies , although the trend of pa is usually downward with advancing age in older adults,14 pa has been generally considered to have an inverse correlation with depressive symptoms and to be effective in reduce the risk of such symptoms.15,20 our study found similarly encouraging evidence . both with and without adjustment , there was a decreased risk of prevalent depressive symptoms in physically active subjects compared with their physically inactive counterparts . moreover , of all the factors independently associated with depressive symptoms , we found that pa was the only protective one , even in the oldest - old and when of light intensity . thus , differences in the ability to engage in pa ( eg , very old age , requiring assistance with self - care , multiple comorbidities , and cognitive dysfunction are likely to hamper the ability to perform pa ) did not appear to explain the difference in depressive symptoms between physically active elderly and physically inactive elderly . social support has been demonstrated to be advantageous in improving mental health in the older population , and may be a confounder.16,47 all our older veterans enjoy chinese military welfare and are generally provided with optimal health care and social support , including free medicine , regular physical examinations , health management , better social insurance , a good socioeconomic level , good housing , and convenient facilities . there was almost no difference in social support between our physically active and inactive subjects , so the influence of social support on the relationship between pa and depressive symptoms is likely to be negligible . it is suggested that any difference may result from physiological and psychological differences derived from pa , or from other pleasurable pursuits available to physically active subjects . the level of social support was not measured , and some pleasurable activities were not considered , and these need to be studied further . it is often questioned whether pa has a beneficial effect on depressive symptoms in the oldest - old . a paper by steffens published in the lancet pointed out that the lack of consistency of results across studies on this topic results at least in part from the difference in mean age between samples.4 a study sample with a mean age < 75 years is more representative of younger old people , and is likely to yield a significant result ; however , a study sample with a mean age of > 80 years often fails to do so . our sample had a median age of 82.29 years , so mostly represented the oldest - old . however , we obtained a surprising result , in that pa was found to protect against depressive symptoms even in the oldest - old . the distinctive condition was inferred to result from the large sample size of the oldest - old ( 6,069 , 70.7% ) making relatively weak statistical effect more powerful . most recommendations have reached a consensus that moderate - vigorous - intensity pa has a generally beneficial effect on health.28,29 in this study , light - intensity pa , such as walking , was more popular among subjects , and was also shown to be achievable for depressive symptoms . the effect of light - intensity pa in older adults was a distinctive finding and has not been fully studied , except for one study conducted by loprinzi that found light - intensity pa was inversely associated with depressive symptoms in older american adults.30 another finding that supervised low - intensity exercise was more effective in reducing depressive symptoms than moderate - intensity or high - intensity exercise in a meta - analysis of outcomes of pa interventions among healthy adults48 also does not support the common contention that insufficient pa intensity was the reason for poor outcomes.28 it has been hypothesized that moderate - intensity to vigorous - intensity pa is required for general health ( as per the recommendations ) , whereas light - intensity pa is probably enough for mental health . further , light - intensity pa may elicit immediate positive feelings that may not be as apparent with more vigorous pa.3 this deserves more investigation , and if confirmed , promotion of light - intensity pa may be more practical for older adults . it is stressed that hobbies and interests such as calligraphy , painting , photography , collectibles , rearing animals , handcrafts , reading , writing a diary , creating writing , listening to the radio , and playing cards were not found to have an association with depressive symptoms in this study , although they were thought to be good for maintaining mental health . it is speculated these pastimes are sedentary behaviors , so have poorer physiological effects compared with pa . multi - tasking ( eg , listening to the radio while walking , performing stretches while watching television , going outdoors for painting or photography , visiting exhibitions of calligraphy or collectibles , going to the library or reading room to read , going to a senior center or community center to play cards ) is suggested for the elderly to decrease their sedentary behavior and increase pa at the same time , which should be feasible for older adults . here we report the first and largest national epidemiological study in the older chinese population that explores the relationship between pa and depressive symptoms , and its results are encouraging . the major strengths of this study are its large sample size and high response rate , resulting in good statistical power . another of its unique characteristics of significantly predominant proportion and absolute number of the oldest - old and the light - intensity pa patterns provided stronger evidence to pa protective effect on depressive symptoms in the two special conditions . first , pa was not measured with a high - quality assessment , so that it may be relatively less reliable . this should be done using either an objective measure ( eg , monitor , accelerometer , pedometer ) or a more specific questionnaire ( eg , the community healthy activities model program for seniors questionnaire,49 the international physical activity questionnaire,50 the global physical activity questionnaire51 ) , all of which result in a quantified level of pa . in this study , pa was categorized as being of light intensity in general , instead of accurate calculation of its energy expenditure . some other studies have reported the level of pa as estimation of energy expenditure in metabolic equivalents of task,52,53 or classified it as the low , moderate , or high intensity according to the total pa score shown as continuous variable . the second limitation of this study concerns its design ; as with other self - reported questionnaire surveys , there is the possibility of recall and reporting bias . given the cross - sectional nature of our study , causality between pa or other factors and depressive symptoms could not be inferred . other variables that may be possible confounders need to be further explored in a future study . because of the widely differing sociodemographic backgrounds between older veterans and the general population of older adults , the generalizability of our study findings to the aged population in general is uncertain . this nationally epidemiological study demonstrated an inverse relationship between pa and depressive symptoms in a large cohort of older chinese veterans living in the community . of all the variables studied , pa was found to be the only independent factor protecting against depressive symptoms , after controlling for a range of confounders . it also indicated that the antidepressant effect of pa probably extended to the oldest - old , and the light - intensity pa was probably available for the same protective effect . although there are some limitations to this research , the findings are encouraging and could provide useful information for further intervention protocols to prevent or reduce depressive symptoms in older chinese veterans living in the community . in particular , it might inspire research concerning the impact of light - intensity pa on depressive symptoms in the oldest - old . the conclusion can not be extended beyond the study population , but it highlights a direction for further research . pa may be a good way of preventing and treating depressive symptoms and be of great importance in reducing the burden on public health .

backgroundphysical activity is generally considered to be effective in reducing the prevalence of depression and promoting remission of its symptoms . however , large - scale epidemiological research on this issue is lacking in older chinese adults . we performed a nationwide epidemiological survey to determine the relationship between physical activity and depressive symptoms in older chinese veterans in the community , with adjustment for potential confounders.methodsa cross - sectional study was conducted in a representative sample of 9,676 community - dwelling older chinese veterans . depressive symptoms were identified using the center for epidemiological studies depression scale . physical activity was self - reported using a one - year physical activity questionnaire . information about covariates was obtained by questionnaire - based interview . relationships between study variables and symptoms of depression were estimated using unadjusted and adjusted analyses.resultsthe median age was 82.29 ( interquartile range 80.2584.60 ) years . in total , 81.84% of the study participants engaged in physical activity that was predominantly light in intensity . in unadjusted analyses , physical activity was associated with a significantly decreased likelihood of depressive symptoms ( 5.43% versus 18.83% , p<0.0001 ) . multivariate logistic regression with adjustment and controlling for confounders , physical activity was still inversely associated with depressive symptoms and was the only independent protective factor ( odds ratio 0.57 , 95% confidence interval 0.440.72 , p<0.0001 ) among the associated factors in this study . in a univariate general linear model , there was a significant difference in center for epidemiological studies depression scale score between subjects participating in active physical activity and those who did not ( f=59.07 , p<0.0001).conclusionthis study found an inverse relationship between physical activity and symptoms of depression in older chinese veterans in the community . it was also indicated that the antidepressant effect of physical activity probably extended to the oldest - old , and the light - intensity physical activity was probably available for the same protective effect . this information could be used to devise further interventions to prevent or ameliorate symptoms of depression .

Introduction Subjects and methods Study design Participants and procedure Measurement of depressive symptoms Assessment of physical activity Covariates Statistical analyses Results Description of sample Physical activity and depressive symptoms Discussion Key findings Mechanisms and analyses Strengths and limitations Conclusion

PMC3881768

in the last few years , numerous new dental restorative materials have been introduced in response to an increasing demand for esthetic and biocompatible materials . the high - strength all - ceramic materials that are currently used in dentistry consist of alumina , zirconia , pressed , castable or machinable glass ceramics . several developments have taken place in these areas resulting in the production of ceramic materials for clinical use . these include the aluminous porcelain crown ( vitadur , vita zahnfabrik , bad sckingen , baden - wrttemberg , germany ) , the non shrink ceramic crown ( cerestore , johnson and johnson , east windsor , nj , usa ) , the castable mica glass - ceramic crown ( dicor , caulk / dentsply , milford , de , usa ) and the leucite - reinforced glass ceramics ( ips empress , ivoclar vivadent , schaan , liechtenstein ) . all these all - ceramic systems exhibit low flexural strengths ( 100 - 150 mpa ) , which make them at risk of failure when used for the construction of either posterior crowns or fixed partial dentures . due to the relatively low strength of the early types of ceramics employed in the conventional porcelain jacket crowns , an alumina - reinforced porcelain core material was developed by mclean for the fabrication of such crowns . a veneer porcelain placed on a core containing approximately 50% fused alumina crystals , compared to the conventional feldspathic porcelain level of about 19% , resulted in a dental ceramic with flexural strength from 100 to 130 mpa . the popularity of high - strength ceramic systems is increasing , and the range of their clinical indications is expanding constantly . lithium disilicate ceramics ( ips empress 2 , ivoclar vivadent , schaan , liechtenstein ) , infiltrated alumina ceramic ( in - ceram alumina , vita zahnfabrik , bad sckingen , baden - wrttemberg , germany ; turkom - cera fused alumina , turkom - ceramic ( m ) sdn . puchong , selangor , malaysia ) , densely sintered aluminum oxide ceramic ( procera ) , and zirconium oxide ceramic ( procera allzirkon , nobel biocare , gteborg , sweden ; lava , 3 m espe , st . paul , mn , usa ) are popular high - strength ceramic materials that offer favorable esthetic characteristics , mechanical properties and biocompatibility . in - ceram alumina has a high strength ceramic core fabricated through the slip - casting technique . a slurry of densely packed al2o3 ( 8082 wt% ) is applied and sintered to a refractory die at 1120c for 10 h. this produces a porous skeleton of alumina particles which is infiltrated with a low - viscosity glass in a second firing at 1100c for 4 h. this system is conventionally used as a core material in conjunction with a more translucent ceramic to enhance esthetic properties . advances in dental ceramics include the introduction of a high - strength all - ceramic core material ( turkom - cera ) containing primarily aluminum oxide ( 99.98% ) . a stone die is covered by a 0.1-mm - thick red plastic foil and dipped in the turkom - cera alumina gel ( 99.98% ) following the manufacturer 's instructions . after drying of the alumina gel , the coping with the red plastic foil is removed from the stone die and sintered for 5 min at 1150c . the sintered coping is crystal hardened in a second firing process using turkom - cera crystal powder for 30 min at 1150c . like all other infiltration ceramics , this core is then veneered with porcelain adjusted to have the correct coefficient of thermal expansion ( 6.5 - 7.2 ) . many ceramics are currently available and marketed for use as dental crown and bridge materials . it has not been ascertain whether the properties of the newer dental materials enable their clinical use to be extended to crowns and bridges in the posterior region . the maximum biting forces that may occur in the posterior area vary between 300 and 880 n. therefore , it is important for the posterior restorations to be able to withstand the maximum biting forces created in this region . although long - term clinical studies constitute the ultimate basis on which to reliably predict the long - term performance of such restorations , several physical and mechanical properties are essential to support the correct indication of these materials . because of different compositions and manufacturing techniques , dental ceramics vary in their physical and mechanical properties . one important property is the strength of the materials , and specially the flexural strength , because of the brittle nature of ceramics . therefore , the aim of this study was to evaluate the flexural strength of turkom - cera compared to two other all - ceramic materials . three different types of ceramic materials , turkom - cera [ ( turkom - ceramic ( m ) sdn . puchong , selangor , malaysia ) ] , in - ceram ( vita zahnfabrik , bad sckingen , baden - wrttemberg , germany ) and vitadur - n ( vita zahnfabrik ) were used in this study . perspex split mold with five circular openings of 16 mm diameter and 2 mm thickness was used for the preparation of the turkom - cera disc specimens . the turkom - cera alumina gel was mixed to an optimum consistency and placed into the disc - shaped perspex mold . the turkom - cera alumina gel was left in the mold for 24 h. after drying of the alumina gel , the discs were taken from the mold and fired ( sintered ) in the furnace ( ivoclar vivadent , programat p300 , ivoclar vivadent ag , schaan , liechtenstein ) for 5 min at 1150c . the turkom - cera crystal powder was mixed with water and the sintered discs were crystal - hardened in a second firing process in the same furnace for 30 min at 1150c . a total of 10 turkom - cera discs ( 16 mm diameter and 2 mm thickness ) were fabricated . perspex split mold with an open top and bottom 5 circular openings ( 16 mm diameter and 2 mm thickness ) was used for the preparation of the in - ceram disc specimens . the mold was rested and secured on a base made from gypsum die material ( densite , shofu inc . , the in - ceram alumina slip was prepared by mixing in - ceram alumina powder with in - ceram mixing fluid and additive supplied by the manufacturer . the slip was poured into the mold and dried for 24 h. after drying , the in - ceram alumina discs were taken from the mold and fired using the in - ceramat furnace ( vita zahnfabrik ) for 6 h at 120c and 4 h at 1120c . the in - ceram glass powder was mixed with water and the sintered in - ceram alumina discs were glass - infiltrated in a second firing process in the same furnace for 30 min at 200c and 4 h at 1100c . a total of 10 in - ceram discs ( 16 mm diameter and 2 mm thickness ) were fabricated . according to the results of a preliminary study , vitadur - n porcelain discs of initial diameter 18 mm shrunk to 15.5 - 16 mm in diameter when fired . therefore , a brass split mold with five circular openings of 18 mm diameter was used for the preparation of the vitadur - n disc specimens . vitadur - n aluminous core porcelain powder was mixed with vita modeling liquid p to an optimum slurry consistency . the slurry was placed into the disc - shaped brass mold and vibrated to reduce air bubbles . a brass compactor was also machined and used to condense the slurry into the mold in order to obtain flat surface . the condensed slurry was left in the mold for 30 min and excess liquid was blotted away with absorbent tissue . a layer of vita modisol ( vita zahnfabrik ) separating medium was applied to the mold before the porcelain mixture was poured to facilitate removal of the set porcelain without any distortion . the disc specimens were then fired according to the manufacturer 's recommendation in a multimat - touch vacuum furnace ( dentsply , dreieich , hessen , germany ) . the furnace was programmed to give a temperature of 1120c for 60 s under vacuum followed by a further 60 s at atmospheric pressure . a total of 10 vitadur - n aluminous core porcelain discs with 15.5 - 16 mm diameter were fabricated . in order to meet the exact requirements of the biaxial testing protocol recommended by iso ( 1995 ) , all specimens were subsequently grinded to a parallel shape using the grinder / polisher machine ( metaserv 2000 , buehler , coventry , west midlands , uk ) . a custom made specimen holder made from aluminum was designed and used for the grinding purpose . eight specimens were fixed into the specimen holder using modeling wax ( figure 1 ) . the initial grinding was performed under running water using a diamond grinding disc with a grain size of 70 m , followed by fine - grinding using a grain size of 30 m . after that , the specimens were polished with a 15 m diamond polishing paste on a polishing cloth for two min . they were then rinsed thoroughly with running water for 20 s and dried in air . specimens mounted in the specimen holder in compliance with iso ( 1995 ) , the specimens were trimmed to 1.20.2 mm in thickness with parallelism of 0.05 mm measured using the digital caliper ( mitutoyo corp , tokyo , japan ) . piston - on - three - ball test was used for the testing . in order to carry out the test , a loading pin and mounting jig were designed and used with the instron testing machine ( instron 4302 , instron corporation , england ) . the mounting jig had a circular opening of 16 mm in diameter with three depressions positioned at equal distances from each other ( 120 apart ) and 5 mm from the center forming a tripod . these depressions were the sites for the 3.2 mm stainless steel ball bearing supports . the specimens were placed in the mounting jig which ensured the same relation between the supports and the applied load for all specimens . the 1.6 mm diameter loading pin was mounted to the crosshead of the instron testing machine and applied the load at the center of each specimen ( figure 2 ) . the test was carried out at a crosshead speed of 0.5 mm / min . the definitive fracture load was recorded for each specimen and the biaxial flexural strength was calculated from the following equation : biaxial flexural strength=-0.238 7p(x - y)/d ; x=(1+v)ln(r2/r3)+[(1-v)/2](r2/r3 ) ; y=(1+v)[1+ln(r1/r3)2]+(1-v)(r1/r3)2 , where p is the total load causing fracture ( n ) , v is poisson 's ratio ( 0.25 ) , r1 is the radius of the support circle ( 5.0 mm ) , r2 is the radius of the loaded area ( 0.8 mm ) , r3 is the radius of the specimen ( 8 mm ) , d is the specimen thickness at the origin of fracture ( mm ) . specimen during testing in the universal testing machine the results of the study were statistically analyzed with the spss software ( v. 11.5.0 for windows , spss , chicago , il , usa ) using levene 's test and dunnett 's t3 post - hoc test at a preset significance level of 5% to determine if significant differences between tests groups were related to the ceramic material used for each group . three different types of ceramic materials , turkom - cera [ ( turkom - ceramic ( m ) sdn . puchong , selangor , malaysia ) ] , in - ceram ( vita zahnfabrik , bad sckingen , baden - wrttemberg , germany ) and vitadur - n ( vita zahnfabrik ) were used in this study . perspex split mold with five circular openings of 16 mm diameter and 2 mm thickness was used for the preparation of the turkom - cera disc specimens . the turkom - cera alumina gel was mixed to an optimum consistency and placed into the disc - shaped perspex mold . the turkom - cera alumina gel was left in the mold for 24 h. after drying of the alumina gel , the discs were taken from the mold and fired ( sintered ) in the furnace ( ivoclar vivadent , programat p300 , ivoclar vivadent ag , schaan , liechtenstein ) for 5 min at 1150c . the turkom - cera crystal powder was mixed with water and the sintered discs were crystal - hardened in a second firing process in the same furnace for 30 min at 1150c . a total of 10 turkom - cera discs ( 16 mm diameter and 2 mm thickness ) were fabricated . perspex split mold with an open top and bottom 5 circular openings ( 16 mm diameter and 2 mm thickness ) was used for the preparation of the in - ceram disc specimens . the mold was rested and secured on a base made from gypsum die material ( densite , shofu inc . , the in - ceram alumina slip was prepared by mixing in - ceram alumina powder with in - ceram mixing fluid and additive supplied by the manufacturer . the slip was poured into the mold and dried for 24 h. after drying , the in - ceram alumina discs were taken from the mold and fired using the in - ceramat furnace ( vita zahnfabrik ) for 6 h at 120c and 4 h at 1120c . the in - ceram glass powder was mixed with water and the sintered in - ceram alumina discs were glass - infiltrated in a second firing process in the same furnace for 30 min at 200c and 4 h at 1100c . a total of 10 in - ceram discs ( 16 mm diameter and 2 mm thickness ) were fabricated . according to the results of a preliminary study , vitadur - n porcelain discs of initial diameter 18 mm shrunk to 15.5 - 16 mm in diameter when fired . therefore , a brass split mold with five circular openings of 18 mm diameter was used for the preparation of the vitadur - n disc specimens . vitadur - n aluminous core porcelain powder was mixed with vita modeling liquid p to an optimum slurry consistency . the slurry was placed into the disc - shaped brass mold and vibrated to reduce air bubbles . a brass compactor was also machined and used to condense the slurry into the mold in order to obtain flat surface . the condensed slurry was left in the mold for 30 min and excess liquid was blotted away with absorbent tissue . a layer of vita modisol ( vita zahnfabrik ) separating medium was applied to the mold before the porcelain mixture was poured to facilitate removal of the set porcelain without any distortion . the disc specimens were then fired according to the manufacturer 's recommendation in a multimat - touch vacuum furnace ( dentsply , dreieich , hessen , germany ) . the furnace was programmed to give a temperature of 1120c for 60 s under vacuum followed by a further 60 s at atmospheric pressure . a total of 10 vitadur - n aluminous core porcelain discs with 15.5 - 16 mm diameter were fabricated . in order to meet the exact requirements of the biaxial testing protocol recommended by iso ( 1995 ) , all specimens were subsequently grinded to a parallel shape using the grinder / polisher machine ( metaserv 2000 , buehler , coventry , west midlands , uk ) . a custom made specimen holder made from aluminum was designed and used for the grinding purpose . eight specimens were fixed into the specimen holder using modeling wax ( figure 1 ) . the initial grinding was performed under running water using a diamond grinding disc with a grain size of 70 m , followed by fine - grinding using a grain size of 30 m . after that , the specimens were polished with a 15 m diamond polishing paste on a polishing cloth for two min . they were then rinsed thoroughly with running water for 20 s and dried in air . specimens mounted in the specimen holder in compliance with iso ( 1995 ) , the specimens were trimmed to 1.20.2 mm in thickness with parallelism of 0.05 mm measured using the digital caliper ( mitutoyo corp , tokyo , japan ) . piston - on - three - ball test was used for the testing . in order to carry out the test , a loading pin and mounting jig were designed and used with the instron testing machine ( instron 4302 , instron corporation , england ) . the mounting jig had a circular opening of 16 mm in diameter with three depressions positioned at equal distances from each other ( 120 apart ) and 5 mm from the center forming a tripod . these depressions were the sites for the 3.2 mm stainless steel ball bearing supports . the specimens were placed in the mounting jig which ensured the same relation between the supports and the applied load for all specimens . the 1.6 mm diameter loading pin was mounted to the crosshead of the instron testing machine and applied the load at the center of each specimen ( figure 2 ) . the test was carried out at a crosshead speed of 0.5 mm / min . the definitive fracture load was recorded for each specimen and the biaxial flexural strength was calculated from the following equation : biaxial flexural strength=-0.238 7p(x - y)/d ; x=(1+v)ln(r2/r3)+[(1-v)/2](r2/r3 ) ; y=(1+v)[1+ln(r1/r3)2]+(1-v)(r1/r3)2 , where p is the total load causing fracture ( n ) , v is poisson 's ratio ( 0.25 ) , r1 is the radius of the support circle ( 5.0 mm ) , r2 is the radius of the loaded area ( 0.8 mm ) , r3 is the radius of the specimen ( 8 mm ) , d is the specimen thickness at the origin of fracture ( mm ) . the results of the study were statistically analyzed with the spss software ( v. 11.5.0 for windows , spss , chicago , il , usa ) using levene 's test and dunnett 's t3 post - hoc test at a preset significance level of 5% to determine if significant differences between tests groups were related to the ceramic material used for each group . the objective was to test if the mean biaxial flexural strengths of turkom - cera , in - ceram and vitadur - n differ from each other . the mean biaxial flexural strength and standard deviation of ten specimens were calculated for each of the three groups tested ( table 1 ) . because of violation of the assumption of homogeneous variances ( levene 's statistic=13.212 , p<0.05 ) , multiple comparisons were performed with dunnett 's t3 post - hoc test at a pre - set significance level of 5% ( table 2 ) . mean and standard deviation ( sd ) of biaxial flexural strength of the three groups multiple comparisons between the three all - ceramic systems tested based on observed means , the mean difference is significant at the 0.05 level the mean biaxial flexural strength values for turkom - cera ( 506.887.01 mpa ) , in - ceram ( 347.428.83 mpa ) and vitadur - n ( 128.712.72 mpa ) differed significantly from each other ( table 2 ) ( p<0.05 ) . strength is an important mechanical property that can assist in predicting the performance of brittle materials . the uniaxial flexural strength tests , including three - point , and four - point bending tests , and biaxial bending tests are the most commonly applied methods for evaluating the strength of dental restorations . for uniaxial flexural strength tests , the principal stress on the lower surfaces of the specimens is tensile , and it is usually responsible for crack initiation in brittle materials . however , undesirable edge fracture , which can increase the variance of the failure stress value , might occur . the method adapted in this study was the one recommended by iso(1995 ) since the test standardizes specimen thickness , diameter , shape and roughness . in addition , the measurement of the strength of brittle materials under biaxial flexural strength conditions rather than uniaxial flexural strength is often considered more reliable because the maximum tensile stresses occur within the central loading area and edge failures are eliminated . therefore , the biaxial flexural test gave less variation in the strength data . according to iso(1995 ) , the biaxial flexural strength is determined by support of a disc specimen on three metal spheres positioned at equal distances from each other and from the center of the disc . the load is applied to the center of the opposite surface by a flat piston . furthermore , the flat surface of the test specimen can be easily controlled by conventional metallographic polishing methods and typical dental finishing techniques . different researchers have studied the biaxial flexural strength of in - ceram core using the same methods as the current study . the biaxial flexural strength of in - ceram has been found to be 337.5 mpa on the average . the mean biaxial flexural strength value for in - ceram ( 347.4 mpa ) obtained in the present study is in agreement with this result . the biaxial flexural strength of vitadur - n core material has been investigated using the same methods as the current study and found to vary from 141.2 to 155 mpa . the mean biaxial flexural strength value for vitadur - n ( 128.7 mpa ) achieved in the current study is in agreement with these results . the higher flexural strength obtained with turkom - cera and in - ceram may be attributed to the followings : 1 . decrease of the total porosity by initial firing ( sintering ) of turkom - cera alumina gel and in - ceram alumina slip ; 2 . the alumina particles increase the strength of the material and limit potential sites for crack propagation ; 3 . prevention of the growth of cracks by crack bridging , as the crystals and glass powders in combination with alumina may bridge the opening created by a crack after the crack front passes ; 4 . compressive stresses , which further improve the strength , are also introduced due to the differences in the coefficient of thermal expansion of the alumina and crystals / glass . despite the high strength reported with high alumina - based ceramics , they are susceptible to fatigue failure that can considerably reduce their strength over time . in this study , therefore , further studies are highly recommended to evaluate the fracture analysis and fatigue behavior of new dental ceramics . in this study , the biaxial flexural strength of three all - ceramic core materials was tested in vitro . the new high - strength all - ceramic core material containing primarily aluminum oxide had significantly higher flexural strength ( 506.887.01 mpa ) than the other ceramic core materials ( in - ceram : 347.428.83 mpa and vitadur n : 128.712.72 mpa ) . this study was supported by a grant ( p019/2006c ) ; university of malaya , kuala lumpur , malaysia . the authors thank turkom - ceramic ( m ) sdn bhd , puchong , selangor , malaysia for supplying the materials used for the preparation of turkom - cera specimens .

advances in all - ceramic systems have established predictable means of providing metal - free aesthetic and biocompatible materials . these materials must have sufficient strength to be a practical treatment alternative for the fabrication of crowns and fixed partial dentures.objectivesthe aim of this study was to compare the biaxial flexural strength of three core ceramic materials . material and methodsthree groups of 10 disc - shaped specimens ( 16 mm diameter x 1.2 mm thickness - in accordance with iso-6872 , 1995 ) were made from the following ceramic materials : turkom - cera fused alumina [ ( turkom - ceramic ( m ) sdn bhd , puchong , selangor , malaysia ) ] , in - ceram ( vita zahnfabrik , bad sckingen , baden - wrttemberg , germany ) and vitadur - n ( vita zahnfabrik , bad sckingen , baden - wrttemberg , germany ) , which were sintered according to the manufacturer 's recommendations . the specimens were subjected to biaxial flexural strength test in a universal testing machine at a crosshead speed of 0.5 mm / min . the definitive fracture load was recorded for each specimen and the biaxial flexural strength was calculated from an equation in accordance with iso-6872 . resultsthe mean biaxial flexural strength values were : turkom - cera : 506.887.01 mpa , in - ceram : 347.428.83 mpa and vitadur - n : 128.712.72 mpa . the results were analyzed by the levene 's test and dunnett 's t3 post - hoc test ( spss software v11.5.0 for windows , spss , chicago , il , usa ) at a preset significance level of 5% because of unequal group variances ( p<0.001 ) . there was statistically significant difference between the three core ceramics ( p<0.05 ) . turkom - cera showed the highest biaxial flexural strength , followed by in - ceram and vitadur - n . conclusionsturkom - cera core had significantly higher flexural strength than in - ceram and vitadur - n ceramic core materials .

INTRODUCTION MATERIAL AND METHODS Materials Preparation of disc-shaped specimens Grinding of specimens Biaxial flexural strength testing Statistical analysis RESULTS DISCUSSION CONCLUSION ACKNOWLEDGEMENTS

PMC3010690

alzheimer 's disease ( ad ) is the most prevalent form of dementia , which affects more than 37 million people worldwide , with an estimated cost of $ 422 billion in 2009 [ 1 , 2 ] . moreover , the incidence of the illness and the prospect of an aging population will result in rising social and economic demands . ad is characterised by the presence of extracellular amyloid plaques and intracellular neurofibrillary tangles within the afflicted brain , which cause neuronal loss in the neocortex , hippocampus , and basal forebrain , leading to progressive cognitive and behavioural decline . zinc , in addition to copper and iron , has been shown to be involved in ad . here , we review the current literature relating to neuronal zinc metabolism and the way in which zinc can modulate normal brain activity . we discuss also the contribution of zinc to the formation , aggregation , and degradation of the amyloid- ( a ) peptide and the contribution of zinc to the pathogenesis of ad . as the most abundant trace metal in the brain , zinc is found tightly associated with numerous proteins conferring either structural or catalytic properties upon them . however , a significant amount of loosely bound , chelatable zinc can be found sequestered in presynaptic vesicles forming a subpopulation of zinc enriched ( zen ) neurones [ 5 , 6 ] , which co - release zinc with the neurotransmitter glutamate upon excitation . neurones have their cell bodies located in either the cerebral cortex or the limbic structures of the forebrain , and so an extensive network uniting limbic and cerebrocortical functions is created . this connection between zinc and glutamatergic neurotransmission allows the ion to modulate the overall excitability of the brain and also influence synaptic plasticity . the identification of synaptic zinc was first made by mclardy over fifty years ago who identified that a band of zinc dithizonate staining correlated with hippocampal mossy fibre axons . since then , many more cerebrocortical pathways have been identified which contain zinc - rich synaptic vesicles ; indeed nearly 50% of the glutamatergic synapses are actually gluzincergic in some parts of the cerebral cortex . significantly , only small amounts of chelatable zinc can be determined in glutamatergic pathways which originate outside the cerebral cortex or limbic systems . despite this extensive network of zinc - containing neurons , there are two families of zinc transporters : the znt family , which act to decrease intracellular zinc concentrations by exporting zinc from the cytoplasm to the lumen of organelles or the extracellular space , and the zip family , which import the metal from the extracellular space or organellar lumen into the cytoplasm . whilst many of the transporters have particular distribution patterns , only znt3 expression is restricted to the brain and the testis . it is located in the vesicular membrane and is necessary to transport zinc from the cytoplasm into the synaptic vesicle of the neuron . targeted disruption of znt3 in a mouse model resulted in a complete lack of chelatable zinc . a number of approaches have been taken to confirm that zinc is coreleased with glutamate from the presynaptic bouton during neuronal excitation : imaging of zinc in boutons before and after stimulation , analytical detection of zinc released into perfusates , and direct imaging of released zinc using fluorescent extracellular probes [ 18 , 19 ] . stimulation of organotypic cultures from rat hippocampus produced a cloud of green fluorescence as the released zinc bound an apometalloenzyme confirming the release of zinc from the culture . a later study by quinta - ferreira and colleagues demonstrated a release of zinc with each pulse of an action potential . whilst there is now no doubt that zinc is released during synaptic activity , there is little consensus on the amount or duration of its existence in the synaptic cleft . following an intense burst of neuronal activity , the release of glutamate and postsynaptic membrane depolarisation open a variety of zinc - permeable ion channels which contribute to removing the ion from the extracellular fluid . these include n - methyl - d - aspartate ( nmda ) channels and calcium permeable -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid ( ampa)/kainate channels . the consequences of zinc acting on these receptors are diverse and demonstrate the significance zinc has in modulating fast excitatory glutamatergic transmission . zinc can act to either enhance or depress synaptic activity with varying degrees of potency . initially , zinc was thought to selectively inhibit nmdar - mediated neuronal activity by inducing a voltage - independent noncompetitive inhibition that decreased the probability of the channel being open [ 2224 ] . a voltage - dependent inhibition of nmdar could be observed at higher concentrations of zinc ( > 20 m ) and was believed to be due to binding of the cation within the channel pore . with the cloning of nmdar subunits , it was confirmed that zinc could cause both voltage - independent and voltage - dependent inhibition . the exceptional sensitivity of the glun2a subunit towards zinc suggests that even contaminating ions found in routine laboratory solutions are sufficient to cause inhibition . significantly , despite being responsible for inhibitory effects at nmdar , it has also been shown that nmdar activation may provide a route of influx for zinc contributing to toxic effects of exposure ( figure 1 ) . whilst it is thought that zinc exposure generally attenuates nmdar - mediated neurotoxicity , zinc has been shown to potentiate ampar - mediated toxicity at suggested physiological concentrations ( 50 m ) . originally , it was proposed that the toxic effect was due to zinc influx via voltage - gated calcium channels , with lin et al . subsequently , it has been demonstrated that zinc can carry currents directly via ampar , mostly via the calcium - permeable subtype ( figure 1 ) . at high supraphysiological doses ( 1 mm ) a few studies have also looked directly at zinc - mediated inhibition of voltage - gated calcium channels , as their proximity to vesicular release sites on the presynaptic membrane suggests they could interact [ 33 , 34 ] . most recently , neurotoxicity has been attributed to transient receptor potential metastatin 7 ( trpm7 ) channel activation resulting in increased intracellular zinc ( figure 1 ) . the significance of synaptically released zinc centres on the amount that is released into the synaptic cleft upon excitation . some authors argue that the amount of zinc ( 10100 m ) released following an action potential arriving at the glutamatergic bouton is high enough to bring about the voltage - dependent inhibition of nmdar and that there would be no spillover onto neighbouring cells [ 36 , 37 ] . other authors disagree , suggesting that the zinc concentration would be sufficient to affect nearby cells . it has also been demonstrated that the zinc level ( low nm ) is only sufficient to block the voltage - independent component of nmdar activity . alternatively , it could be that there is little or no diffusible zinc released , supporting the notion that zinc behaves in a tonic mode . kay and tth proposed that zinc is exocytosed from the presynaptic membrane and that instead of diffusing into the extracellular space it remains tightly bound to an as - yet unidentified presynaptic component . this would create a veneer of zinc ions which would build up with synaptic activity or erode with quiescence to modulate plasticity . first , synaptic zinc acts as a conventional neurotransmitter , is contained within presynaptic vesicles , and is released upon excitation and binds to a variety of receptors on the postsynaptic membrane . the downstream consequence of receptor binding is one of tonic modulation of glutamatergic excitatory synapses . the second type is similar to calcium signalling and occurs in conjunction with synaptic zinc signalling . a transmembrane flux of synaptic zinc from the extracellular space passes through post - synaptic zinc - permeable ion channels . this class is difficult to define and as yet has not been identified in neurons but has been demonstrated in mast cells . it is found in synaptic vesicles , released upon excitation , and has multiple synaptic targets . the significant inhibitory effect of zinc on nmdar , alongside the crucial function of nmdar in both neurophysiology and pathophysiology , advocates a vital role for zinc in both healthy and diseased brains . the most prominent lesions in the brain of ad sufferers are the amyloid or senile plaques , which predominantly consist of a peptides derived from the proteolytic processing of the amyloid precursor protein ( app ) . app is an ubiquitously expressed glycosylated transmembrane protein with a large n - terminal extracellular domain , a single hydrophobic transmembrane domain , and a small c - terminal cytoplasmic domain . a specific and saturable binding site for zinc ( kd = 750 nm ) has been reported in the cysteine - rich region on the ectodomain of app [ 44 , 45 ] . it is hypothesised that zinc could have a role in sustaining the adhesiveness of app during cell - cell and cell - matrix interactions [ 46 , 47 ] . app can be processed by one of two pathways : the amyloidogenic pathway , leading to the production of a , and the non - amyloidogenic pathway ( figure 2(a ) ) , reviewed in . in the amyloidogenic pathway , app is sequentially cleaved by the aspartyl protease , -site app - cleaving enzyme 1 ( bace1 ) forming the secreted app ( sapp ) fragment and a membrane bound c - terminal fragment of 99 amino acids ( c99 ) . the c99 fragment is then further processed by the -secretase complex into app intracellular domain ( aicd ) and a peptides , predominantly 40 and 42 amino acids in length . it is these aggregation - prone a peptides which form oligomeric and fibrillar structures which deposit in the brain and over time cause ad . the -secretase complex comprises four components : presenilin ( ps ) 1 or 2 , nicastrin ( nct ) , presenilin enhancer 2 ( pen-2 ) , and anterior pharynx defective-1 ( aph-1 ) . sapp is involved in the pruning of synapses during the development of central and peripheral neurons and aicd is known to be a transcription factor for several genes , including the upregulation of the a-degrading enzyme , neprilysin ( nep ) . the predominant app - processing pathway in healthy brain is the nonamyloidogenic pathway where app is cleaved by the -secretase within the a region forming the secreted app ( sapp ) fragment and the membrane - bound c - terminal fragment of 83 amino acids ( c83 ) ( figure 2(a ) ) . c83 is subsequently cleaved by the -secretase complex generating aicd and p3 ( figure 2(a ) ) . -secretase activity is attributed to the disintegrin and metalloprotease ( adam ) family of zinc metalloproteases ( reviewed in [ 48 , 52 ] ) . the adams , along with the matrix metalloproteases ( mmps ) , are members of the metzincin clan of metalloproteases as they have a long zinc - binding consensus sequence hebxhxbgbxh ( h , histidine , zinc ligand ; e , catalytic glutamate ; g , glycine ; b , bulky apolar residue ; x , any amino acid ) which contains three zinc ligands . structurally , the catalytic domain is globular and divided into two subdomains , with the active site cleft running between the two . the defining feature of the metzincins is the conserved methionine residue which creates a 1,4--turn ( met - turn ) , creating the catalytic cleft . the catalytic zinc atom sits at the bottom of the groove between the subdomains , with the subsites in the groove determining specificity for particular amino acid sequences in the substrate ( reviewed in ) . a number of enzymes in this family , namely , adam9 , 10 , 17 ( also known as tnf- converting enzyme , tace ) and 19 , are known to exert -secretase activity , but it is unclear which enzyme or enzymes are responsible for the -cleavage of app in vivo [ 5558 ] . adam10 appears to be the predominant enzyme as overexpression of functional adam10 in ad transgenic mice led to an increase in sapp and reduced a production , plaque deposition , and cognitive deficits . although fibroblasts from adam10 mice showed no deficiency in -secretase activity , probably due to compensation by adam17 , a recent detailed study has provided strong evidence that adam10 is the physiologically relevant constitutive -secretase in primary neurons . in a healthy brain , the relatively small amount of a-constitutively being produced is rendered safe by a degrading enzymes . the steady state levels of a synthesis and clearance in cerebrospinal fluid ( csf ) are 7.6% and 8.3% per hour , respectively . a large number of candidate a-degrading enzymes have been identified , with the majority being zinc metalloproteases . these include nep , insulin - degrading enzyme ( ide ) , endothelin - converting enzyme ( ece ) 1 and 2 , mmp2 , 3 , and 9 , and prep ( reviewed in ) . nep , ece1 and 2 , and mmps have the conserved zinc - binding motif hexxh ( h , histidine , zinc ligand ; e , catalytic glutamate ; x , any amino acid ) , and ide and its homolog prep are both inverzincins , as they contain the inverted zinc - binding motif hxxeh . nep appears to be the dominant a protease [ 6366 ] , and is capable of degrading monomeric and oligomeric a [ 63 , 67 ] ( figure 2(b ) ) . nep knockout mice have significantly elevated a levels [ 68 , 69 ] and more importantly , human postmortem tissue analysis suggests that nep expression is inversely related to the extent of ad pathology [ 7072 ] . ide is predominantly a cytosolic enzyme but has been found extracellularly and can only degrade monomeric a and not oligomers ( figure 2(b ) ) , although it did lower the net production of oligomers by cultured cells . even though there are conflicting data regarding ide and ad , ide knockout mice have elevated levels of endogenous a [ 76 , 77 ] . there are two isoforms of ece , 1 and 2 , both of which have similar catalytic activity , with ece 1 being the predominant isoform within the brain [ 65 , 78 ] . however , both eces are primarily localised to endothelial cells but have been detected in neurons and glial . the mmps , specifically mmp2 and mmp9 , degrade fibrillar a , whereas prep degrades monomeric a. however , prep is confined to mitochondria , so it would therefore have limited action on the majority of a which is extracellular . high concentrations of zinc ( up to 1 mm ) have also been found within amyloid plaques , which is thought to have been released from glutamatergic synapses . aggregation of the a peptides can be rapidly induced in the presence of zinc ions under physiological conditions in vitro [ 83 , 84 ] and studies on ad tissues show that zinc colocalised with a deposits . the metal binding site for zinc on a is the same as the copper binding site and lies within the n - terminal hydrophilic region , the first 16 residues ( figure 3 ) . the majority of studies on zinc binding to a have utilised truncated peptides of 16 ( a116 ) and 28 ( a128 ) amino acids , as the binding region is situated within these sequences . a116 does not aggregate under moderate conditions [ 8790 ] , making it an ideal model system , and a128 can undergo aggregation and fibril formation , but at significantly reduced rates , to the full - length peptides . zinc binds to a in a 1 : 1 stoichiometry with a mononuclear binding site [ 84 , 8690 , 9294 ] . nmr studies have shown that all three histidine residues ( residues 6 , 13 and 14 ; see figure 3 ) are involved in zinc binding [ 87 , 88 , 90 , 93 , 95 ] , with confirmation from mutational studies [ 96 , 97 ] . other possible ligands have also been reported as zinc coordination classically involves four to six ligands . the candidate ligands are asp1 [ 91 , 93 ] , arg5 , ser 8 , glu11 , and tyr10 ( figure 3 ) however , tyr10 has been ruled out and arg5 has been deemed highly unlikely . the carboxyl side chain of glu11 is a zinc ligand [ 88 , 90 ] however , asp1 is considered the most attractive zinc ligand , either through its n - terminal amino group and/or its side - chain carboxylate group [ 87 , 88 , 91 , 93 ] . raman spectroscopy has shown zinc binding to the n site of histidine side chains in senile plaques taken from ad brains , but it is unclear if zinc only binds to free a peptides that subsequently aggregate or whether zinc binds to a in preformed plaques . the reported apparent binding constants ( kd ) of zinc to a peptides vary from 1 to 300 m ( reviewed in ) . the published kd values vary greatly due to the in vitro conditions ( e.g. , buffer composition , ph ) , a fragment , and experimental method . the high kd ( 20300 m ) values come from tyrosine fluorescence experiments however , even these are contentious and hard to reproduce [ 99101 ] , and any change in tyrosine fluorescence could be due to a aggregation rather than zinc binding . discounting the tyrosine fluorescence measurements , the most likely apparent kd value for zinc binding to a peptides is a range of 120 m . the binding affinity of zinc for preformed a fibrils is approximately the same as of the peptides with a kd of 120 m [ 89 , 101 ] . it has been shown that there is a significant increase in serum and hippocampal zinc in ad patients compared to age - matched controls . . demonstrated a significant decrease in csf zinc but could find no difference in serum zinc levels between ad and age - matched controls . a decrease in serum zinc has also been reported , though it is possible that some of the ad patients included in one study were malnourished [ 104 , 105 ] . overall , there is currently no consensus on what happens to zinc concentrations in ad subjects though much of the discrepancy could be put down to differences in patient allocation , sample type , postmortem interval , or type of analysis used . lovell and coworkers [ 106 , 107 ] have mapped the expression levels of a number of the znt zinc transporters in ad . znt-1 , 4 , and 6 were all found to show increased expression in early stages of disease , though znt-1 expression was decreased during mild cognitive impairment [ 106 , 107 ] . although it is unknown whether increased expression necessarily correlates with increased activity , these changes in transporter level could result in modified subcellular zinc concentrations . an increase in znt6 would lead to an increase in zinc in the tgn which could reduce -secretase activity . it is well established that amyloid plaques contain increased concentrations of copper , iron , and zinc [ 98 , 109 ] . whilst copper and iron appear to be primarily responsible for the toxicity of a via oxidative - stress - type mechanisms [ 109 , 110 ] , zinc has a crucial role in a aggregation which is the most well - established contribution that zinc may have in ad pathogenesis . whilst the concentration of zinc required for fibrillisation to occur is contentious , with concentrations differing by 100-fold being suggested , zinc is an unequivocal partner in the process [ 83 , 111 , 112 ] . in 2006 , dong and co - workers were able to show that zinc could control the rate of self - assembly of the a peptides and go on to regulate the amyloid morphology via specific coordination sites . furthermore , it has been demonstrated that zinc can spontaneously coordinate both intra - and inter - molecular bridging between two peptides to promote a aggregation and that synaptic zinc promotes a oligomer formation and their accumulation at excitatory synapses . studies with synthetic a showed that chelation chemistry could help solubilise amyloid plaques , with depletion of zinc having a more marked effect on extracting a than depletion of copper . oral treatment with 5-chloro-7-iodo-8-hydroxyquinoline ( clioquinol cq ) in tg2576 mice resulted in a 49% reduction in cortical amyloid deposition . although cq has a fairly low affinity ( nm ) for both copper and zinc , it was still able to release the ions from the a binding site . a pilot phase ii trial in humans showed a decrease in cognitive decline and a reduction in plasma a1 - 42 in moderately severe ad compared with placebo control . it has been suggested that although cq may chelate copper and zinc from metallated a and promote disaggregation , it may not completely holt the aggregation process . a second generation chelator ( pbt-2 ) , with improved blood brain barrier penetration , has just completed a phase ii clinical trial in early ad with promising results showing good tolerance , a reduction in csf a and neuropsychological testing . recently , it has been shown that zinc can also accelerate the aggregation of a tau peptide under reducing conditions . zinc inhibited the formation of intramolecular disulphide bonds but promoted intermolecular bonds between key cysteine residues . furthermore , zinc exposure has been shown to increase the phosphorylation of pi3k and mapk - dependent pathways which are key players in tau modifications . the essential requirement for znt3 in loading zinc into synaptic vesicles would suggest that this transporter could have a major impact on zinc signalling in the neuron , even regulating cognitive function . whilst a lack of zinc signalling in brain slices from znt3 mice confirmed the vesicular origin of the released zinc , the mice failed to express a cognitive phenotype . initial studies detailed a 20% reduction in total zinc level and a loss of histochemically reactive zinc in the synaptic vesicle ; however , there was no impairment of spatial learning , memory , or sensorimotor function . the implication being that the vesicular zinc is not required for cognitive function or that compensatory mechanisms made up for the deficits . however , a follow - up study demonstrated marked differences in learning and memory when an older ( 6 month ) cohort of mice was used , suggesting that the lack of effect in the previous study was due to the young age ( 610 weeks ) of the mice and highlights the importance of aging ( the most significant risk factor ) when modelling ad pathology . the results obtained from the older znt3 cohort established a requirement for zinc in memory function and the maintenance of synaptic health upon aging . adlard and colleagues proposed that -amyloid pathology could cause cognitive impairment by trapping zinc within plaques rather than via a directly toxic mechanism . the zinc immobilisation by amyloid would then have similar consequences as a loss of znt3 activity with a loss of zinc - dependent synaptic modulation promoting cognitive decline . an alternative approach to minimising the consequences of released zinc could be to promote mechanisms which enhance reuptake . recently , it has been shown that the cellular form of the prion protein ( prp ) is an evolutionary descendent of the zip family of divalent metal transporters . in particular , zips 5 , 6 , and 10 were found to have a prion - like domain with significant structural similarity . as both prp and the zips bind divalent metal ions via histidine - rich motifs contained with n - terminal repeating sequences , this could suggest that prp is involved in zinc sensing , scavenging , or transport . in agreement with that possibility , we have shown that prp promotes zinc uptake ( watt et al . , unpublished ) . ensuring efficient clearance of extracellular zinc from the synaptic cleft via prp would exploit an existing physiological process . furthermore , enhancing zinc uptake would help prevent its ability to contribute to the synaptic targeting of a oligomers , thus preserving synaptic function and maintaining the proposed ferroxidase activity of app . as prp levels decrease with age and in sporadic ad , it is possible that zinc is cleared less efficiently from the synaptic cleft enhancing aggregation of a and inhibiting app ferroxidase activity to promote a pro - oxidative environment . this would suggest that preserving prp function during ad could provide multifactorial benefits , an inhibition of bace1 which would reduce a formation and ensure efficient clearance of zinc from the synaptic cleft to prevent aggregation of a peptides , as well as provide protection against oxidative stress [ 130 , 131 ] . it is clear that zinc not only plays critical roles in the structural and functional integrity of many proteins , but that it also modulates the activity of glutamatergic synapses and indeed may act as a neurotransmitter in its own right . several of the enzymes involved in processing app and a are zinc metalloproteases , with an essential requirement for zinc in their catalytic activity . although zinc dyshomeostasis may contribute to the development of ad , further work is required to clarify the molecular and cellular mechanisms affected by zinc under both normal and disease situations .

zinc , the most abundant trace metal in the brain , has numerous functions , both in health and in disease . zinc is released into the synaptic cleft of glutamatergic neurons alongside glutamate from where it interacts and modulates nmda and ampa receptors . in addition , zinc has multifactorial functions in alzheimer 's disease ( ad ) . zinc is critical in the enzymatic nonamyloidogenic processing of the amyloid precursor protein ( app ) and in the enzymatic degradation of the amyloid- ( a ) peptide . zinc binds to a promoting its aggregation into neurotoxic species , and disruption of zinc homeostasis in the brain results in synaptic and memory deficits . thus , zinc dyshomeostasis may have a critical role to play in the pathogenesis of ad , and the chelation of zinc is a potential therapeutic approach .

1. Introduction 2. Physiological Role of Zinc in the Brain 3. Role of Zinc in APP Processing 4. Role of Zinc in A 5. Zinc Binding to A 6. Role of Zinc in Alzheimer's Disease 7. Conclusions

PMC3719162

proteins are essential components of all organisms , carrying out tasks defined by the information encoded within genes , for example catalyzing biochemical reactions , mediating cell signaling , or providing structural rigidity . simulations range from elucidation of enzymatic reaction mechanisms , to the study of folding pathways , to design of therapeutic molecules against disease . in biomolecular simulations such as these , molecular mechanics ( mm ) force fields are often used in which electrostatic interactions are described by atom - centered point charges . however , there is no unique method for partitioning the rigorously calculated quantum mechanical ( qm ) electron density among the individual atoms and different charge derivation schemes often lead to very different results . in commonly used force fields such as amber , the mm partial charges of protein molecules are optimized by fitting them to reproduce the qm electrostatic potential ( esp ) of small molecules . these esp charges are well - suited for mm force fields , as they reproduce ab initio multipole moments and electrostatic interactions between molecular fragments . a disadvantage of such techniques is the neglect of polarization by the environment indeed , a recent density functional theory ( dft ) natural population analysis of an entire protein in water found that net charges of residues can vary by up to 0.5 e from their putative integer values . while mean field approaches for charge fitting are the most appropriate for deriving transferable force field parameters , often , as in the example of the study of protein ligand binding , we are only interested in sampling in the vicinity of the protein s native state . in these cases , it would be ideal to incorporate electrostatic polarization that is specific to that native state into the charge fitting procedure . recent studies have calculated atom - centered charges for entire proteins , accounting for native state polarization by including background point charges in a series of iterative fragment - based esp fits . the resulting polarized protein - specific charges perform better than standard amber charges in determining free energies of ligand binding , in pka calculations , and in comparisons with nmr data . these results point to the potential improvements that can be made by using polarized protein - specific point charges . however , such an approach potentially requires a large number of qm calculations to iterate all charges in the system to self - consistency and requires restraints or conformational averaging to treat buried atoms and to address the sensitivity of the charges to small conformational changes . an ideal charge derivation scheme should efficiently account for the surrounding environment , while the resulting charges should be chemically intuitive , reproduce ab initio electrostatic properties , be robust with respect to conformational changes , and be insensitive to buried atoms . the charges should be derived from first principles , with no empirical parameters , applicable to a wide range of systems without requiring specialized treatments based on specific chemical knowledge of a particular molecule , and preferably computable from a single qm calculation of the whole system . recently , there has been renewed interest in electronic density - based atoms - in - molecule ( aim ) charge partitioning based on the hirshfeld approach . such methods differ conceptually from esp in that the net atomic charges are assigned by dividing a converged , qm electronic density into a union of overlapping atomic basins . the density derived electrostatic and chemical charges ( ddec ) method , developed by manz and sholl , combines two aim approaches , iterative hirshfeld ( ih ) and iterated stockholder atoms ( isa ) , to assign atomic charges from the electron density . the resulting charges have already been shown to be suitable for force field development . they adapt to the atom s environment , reproduce the ab initio electrostatic potential , and , where applicable , correlate well with x - ray diffraction and x - ray absorption near - edge spectroscopy data . the method can be applied with no adjustable parameters to buried atoms and to either periodic or nonperiodic systems . ddec charges have already been used to develop force fields for molecular adsorption inside metal organic frameworks . the ddec method is implemented in a freely available code ( http://ddec.sourceforge.net/ ) , which is interfaced with codes such as vasp and gaussian among others . thus , ddec charges are suitable for environment - specific , flexible force field development for biomolecular simulations but are limited by the unfavorable computational scaling of the underlying qm calculation to systems of a few hundred atoms . in this paper , we overcome this limitation by implementing the ddec scheme in the onetep linear - scaling dft code . onetep combines high basis set accuracy , comparable to that of plane - wave dft methods , with a computational cost that scales linearly with the number of atoms , which allows for an accurate , fully qm description of systems of thousands of atoms , including entire proteins . we begin by outlining the various underlying aim schemes , followed by a brief description of the linear - scaling dft code onetep . we validate our internal implementation of the ddec methodology against quantum chemistry calculations in gaussian09 for a benchmark set of 25 representative small molecules and show that charges are derived with linear - scaling computational cost , allowing analysis of proteins comprising thousands of atoms from a single dft calculation . we demonstrate that , for these large systems , the features of the ddec scheme that make the charges desirable for flexible force field development are maintained . namely , the charges ( i ) respond to their environment in a chemically intuitive manner ; ( ii ) reproduce electrostatic properties of the dft calculation ; and ( iii ) are not overly sensitive to small conformational changes or the presence of buried atoms . finally , we construct a mm force field based on the ddec charges for three proteins and compare the results of our mm simulations with experimentally measured nmr dynamic observables and a standard biomolecular force field . the ddec method is based upon two recently developed extensions to the original hirshfeld aim scheme . in the original formulation , an electronic density n(r ) is divided into overlapping atomic basins na(r ) for each atom a according to the weighting formula:1where na0(r ) is the atomic reference density , whose overlapping sum over all atoms in the system bnb0(r ) is termed the promolecular density . this form of stockholder partitioning , where the electronic density at each point r is distributed based on the proportional contribution from the reference densities of each atom at that point , has been shown to minimize the information distance faim ( kullback liebler entropy ) between the real and promolecular density , maximizing the information retained in the reference atomic states na0(r ) when transferring to the molecular environment:2subject to the constraint that ana(r ) = n(r ) ; that is , the electronic density is completely partitioned . in other words , the stockholder partitioning exhaustively divides the real electronic density into a set of overlapping atomic densities na(r ) in such a way as to maximize the density distribution similarity to their respective reference density counterparts na0(r ) . shortcomings of the original hirshfeld method included an arbitrariness in the choice of reference atomic densities na0(r ) . neutral , gas - phase atomic densities were often chosen as references , although these commonly led to atomic populations that were too close to zero . such problems are addressed in recently proposed iterative extensions to the hirshfeld method , in which reference densities are successively improved until self - consistency is achieved . in the iterative hirshfeld ( ih ) , the ih reference densities na0(i , r ) are derived from the partitioned atomic density at iteration i , na(i , r ) , by the following procedure . first , the ( noninteger ) electronic populations of each atom qa(i ) are computed in the usual way from the partitioned atomic densities:3instead of choosing neutral reference states as in the original hirshfeld method , new reference states are generated by linear interpolation between densities of free atoms or ions with the next lowest integer ( = lint(qa(i ) ) and the next highest integer ( + 1 ) number of electrons:4the purpose of the interpolation is to obtain a suitable gas - phase reference density for a hypothetical ion comprising qa(i ) electrons . the partitioned atomic densities for the next iteration i + 1 are then derived from the reference states generated at iteration i:5the procedure is iterated until the changes in the set of ih charges fall below a specified threshold . the resulting charges have been shown to reproduce ab initio electrostatic properties of small polypeptides and to be relatively insensitive to small conformational changes . an alternative approach by lillestolen and wheatley , meanwhile , named iterated stockholder atoms ( isa ) , takes the spherical average of the partitioned atomic density na(i , r ) at iteration i as the ( isa ) reference density that enters into eq 5 , where a denotes spherical averaging about the center of atom a:6 in practice , the averaging is performed on a set of discrete radial shells up to a maximum radius rmax . the isa scheme is argued to be less empirical than ih , as the latter still relies on a library of externally generated ionic densities . isa also produces a better fit to the esp due to the low - order multipoles possessed by the converged na(r ) resulting from the spherical - averaging procedure used to generate the reference densities . the ddec scheme by manz and sholl combines the ih and isa methods by minimizing a combined information entropy functional ( eq 2):7where fih / isa are constructed with ih / isa reference densities and weighted by an adjustable parameter . minimizing eq 7 with respect to na(r ) with the same constraint as eq 2 leads to a partitioning of the form:8where naih / isa(i , r ) are the respective reference densities given by eqs 4 and 6 . by allowing a fraction of fih to contribute toward curvature in regions that otherwise have shallow optimization landscapes , for example , buried atoms , this technique alleviates the slow convergence of the isa method for such regions while retaining the appealing attributes of the isa scheme . an important addition to the ddec scheme is the employment of charge - compensated reference ih densities , referred to as the ddec / c2 scheme . these densities are generated from dft ground - state ionic calculations in the presence of a charge compensation sphere , akin to a conductor - like polarizable continuum model ( cpcm ) treatment with a solvent of infinite dielectric constant . the justification for using such compensated densities instead of free ionic states , as in the original ih scheme , stems from the dielectric screening experienced by an ion embedded within a molecule , whereby its density profile can be modified by the effective dielectric constant . the charge compensation sphere acts to expand ( contract ) the reference density in the case of cations ( anions ) . onetep is a linear - scaling dft package based on a reformulation of conventional kohn sham dft in terms of the single - particle density matrix:9where { (r ) } are nonorthogonal generalized wannier functions ( ngwfs ) that are localized in real space , and k is a representation of the density matrix in the biorthogonal duals of the ngwfs . onetep achieves linear - scaling by exploiting the nearsightedness of the single - particle density matrix in nonmetallic systems . in practice , linear - scaling arises from enforcing strict localization of the ngwfs onto atomic regions and through the optimization of the density kernel and ngwfs , subject to localization constraints . optimizing the ngwfs in situ allows for a minimal number of atom - centered orbitals to be used while maintaining plane - wave accuracy . the ngwfs are represented in a basis of highly localized periodic cardinal sine ( psinc ) functions ( otherwise known as fourier the psinc functions are related to plane waves via a fourier transform , meaning that systematic improvement is possible through adjustment of the psinc grid spacing , analogous to converging the kinetic energy cutoff in traditional ( n ) plane - wave dft codes . in order to expedite optimization , the ngwfs can be initialized closer to their ground states by using an in - built pseudoatomic solver , which self - consistently solves the dft kohn sham equations for isolated atoms using the same pseudopotentials and exchange - correlation functional as the full calculation . implicit solvation , whose inclusion is essential both for an accurate description of the protein s aqueous environment and to aid optimization of the density kernel , is implemented within onetep . this is a minimal parameter , self - consistent model based on direct solution of the inhomogeneous poisson equation for a solute cavity defined by the isosurface of the electron density . we have implemented the ddec method within onetep as a postprocessing module to take advantage of its parallel and efficient algorithms for sparse matrix algebra and operations with localized orbitals and electron distributions . a single dft calculation is performed on the system to obtain the ground - state electronic density , which is then processed to extract the ddec net atomic charges . reference densities for the ih part of the calculation are generated internally at run time using the same exchange - correlation functional , pseudopotentials , and ngwf cutoff radius as the full dft calculation , with the c2 charge compensation scheme . specifically , using the pseudoatomic solver module , as described in section 2.2 , the kohn sham equations for a set of atomic orbitals of an isolated atom are solved self - consistently on a radial grid with an additional spherical surface of compensation charge . the effect of this charged surface is expressed equivalently via gauss law as an additional radial electrostatic potential term vcomp in the hamiltonian:10where the first three operators are the kinetic energy , local and nonlocal potentials , and vcomp = qcomp/|r| for |r| > rcomp , with qcomp and rcomp the charge and radius of the compensation sphere . following manz and sholl , for cations , the magnitude of the compensation charge ( qcomp ) is chosen to neutralize the ion , with the compensation sphere radius ( rcomp ) set to the average of nlm|r|nlm expectation values of all occupied orbitals { |nlm } in the neutral species that are vacant in the cation . for anions , the compensation charge is chosen to make the electrostatic potential at an infinitesimal distance outside the compensation sphere zero . the radius of the compensation sphere is incrementally adjusted in 0.1 bohr steps until the total energy of the system is minimized . whole - molecule densities ( total and promolecular ) are stored on regular cartesian grids , while spherically averaged promolecular density profiles for individual atoms are computed and stored on atom - centered sets of equally spaced radial shells up to a predefined maximum cutoff radius . linear - scaling computation with respect to the number of atoms ( for each iteration ) is achieved at a modest cost of computational memory by computing and storing the total promolecular density ( bnb0(i , r ) ) once per ddec iteration , on the same grid as the molecular density . figure 1a shows the computational scaling of our ddec implementation for periodic supercells of bulk water of increasing size , up to around 2500 atoms ( supporting information ) . even for the largest system , the computation time of the ddec analysis is under 20 min . the dft calculation itself requires around 12 h on 160 cores , making the calculation of ddec charges for systems comprising thousands of atoms feasible on a near - routine basis . ( a ) computation time required for ddec postprocessing calculations of bulk water . ( b ) ddec atomic and molecular charge distributions of bulk water for three system sizes . importantly , the number of iterations required for charge convergence remains virtually constant for increasing numbers of atoms , ensuring that the total computational effort remains linear - scaling with respect to system size . figure 1b reveals that the distribution of charges obtained for bulk water is independent of system size . the atomic charges distributions are centered on values close to those typically used in mm force fields ( tip3p ) , while they show some spread as one might expect for atoms in a liquid with differing local environments . recently , manz and sholl introduced an update to the ddec method , named ddec / c3 . the ddec / c3 method improves convergence of charges for nonporous solids with short bond lengths between diffuse atoms by enforcing exponential decay of atomic electron densities and conditioning the reference ih densities naih(i , r ) that enter eq 8 to the material of interest . in our implementation , we use the conditioned reference densities but do not enforce radial decay of the partitioned atomic densities na(r ) . our reasoning is that our studies are aimed at biomolecular systems , which are porous materials lacking compacted regions , and we anticipate that the partial weighting by ih reference densities during optimization will be sufficient to ensure the stability of atomic charges for embedded atoms . in addition , in our implementation , core charge handling has been completely excluded , as we employ norm - conserving pseudopotentials . literature studies have found little dependence of ih charges on the treatment of core electrons . nevertheless , in the following section , we test both of these assumptions by validating onetep - calculated ddec charges against the full ddec / c3 method employing the all - electron code gaussian09 . the validity of our ddec implementation was tested using a set of 25 diverse , neutral , small , organic molecules . benchmarking was performed against the standard ddec method as implemented in the chargemol package ( version 2.1 beta , obtained from http://ddec.sourceforge.net/ ) , employing 100 radial shells ( nrad ) up to a maximum cutoff radius ( rmax ) of 5 , together with the supplied c3 charge - compensated reference density library ( ddec / chargemol ) . molecular geometries were optimized in vacuo with a 6 - 311g(d , p ) basis set , and electronic densities were generated with an aug - cc - pvqz all - electron basis set in gaussian09 . the mixing parameter was set to /14 , which has been shown to give the optimal balance between minimizing the atomic multipoles and maximizing chemical accuracy . the set of net atomic charges was considered converged when the maximum absolute change for any atom for three successive iterations was less than 2 10e . onetep calculations were performed in vacuum using a cubic simulation cell of 30 , with the spherical cutoff coulomb approach to avoid electrostatic interactions between molecules and their periodic images . ngwfs were initialized as orbitals obtained from solving the kohn sham equation for free atoms , with a 1s configuration for h , a 2s2p configuration for c , n , o , and f , and a 3s3p configuration for s and cl . the ngwfs were expanded in a psinc basis with an energy cutoff of 1000 ev , corresponding to a grid spacing of 0.45 bohr , and were localized in real space with radii of 10 bohr . convergence of the atomic charges generated by our implementation of the ddec method in onetep ( referred to as ddec / onetep ) with respect to the onetep psinc grid spacing and ngwf cutoff radii , as well as the ddec parameters rmax and nrad was investigated for nitroethane and discussed in supporting information figure s1 . figure 2 shows the correlation between the ddec / onetep and ddec / chargemol charges for every atom in the benchmark set . despite the different approaches used in obtaining the ground - state electronic densities and the subsequent charge analysis , the difference between the two charge sets is very small with a mean absolute deviation ( mad ) of less than 0.02 e. correlation between ddec / chargemol and ddec / onetep charges for all atoms in the 25 molecule benchmark set . also shown is the mean absolute deviation ( mad ) between the two sets . atomic charges for use in force fields should approximately reproduce the ab initio electrostatic potential outside the molecule s electron density . we can measure the error v in the coulombic potential of the ddec charges , compared with dft , as11where the sum is performed for all points i lying within 1.4 and 2.0 times the van der waals radii of the nuclei , on the same grid mesh used to calculate the electron density . in order to remove the arbitrary vacuum level of the dft potential v(r ) , the potentials are displaced by the averaged difference over the included grid points . our calculations of v are performed for a test charge of 1 e in vacuum . typical errors in , for example , interaction energies of neutral ligands with proteins in solution will tend to be lower than this error estimate . nevertheless , it is a good indicator of the ability of a method to reproduce the ab initio esp . figure 3 ( bottom ) shows that ddec charges reliably reproduce the electrostatic potential around the molecule , with v errors of approximately 1 kcal / mol for both ddec / chargemol and ddec / onetep . for neutral molecules , the dipole is the leading order term in the multipole expansion and , thus , is the most significant in determining intermolecular electrostatic interactions . in figure 3 ( top ) , we compare the ddec vector dipole moments with those computed directly from their respective ab initio electronic densities . again , onetep performs as well as chargemol . for comparison , we have also plotted the error in the dipole moment and v ( eq 11 ) for esp charges . in agreement with previous charge comparisons , the esp charges reproduce the ab initio electrostatic potential and dipole moment more closely . despite this better performance of esp charges for small molecule electrostatics , ddec charges will be suitable for polarized protein - specific charges if , as we shall investigate in the next section , they are able to reproduce the electrostatic properties of much larger molecules containing buried atoms better than standard mm force fields . we conclude that , at least for organic systems comprising light atoms , our use of ddec charge analysis within the pseudopotential approximation and without enforcing radial decay of the partitioned atomic densities is valid . it should be emphasized that the ddec analysis can only be as accurate as the electron density output by the underlying qm calculation . in the current paper , we have used the pbe exchange - correlation functional in our onetep calculations , although future advances will allow alternative treatments of electron electron interactions , such as b3lyp and dynamical mean field theory . for comparison , supporting information figure s2 compares gaussian09 ddec charges calculated using pbe and b3lyp exchange - correlation functionals . the mad is less than 0.02 e , implying that the pbe functional gives a reliable electron density for the calculation of ddec charges . in order to aid in the interpretation of qm simulations , atomic point charges should respond in a chemically intuitive manner to their environment . as an example , figure 4 , b and c , shows onetep / ddec charges for the phenol molecule in two different environments : first in water and , second , in a small model cluster representing the negatively charged binding pocket of the l99a / m102e mutant of t4 lysozyme ( supporting information ) . the two environments are typical of those simulated in , for example , the optimization of small molecule inhibitors for drug design . for comparison , figure 4a shows the resp charges for phenol , calculated in vacuum at the hf/6 - 31 g * level to approximate aqueous polarization , the same level of theory that has been used to parametrize the amber force field that we employ as a benchmark in later sections . the ddec charges in water agree with the resp charges with a root - mean - square ( rms ) deviation of 0.04 e. similar results are obtained by comparing with resp charges derived using a polarizable continuum model ( pcm ) to model solvation ( supporting information figure s3 ) . there are no large changes in the ddec charges of the phenol molecule on moving to the protein binding pocket . charges on the aromatic ring change by up to 0.05 e in the more hydrophobic environment , while the oh group , which is hydrogen - bonded to residue e102 , becomes more strongly polarized . ( a ) resp charges for phenol calculated in vacuo at the hf/6 - 31 g * level . ( b ) ddec / onetep charges calculated in a 20 molecule water cluster using with identical simulation parameters as section 2.4 . ( c ) same as in part b , but within a 86 atom cluster representing the t4 lysozyme l99a / m102e binding pocket . standard error of the mean is less than 0.01 e for the oh group , and the maximum on any atom is 0.018 e. we have utilized the linear - scaling nature of our ddec implementation to calculate ddec charges of three proteins : ubiquitin , the smn tudor domain , and hen egg lysozyme ( pdb : 1ubq , 1mhn , 6lyt ) . initial structures were prepared by protonation using the amber11 tleap module or by the molprobity software . dft calculations were performed using the minimal parameter continuum solvation model of onetep with a relative dielectric permittivity of 80 to represent water solvent . a postprocessing calculation was performed on the converged electronic density to extract ddec charges ( supporting information ) . it should be emphasized that the entire charge set is derived from a single dft calculation , on systems up to 1960 atoms , and thus naturally incorporates native state polarization . as we found for the small test systems , ddec charges for the proteins are similar to the mean field amber ff99sb charges , with a correlation coefficient of 0.96 ( figure 5a ) . however , the rms deviation between the charge sets is 0.11 e , as expected since the aims were optimized based on the ab initio electronic density for atoms embedded in their local environment . comparisons with the amber ff03 force field charges yield similar conclusions ( supporting information figure s4 ) . for chemically identical atoms that share the same charges in amber , the ddec charges span a wider range , which is indicated by the vertical spread of points . for example , ddec charges for backbone o atoms of leucine vary between 0.72 and 0.56 e , with an average of 0.63 e , compared to the constant amber value of 0.57 e. the prominent outliers in figure 5a ( with absolute charge disagreement larger than 0.25 e ) are mostly nonbackbone sp carbon atoms , most of which are close to neutral in amber , but adopt a range of values in ddec , depending on the residue type and their particular local environments . for example , the c atom in glutamine has a charge of 0.06 e in amber , but ranges from 0.41 to 0.37 e in ddec . similar deviations in c charges from amber values have been observed in conformationally averaged esp charges derived from dipeptide fragments . the largest discrepancy between the two charge sets is in the terminal nitrogen atom , which is positively charged in amber ( 0.16 e ) but negatively charged in ddec ( 0.52 e ) . several esp studies have indicated that our ddec value is more appropriate for the charge of the n - terminus . ( a ) correlation between ddec partial atomic charges and the amber ff99sb force field for ubiquitin . ( b ) electrostatic contribution to the backbone hydrogen bond energies in the x - ray crystal structure of ubiquitin . backbone hydrogen bonds calculated by ddec / onetep are more polarized than in the amber force field . following ji et al . , we define the electrostatic contribution to the hydrogen bond between backbone nh and co groups as the coulombic interaction between atoms carrying point charges q:12figure 5b reveals that the resulting dipole it is possible that the enhancement of the interaction is a systematic bias in the potential of the ddec charges , though it does support previous hypotheses that proteins are stabilized in their native environment via electronic polarization . we investigate this effect further in section 4 by comparing the dynamics of backbone hydrogen bonds with experimental nmr observables . the correct treatment of electrostatics is vital in the accurate determination of molecular interactions in biological systems . in particular , if ddec charges are to be useful as polarized protein - specific charges , they should reproduce ab initio electrostatics for large molecules better than standard force fields that are based on esp charges . we have calculated the electrostatic potential and the dipole moments of the three proteins studied in the previous section with full dft , the derived ddec charges , and with a standard amber force field ( supporting information figure s4 gives the same information for the amber ff03 force field ) . for all charge distributions , the electrostatic potential was computed in vacuum , in order to better observe the discrepancy between ab initio and point charge values that would otherwise be dampened by dielectric screening . we note , however , that the ab initio , ddec and amber charge distributions have already incorporated solvation effects in their calculation , and the electrostatic comparisons are , therefore , equivalent . table 1 shows that ddec / onetep aims reproduce the total ab initio potential and dipole moments dft for all three proteins , vastly outperforming the standard mm charges . figure 6 shows the difference in electrostatic potential between the two charge sets and the dft calculation . although the standard mm charges are fit to closely reproduce the esp of small molecules , they neglect large - scale electronic polarization that is present in protein - specific charges , and hence , the error in the computed esp is large . the computed errors in the mm electrostatic potential are consistent with literature studies of errors in interaction energies between charged biotin ligands and the avidin protein in vacuum ( 11 kcal / mol on average ) . for more realistic simulations , the same study found that the error was reduced to approximately 2 kcal / mol for neutral ligands and to 0.7 kcal / mol if solvent screening is also included . the esp difference is calculated at the solvent - accessible surface of the protein . also shown as gray , blue , and red arrows are the calculated dft , ddec , and amber dipole moments , respectively . also shown are errors in the protein dipole moment vectors , which have been calculated relative to the center of mass of each protein to remove ambiguity for charged systems . the ddec charges reproduce electrostatic properties of large molecules much better than do the mean field classical point charges . we noted earlier that isa charges are particularly good at reproducing the esp , since the electron density is divided into spherically symmetric partitions . this point is reinforced by figure 7 , which reveals that the deviation from the ab initio dipole moment of ubiquitin decreases with decreasing ( increasing isa fraction ) . however , one of the motivations for including ih reference densities in the ddec information entropy functional ( eq 8) was to alleviate problematic convergence for aim charges of embedded atoms due to the shallow optimization landscapes experienced in such regions . we find that the ddec method with = /14 , as recommended for all systems by manz and sholl , is a good compromise between reproducing accurately the esp of the system , reducing the number of self - consistency iterations required for convergence ( figure 7 ) and , as we shall see in the next section , improving the transferability of the charges . further comparisons between ddec , ih , and isa charges are presented in supporting information figure s5 . dependence of the ddec dipole moment error on the mixing parameter . increasing the isa content reduces the dipole error but also increases the number of iterations required for charge convergence and , hence , the computational time . in order to be of use in parametrizing flexible force fields , atomic charges , even those of buried atoms , must not be overly sensitive to small conformational changes . to study conformational dependencies of the ddec charges for large systems , we have performed ddec analysis of nine experimental nmr conformers of bovine pancreatic trypsin inhibitor ( bpti , pdb : 1pit ) in implicit solvent ( 892 atoms ) . each residue in figure 8 is colored by the average of the standard deviation in the calculated atomic charges over the nine conformations . the first point to note is that the ddec charges are much more stable to conformational change than the isa charges . while some fluctuation due to the changing environment is expected , the protein conformers are all close to the native state and the ddec charges reflect this . this is in stark contrast to resp charges , for which a recent review of atomic charge models found strong fluctuations in partial charges for an ensemble of polypeptide chain conformations . second , there is no discernible difference between charge fluctuations in buried and exposed residues , as would be observed for shallow optimization landscapes . finally , the width of the protein chains in figure 8 reflects the average positional fluctuation of each residue . it is noticeable that ddec charges vary more strongly in residues with a high degree of flexibility , especially when they form intramolecular contacts ( between -sheets , within -helices , and in sections linked by disulfide bridges ) , as one might expect . ddec / onetep charge and conformation fluctuations for nine nmr models of bpti . average positional fluctuation per residue is represented by its putty width and average charge fluctuation ( standard deviation ) per residue by its color . isa charge fluctuations have been clipped at 0.04 e for visual clarity ( ten residues in the isa method have values greater than 0.04 e , with a maximum of 0.06 e ) . isa charges are ddec / onetep charges derived with = 0 ( eq 7 ) . we showed in the previous section that ddec charges are able to accurately reproduce the esp around the structure that they are fit to . here , we investigate whether average charges derived for an ensemble of native state conformations are able to accurately describe the electrostatic properties of each conformation close to that native state . such a test is vital if the charges are to be used in the construction of force fields for simulating the dynamics of proteins . figure 9 shows the discrepancy between dipole moments calculated via atomic point charges and dft . to obtain the best agreement with the dft dipole moment of a particular structure , it is preferable to fit the ddec atomic point charges to that particular structure . however , encouragingly , there is very little degradation in performance if ddec charges that have been averaged over the full ensemble are used instead . this reflects the transferability of ddec charges between conformations close to the native state . the force field charges perform relatively well for this structure but not as well as the polarized protein - specific charges . this behavior is in agreement with previous work showing that esp charges give the best fit to ab initio electrostatics for a given structure , but that ddec charges are generally more transferable to different , similar structures . deviation between charge model and ab initio dipole moment vectors for nine nmr conformers of bpti . ddec / onetep charges have been calculated separately for each structure ( dark blue ) and averaged over all structures ( light blue ) . as we have demonstrated in the previous section , ddec charges derived from the dft electron density of entire proteins have properties that make them desirable for use in flexible force fields . we now put this into practice by performing molecular dynamics ( md ) simulations of the three proteins described previously ( pdb : 1ubq , 1mhn , 6lyt ) using force fields based on our calculated ddec charges . we have followed the procedure of tong et al . by taking the bonded and lennard - jones parameters directly from the amber ff99sb force field but replacing the atom - centered point charges by the ddec / onetep charges . it should be emphasized that torsional and lennard - jones parameters are as important as the charges in determining protein dynamics and a large number of alternative force fields exist , many of which outperform the ff99sb force field in comparison with experiment . nevertheless , here we concentrate on demonstrating the feasibility of using aim approaches to supplement an existing , commonly used force field . figure 10 shows the backbone rms deviations of the three proteins from their x - ray crystal structures over the course of 10 ns simulations , performed at 300 k in explicit water ( supporting information ) . for both force fields , backbone c root - mean - square deviation ( rmsd ) with respect to the initial experimental pdb structure for 1ubq , 1mhn , and 6lyt , with running averages taken over 50 ps windows . the first and last four residues have been excluded due to their high flexibility . to assess the quality of simulated mm dynamics , it is common to use , as metrics , experimental nmr - derived quantities . the square of the generalized order parameter s , based on the lipari szabo model - free approach to analyzing nuclear spin - relaxation measurements , is the plateau value of the time - correlation function c2(t ) of the second - order legrendre polynomial of backbone n h bond unit vectors e(t):13where c2(t ) is time - averaged over a trajectory .s represents the spatial restriction of the motion of n h bond vectors , and a lower s value corresponds to a more flexible residue . s values were calculated from our 10 ns md simulations using the ired method as implemented in the amber11 ptraj module . in this method , h unit bond vector pairs ei , j in a fixed reference frame and averaged over the entire trajectory:14s of residue i is then obtained from solving the eigenvalue equation mm = mm as15where the sum is taken over n 5 eigenvectors excluding five with the largest eigenvalues , and mi is the ith component of eigenvector m. figure 11 compares simulated and experimentals profiles for the standard amber ff99sb force field and the same force field supplemented by ddec / onetep atomic charges . the only difference between the simulation protocols is in the point charges and so any improvement in the calculated order parameters is due to the inclusion of native state polarization in their calculation . table 2 compares the agreement between simulated and experimental s data ( for all residues where experimental data is available ) . it indicates that ddec aim charges perform better than mean field force field charges in providing a suitable electrostatic environment that maintains protein stability throughout the 10 ns trajectory while remaining dynamically consistent with experimental observations . h order parameter s for 1ubq , 1mhn , and 6lyt using amber ff99sb and ddec atomic charges . the agreement of the generalized order parameter with experiment is improved by using the ddec / onetep protein - specific charges . in addition to backbone rigidity , we also compared the nmr scalar coupling jnc of the n backbone hydrogen bonds , which provides a measure of hydrogen bond dynamics within a protein . it was shown by barfield that jnc values , being dependent on the wave function overlap across the hydrogen bond , are strongly geometry dependent and can be parametrized as such by several formulas , with the simplest based on only the ho distance roh and ho = c angle :16equation 16 was used to compute the time - averaged jnc of backbone backbone hydrogen bonds using the amber ff99sb and ddec / onetep mm snapshots over the 10 ns trajectories . the rms deviations between simulation and experiment ( figure 12 , in brackets ) once again indicate that ddec / onetep charges perform at least as well as amber charges , illustrating that backbone n h and c = o bond polarization is also suitably described by the electron density partitioning approach to charge derivation . the ddec atoms - in - molecule scheme for the assignment of atomic charges through partitioning of the ab initio electron density has a number of features that makes it desirable for charge derivation for flexible force fields . however , its use in the analysis of realistic biomolecular systems has , until now , been limited by the computational expense of the underlying qm calculation . in this paper , we have implemented the ddec scheme in the linear - scaling dft code onetep . strict localization of the ngwf basis in onetep onto atomic regions allows derivation of the electronic density of systems comprising thousands of atoms with affordable computational expense . optimizing the ngwfs in situ allows for a minimal number of atom - centered orbitals to be used while maintaining near - complete basis set accuracy . systematic improvement in accuracy is achievable via tuning of a very small number of parameters and , along with a minimal parameter solvation model , allows the user to derive charges for any molecular system with no prior knowledge of its chemical characteristics . the ddec method is also suitable for implementation in other dft codes , as well as for example fragment molecular orbital approaches to generating the electron density of large systems , for which we hope our implementation will provide an accuracy benchmark . we have validated our implementation of the ddec method in onetep against the standard chargemol package with quantum chemistry calculations in gaussian09 . in agreement with previous observations , ddec charges perform less well than esp charges in reproducing the ab initio potential of small molecules . nevertheless , our interest here is in the calculation of polarized charges for entire proteins . indeed , we have found that the errors in the electrostatic potential at the surfaces of three proteins are very similar to those for small molecules ( errors of 1.31.5 kcal / mol ) . furthermore , the system - specific ddec charges outperform standard amber charges for these molecules ( errors of 57 kcal / mol ) . we would expect similar performances for other force fields that have been fit to small molecules without incorporating large - scale polarization . although the esp method is unsuited to the direct calculation of atomic charges from a single qm calculation due to the sensitivity of the charges to buried atoms and small conformational changes , a number of studies have determined protein - specific charges based on this method . by separating the system into a large number of dipeptide fragments , fitting esp charges to the resulting structures and averaging them over several conformations of the protein , stable and transferable charges have been obtained . by also including the effects of the environment , represented by point charges , in an iterative esp fit , it should be possible to further derive polarized protein - specific charges that are stable with respect to small changes in conformation . although the esp charges are preferable to ddec for reproducing the ab initio potential of small molecules , it is not obvious that these conformation - averaged esp charges are the optimum choice for large molecules . in fact , manz and sholl have shown that ddec and conformation - averaged esp charges give essentially the same agreement with the ab initio electrostatic potential of small molecules . it would be interesting , in future work , to make this comparison for larger molecules . further advantages of the ddec charges are that , since they are derived from a single dft calculation of entire biomolecules , environmental polarization is naturally included . moreover , no prior , system - specific chemical knowledge or specialized treatment is required to ensure proper charge derivation , rendering the method extremely versatile . in agreement with previous observations of smaller systems , the charges are insensitive to small conformational changes and buried atoms require no special treatment . in this paper , we are interested in the application of ddec charges to standard mm force fields . therefore , we have restricted the ddec expansion to monopole order in our electrostatic potential comparisons . as discussed by manz and sholl , the electrostatic potential description of ddec charges can be trivially improved by including higher order multipole terms in the partitioned aim densities , without requiring a new fitting procedure . we have incorporated the ddec charges of three proteins into a classical force field and run molecular mechanics simulations to compute nmr order parameters and scalar couplings . our observations indicate that the ddec aim charges perform at least as well , if not better than amber ff99sb in replicating protein dynamics . backbone hydrogen bonds are more polarized in ddec / onetep than in the standard amber force field . nevertheless , they are able to replicate dynamics consistent with experiments when used in mm force fields . our results are therefore consistent with previous hypotheses that electronic polarization is important in stabilization of a protein s native state . it should be noted that these ddec aims were obtained without explicit fitting to replicate electrostatic effects nor parametrization to experimental properties . the potential applications of linear - scaling ddec analysis span a wide range of problems in which accurate determination of electrostatics via atomic point charges is important . ddec charges have already been used to study molecular adsorption inside metal organic frameworks . in the biomolecular sciences , the optimization of protein - inhibitor interactions for drug design , pka calculation , study of ion channel conductivity and elucidation of enzymatic reaction mechanisms all depend critically on an accurate description of the electrostatic potential , for which ddec perform markedly better than standard classical force field charges .

atomic partial charges for use in traditional force fields for biomolecular simulation are often fit to the electrostatic potentials of small molecules and , hence , neglect large - scale electronic polarization . on the other hand , recent advances in atoms - in - molecule charge derivation schemes show promise for use in flexible force fields but are limited in size by the underlying quantum mechanical calculation of the electron density . here , we implement the density derived electrostatic and chemical charges method in the linear - scaling density functional theory code onetep . our implementation allows the straightforward derivation of partial atomic charges for systems comprising thousands of atoms , including entire proteins . we demonstrate that the derived charges are chemically intuitive , reproduce ab initio electrostatic potentials of proteins and are transferable between closely related systems . simulated nmr data derived from molecular dynamics of three proteins using force fields based on the onetep charges are in good agreement with experiment .

Introduction Computational Methodology Properties of DDEC Charges Protein Dynamics Conclusions

PMC3536429

hearing aid validation using aided speech evoked auditory evoked potentials is of research and clinical interest . such measurements involve elicitation of an evoked potential using a speech stimulus that has been processed through a hearing aid . hearing aids , being mostly nonlinear , may have implications for the nature of speech stimulus used as input . the present study focuses on the effect of nonlinear hearing aid processing on speech stimuli used for measurement of cortical auditory evoked potentials ( caeps ) . factors such as input level , duration , crest factor ( ratio of peak to root mean square ( rms ) amplitude ) , modulation depth , and modulation frequency of the input signal may affect the gain applied by the hearing aid , in ways that would not occur with a linear system [ 14 ] . these effects have been attributed to the level - dependent signal processing architecture , which in many hearing aids includes frequency specific compression threshold , compression ratio , compression time constants , number of channels , gain in each channel , expansion threshold , and expansion time constants [ 1 , 512 ] . in addition , hearing aid processing may also consider the frequency characteristics of the input stimulus ( e.g. , [ 13 , 14 ] ) . hence the output of a hearing aid to a specific input is the product of complex interactions between input stimuli and hearing aid features that may or may not be known to or may not be adjustable by the end user . nonlinear hearing aids , being sensitive to features of the input signal , process speech or speech - like stimuli differently from nonspeech stimuli [ 3 , 7 , 10 , 15 ] . since the main goal of hearing aid validation procedures is to assess benefit of hearing aid use while listening to speech , it is preferable that such procedures use speech stimuli in the most natural or frequently encountered form as possible . behavioural validation procedures ( tests that require active participation of the hearing aid user ) such as speech tests , mostly use speech in various natural forms . examples include the use of sentence materials , such as the bamford - kowal - bench sentence test , or materials with less grammatical context such as isolated words or nonsense syllables ( e.g. , the nonsense syllable test ) . but the speech stimuli may need to be modified for use in alternative validation methods such as aided auditory evoked potentials [ 1823 ] . aided auditory evoked potentials are objective and electrophysiological ( they record neural responses to sound ) but historically have not used speech stimuli . of these , one of the reasons caeps have been of interest in the validation of hearing aid fittings is because natural speech sounds can be used as stimuli [ 19 , 2327 ] . often phonemes or syllables excised from running speech or from standard speech tests have been used to record reliable caeps ( e.g. , [ 2729 ] ) . although natural speech can be used as stimuli , caep testing involves presentation of these stimuli with interstimulus intervals ( isi ) . these isis usually range on the order of 1 - 2 seconds ( e.g. , [ 23 , 29 , 30 ] ) optimized for the latency of caeps and refractory periods of the cortical pyramidal neurons [ 3032 ] . these stimuli are repeated 100200 times , with constant or slightly variable isis and caeps elicited to each of the presentations are averaged . presence of a caep elicited by a specific stimulus is interpreted as the stimulus being relayed to the source of caeps , the auditory cortex [ 21 , 24 ] . evidence suggests that caep thresholds ( i.e. , the lowest stimulus level at which a caep is detected ) are closely related to behavioral thresholds ( i.e. , the lowest stimulus level at which the participant detects the stimulus ) [ 33 , 34 ] . therefore , presence of a caep is likely to suggest audibility of the eliciting stimulus . on these premises , recent aided caep protocols for hearing aid validation have used brief segments of speech in the form of phonemes or syllables ( e.g. , [ 2125 ] ) . depending on their length , these brief segments may differ in their representation of certain features cues such as formant transitions , compared to longer segments of these same phonemes embedded in running speech . commercial equipment such as the hearlab uses phonemes , sampled across the speech frequency range presented at their naturally occurring levels within running speech , and presented in isolation to permit averaging of caep across several sweeps . phonemes presented in isolation for caep protocols may differ in several important ways from phonemes presented within running speech . in caep protocols , the target phoneme is preceded by an isi ( a silence period ) whereas the same phoneme in running speech is likely to be preceded by other phonemes . since nonlinear hearing aids continuously and rapidly adjust band - specific gains based on the acoustic input , there is a possibility that the hearing aids may react differently to the same phoneme when presented during aided caep testing as compared to when they occur in running speech . with 1 - 2 seconds of isi preceding every repetition of the stimulus , nonlinear hearing aids may demonstrate an overshoot at the onset of the stimulus consistent with compression circuitry . also , hearing aids of different models and different manufacturers may vary in how quickly they respond to changes in the acoustic input . therefore , verifying that hearing aid output is comparable for phonemes presented in these two contexts ( preceding silent periods / isi versus embedded in running speech ) may be an important step in evaluating the validity of using caep protocols in hearing aid validation . previous reports on non - caep related measures suggest that certain features of nonlinear signal processing in hearing aids may attenuate the level of speech sounds immediately preceded by silence [ 37 , 38 ] . the effects of caep protocols on the gain achieved while processing tone bursts have been reported elsewhere in this issue [ 40 , 41 ] . these studies provide evidence that hearing aid gain differs for tone bursts ( short and long ) presented in isolation versus pure tones that are continuous . specifically , the gain achieved during processing of tone bursts was lower than the verified gain , when measured at 30 ms poststimulus onset and at maximum amplitude . onset level is of interest because the first 30 to 50 ms of the stimulus primarily determines the characteristics of the elicited caep . stimulus level of the hearing aid processed tone bursts was positively related to the caep amplitude , with stimulus level at 30 ms poststimulus onset being a better predictor of caep amplitude compared to maximum stimulus level . these reports [ 40 , 41 ] substantiate the need to verify output levels of caep stimuli across contexts , and to consider stimulus onsets . the present study will focus upon aided processing of phonemes across contexts and measure both overall level ( level measured across the entire duration of the phoneme ) and onset level of the stimuli at the output of the hearing aid . the purpose of this study was to understand if hearing aids process caep phonemes presented in isolation differently to phonemes presented in running speech . the primary outcome measure of interest in this study was the output level of phonemes in both contexts . findings from this study may provide some insights into the design of hearing aid validation protocols that employ aided caep measures , because large differences in hearing aid output arising due to stimulus context may influence interpretation of audibility based on aided caeps . hearing aids were sampled across a representative range of major manufacturers and were behind - the - ear ( bte ) in style . of the 10 hearing aids , six were programmed and verified to meet dsl v5a adult prescription targets for an n4 audiogram . the n4 audiogram represents hearing loss of moderate to severe degree with thresholds of 55 db hl at 250 hz worsening down to 80 db hl at 6 khz . the remaining four hearing aids were programmed and verified to meet dsl v5a targets for an n6 audiogram . the n6 audiogram represents hearing loss of severe degree with thresholds ranging from 75 db hl at 250 hz worsening to 100 db hl at 6 khz . hearing aids appropriate for different audiograms were chosen from different manufacturers to obtain a representative sample of commonly available commercial products . all hearing aids were programmed to function on a basic program with all additional features such as noise reduction , feedback cancellation , and frequency lowering disabled during verification and recording . as such , variance across devices is mainly attributable to the nonlinear characteristics of the devices , in isolation of these other aspects of hearing aid signal processing . stimuli were constructed to have both running speech and phoneme - in - isolation contexts as follows . for the running speech context , eight phonemes ( /a/ , /i/ , /u/ , /s/ , // , /m/ , /t/ , and /g/ ) were identified within a recording of the rainbow passage . the passage was spoken by a male talker and lasted 2 minutes and 14 seconds . aided recordings of this passage were made for each hearing aid , and the level of each phoneme was measured from within the aided passage . for the isolated context , the same phonemes and phoneme boundaries were used , but were excised from the passage for use as individual stimuli . boundaries of these phonemes were chosen such that any transitions preceding and following these phonemes due to coarticulation were excluded . ms , /i/84 ms , /u/124 ms , /s/133 ms , //116 ms , /m/64 ms , /t/26 ms , and /g/19 the durations of these phonemes differed naturally and were not modified in order to allow direct comparisons between the two contexts . these specific phonemes were chosen as the first six of these phonemes are a part of the commonly used ling 5 or 6 sounds test [ 44 , 45 ] . the last three have been commonly used in a series of aided caep studies ( e.g. , [ 26 , 27 , 46 ] ) and are also a part of the stimulus choices available in the hearlab . a silent interval of 1125 ms preceding each phoneme was created using sound editing software goldwave ( v.5.58 ) . this is to simulate a caep stimulus presentation protocol where the isi usually ranges between one and two seconds . recordings of hearing aid output used a click - on coupler ( brel & kjr ( b&k ) type 4946 conforming to ansi s3.7 , iec 60126 fitted with microphone type 4192 ) with an earplug simulator . this was set up in a b&k anechoic box ( box 4232 ) that also housed a reference microphone . the outputs of the reference and coupler microphones were captured in spectraplus ( v5.0.26.0 ) in separate channels using a sampling rate of 44.1 khz with 16-bit sampling precision . spectraplus software was used to record the reference and coupler signals as .wav files for further signal analyses . running speech was presented at overall rms levels of 55 , 65 , and 75 db spl . these levels approximate speech at casual through loud vocal effort levels . since individual phonemes naturally varied in their relative levels within the rainbow passage , the level of each isolated phoneme was matched to the level at which it occurred in the rainbow passage , for each presentation level . with this recording paradigm , the overall input levels of each phoneme were matched between the two contexts . during presentation of phonemes in the isolation context , approximately 10 repetitions of each phoneme ( each preceded by isi of 1125 ms ) were presented during any single recording . two measurements were made per phoneme and per context : the overall level of the phoneme ( db spl rms recorded over the entire duration of the phoneme ) and the onset level of the phoneme ( db spl rms recorded over the first 30 ms of the stimulus phoneme ) . onset measurements could not be completed for phonemes /t/ and /g/ as the duration of these phonemes was shorter than 30 ms . for these phonemes , the first few repetitions were discarded as , in our preliminary recordings using a few hearing aids , interrepetition variability was observed to be high in the first few repetitions . this is likely related to nonlinear signal processing in the hearing aids but these effects were not formally evaluated in this study . figures 1(a ) and 1(b ) illustrate examples of the variability observed in the first few repetitions . repeated measures of analysis of variance ( rm - anova ) were completed using spss ( v. 16 ) with context ( running speech and isolation ) , level ( 55 , 65 , and 75 db spl ) , and phoneme as the three independent factors . for interpretation of these multiple t - tests , sequential bonferroni type corrections that control for false discovery rates were used to determine critical p values [ 49 , 50 ] . phonemes embedded in running speech were measurable for nearly all hearing aids in this study . for one of the hearing aids , the output level of /g/ in isolation at 55 db spl input level could not be measured as it was embedded within the hearing aid noise floor . across the sample , the average overall phoneme level measured in the running speech context was 94.07 db spl ( standard error ( se ) = 1.79 db ) and in the isolation context was 92.43 db spl ( se = 1.94 db ) . on average , the phoneme onset level measured in the running speech context was 94.67 db spl ( se = 1.79 db ) and in the isolation context was 94.44 db spl ( se = 1.83 db ) . the outcome of statistical tests for overall phoneme level and phoneme onset level will be described below . rm - anova revealed a significant effect of context ( f = 10.114 [ 1 , 8 ] , p = 0.013 ) , input level ( f = 834.58 [ 1.02 , 8.12 ] , p < 0.001 ) , and phoneme ( f = 93.26 [ 1.95 , 15.62 ] , p < 0.001 ) . interactions between input level and context ( f = 8.36 [ 1.35 , 10.82 ] , p = 0.011 ) , phoneme and context ( f = 3.38 [ 2.63 , 21.05 ] , p = 0.042 ) , and input level and phoneme ( f = 5.25 [ 2.69 , 21.56 ] , p = 0.009 ) were also significant . the three - way interaction between input level , context , and phoneme was not significant ( f = 1.061 [ 2.48 , 29.79 ] , p = 0.388 ) . paired contrasts comparing overall phoneme levels between contexts at each input level showed significant differences at the 55 and 65 db spl input levels but not at the 75 db spl input level . at input levels of 55 and 65 db spl , the levels of phonemes were significantly higher when they appeared in running speech compared to when they occurred in isolation ( see figure 2(a ) and table 3 for group means ) . in summary , the difference between contexts reduced as input level increased . paired contrasts comparing overall phoneme levels between contexts for each phoneme showed significant differences for all phonemes except /m/ all phonemes except /m/ were higher in level when they occurred in running speech compared to when they occurred in isolation . a similar result was obtained for phoneme onset level . rm - anova revealed a significant effect of context ( f = 7.41 [ 1 , 9 ] , p = 0.024 ) , input level ( 846.94 [ 1.05 , 9.44 ] , p < 0.001 ) , and phoneme ( f = 52.84 [ 1.78 , 16.04 ] , p < 0.001 ) . interactions between input level and context ( f = 17.71 [ 1.20 , 10.81 ] , p = 0.001 ) , and phoneme and context ( 3.95 [ 3.45 , 31.09 ] , p = 0.013 ) were significant . interaction between input level and phoneme ( f = 1.49 [ 2.06 , 18.56 ] , p = 0.250 ) and the three - way interaction between input level , context , and phoneme were not significant ( f = 0.89 [ 3.25 , 29.25 ] , p = 0.473 ) . paired contrasts between phoneme onset levels of both contexts at each input level showed significant differences between contexts at 55 and 65 db spl but not at the 75 db spl input level . at input levels of 55 and 65 db spl , the onset levels of phonemes were significantly higher when they appeared in running speech compared to when they occurred in isolation ( see figure 3(a ) and table 3 for group means ) . similar to overall phoneme level , the difference between contexts reduced with increasing input level . paired contrasts comparing phoneme onset levels between contexts for each phoneme revealed no significant differences for all phonemes except // and /u/ ( see figure 3(b ) and table 4 for group means ) . phonemes // and /u/ were higher in onset level when they occurred in running speech compared to when they occurred in isolation . the mean difference in overall phoneme level averaged across hearing aids , input levels , and phonemes was found to be 1.64 db , where phonemes in running speech measured higher on average . the mean difference in phoneme onset level computed similarly was 0.23 db , onset of phonemes in running speech measuring higher on average . although the mean value suggests a clinically insignificant difference due to context , inspection of individual data highlights the differences observed across hearing aids and phonemes . tables 5(a ) and 5(b ) provide the difference ( in db ) in the output measures ( overall phoneme level and phoneme onset level ) in both contexts , averaged across all three input levels . these differences were obtained by subtracting the level of each phoneme in isolation from the corresponding level in running speech . hence , a positive value indicates that the level of the phoneme is higher when it occurs in running speech , as it would in daily life , versus in isolation , as it would during caep measurement . differences of greater than 3 db are presented in bold . the proportion of difference values greater than 3 and 5 db are presented in table 6 for both overall phoneme levels and phoneme onset levels at each input level . pooled across both directions of differences and input levels , about 24% of the overall phoneme levels ( total of 239 observations across three levels , 10 hearing aids and eight phonemes , 1 missing value ) showed differences of greater than 3 db and 7% showed differences of greater than 5 db . in case of phoneme onset levels , about 33% of the observations ( total of 180 observations across three levels , 10 hearing aids and six phonemes ) showed differences of over 3 db and nearly 13% showed differences of over 5 db . in general , differences greater than 3 db are well outside of test - retest differences in electroacoustic measurement , while differences greater than 5 db are greater than a typical audiometric step size . the latter is likely clinically significant , while the former may have impact for interpretation of research data and calibration . we note that the majority of aided phoneme levels agreed between the two contexts within 3 db . results suggest that hearing aid output level of a phoneme in isolation may either match or may differ from the output level of the same phoneme when it occurs in running speech . agreement was observed in approximately 66% to 75% of cases , while differences exceeding 3 db were observed in 24% to 33% of cases . agreement occurred in more cases ( 75% ) for measures of overall level of phoneme , and in fewer cases ( 66% ) for measures of phoneme onset level . when differences existed , they typically manifested as the hearing aid producing a lower output for the phoneme in isolation than it did for the phoneme in running speech . results from the present study are similar to the findings from other reports in this issue [ 40 , 41 ] . specifically , these reports and the current study show that across measurement strategies and stimulus types , hearing aids may apply lower gain and output ( at onset as well as at maximum amplitude ) to brief stimuli that are immediately preceded by silence , such as those commonly used to elicit the caep . however , one may note that the hearing aids used in these studies [ 40 , 41 ] were set to function linearly , unlike the hearing aids used in the present study . another study has used a nonlinear hearing aid to study the effect of hearing aid processing on the tone burst onset while comparing it with the unaided condition . the aided condition in this study produced a marginal increase in the level at onset due to the presence of an overshoot . in the present study , there were fewer instances of significant overshoot , but recall that the unaided condition was not assessed in this study . therefore , the present results pertain only to the comparison of aided levels between the isolation context and running speech . overshoot may be present in both conditions . also , the effects of overshoot attributable to nonlinear signal processing in hearing aids may vary across devices , with the effects being idiosyncratic to specific devices or stimuli . results similar to the majority of the observations in the present study have also been noted in non - caep related studies of nonlinear signal processing in hearing aids [ 37 , 38 ] . the decrease in differences in overall and onset level of phonemes between contexts with increase in input level could indicate an effect of output limiting . as the output levels of phonemes come close to the maximum power output of the hearing aids , they are subject to compression limiting [ 1 , 5 ] . compression limiting restricts the maximum output level by using a very high or infinite compression ratio in an output controlled compression system . hence , at higher input levels , where the output levels are likely subject to output limiting in both stimulus contexts , the differences seen are smaller compared to lower input levels that are relatively less likely to be affected by output limiting . we did not perform a direct comparison across phonemes because the individual phonemes occur at different levels relative to , the overall rms level of running speech . compression , being a level - dependent nonlinear factor in the hearing aid , may therefore vary the gain applied for each of these phonemes , especially when they are presented in isolation . in addition , compression features such as compression ratio and time constants were likely different across different frequencies due to the slightly sloping configurations of audiograms chosen and the presence of multiple channels in our hearing aid sample . since phonemes varied in their spectral composition and position of spectral peaks one stimulus characteristic that could have been influential in determining overall phoneme output levels is the duration of phonemes . table 5(a ) suggests that differences larger than 3 db occurred more often for /g/ and /t/ relative to other phonemes . among all eight phonemes , /t/ and /g/ were the lowest in level and shortest in duration , measuring 26 ms and 19 ms , respectively . this may have made these phonemes in isolation more susceptible to the dynamic effects of hearing aid nonlinearity [ 1 , 37 , 38 ] . however , this study did not study systematically the effects of duration and level as they interact with context . further study on this may be necessary to determine the effects of phoneme level and duration . also , the preceding context within running speech may have differed in ways crucial to determination of gain / compression characteristics for the target phoneme . tables 5(a ) and 5(b ) illustrate that individual hearing aids may amplify individual phonemes differently , even though they were set to produce similar gain for long - duration signals . hearing aids not only varied in differences due to context but also showed differences for the same phoneme in the same context . differences in other parameters across hearing aid manufacturers have also been reported among hearing aids that were matched in gain characteristics ( e.g. , sound quality comparisons by dillon et al . ) . the finding that hearing aids show large individual variability makes it challenging to predict the nature of differences on a case - by - case basis in clinical practice . parameters such as amplitude and latency of individual peaks reflect changes in stimulus level or sensation level of the stimulus with reference to the behavioral threshold of the caep stimulus . a change in sensation level of the stimulus from a positive ( above threshold ; audible ) to a negative ( below threshold ; inaudible ) value is likely to decrease the probability of eliciting a caep . if output levels of phonemes in running speech are considered to be the reference condition of interest , caep test measures may underestimate audibility when phonemes are presented in isolation . these data indicate that underestimation is minimal ( about 2 db ) on average , but was between 3 and 8 db in over 24% of cases . there were also instances that may result in overestimation of audibility , but these are far fewer in number and magnitude . since the experimental conditions used in this study were limited to one duration of isi and one naturally occurring preceding context per phoneme , generalization to other instances and variation across durations or levels of phonemes may require further investigation . investigation of the effects of hearing aid signal processing on spectral characteristics such as formant transitions may also be possible , but these effects were not evaluated in this study . the effects of other aspects of hearing aid signal processing , such as digital noise reduction , may also be relevant and were not explored in this study . based on this study , we conclude that significant differences in hearing aid functioning between running speech and isolated phoneme contexts occur , along with considerable interhearing aid variability . in over a fourth of aided phonemes , the magnitude of these differences was large enough to impact calibration , or interpretation of group data . this may indicate the need to perform acoustic calibration for individual hearing aids for the purpose of well - defined caep stimuli . in 7%13% of phonemes , the differences exceeded that of an audiometric step size and therefore may be clinically important .

background . functioning of nonlinear hearing aids varies with characteristics of input stimuli . in the past decade , aided speech evoked cortical auditory evoked potentials ( caeps ) have been proposed for validation of hearing aid fittings . however , unlike in running speech , phonemes presented as stimuli during caep testing are preceded by silent intervals of over one second . hence , the present study aimed to compare if hearing aids process phonemes similarly in running speech and in caep testing contexts . method . a sample of ten hearing aids was used . overall phoneme level and phoneme onset level of eight phonemes in both contexts were compared at three input levels representing conversational speech levels . results . differences of over 3 db between the two contexts were noted in one - fourth of the observations measuring overall phoneme levels and in one - third of the observations measuring phoneme onset level . in a majority of these differences , output levels of phonemes were higher in the running speech context . these differences varied across hearing aids . conclusion . lower output levels in the isolation context may have implications for calibration and estimation of audibility based on caeps . the variability across hearing aids observed could make it challenging to predict differences on an individual basis .

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

PMC3515921

carcinoma of the prostate is the most common malignancy in men with increased incidence rates owing to the population ageing and the improvement of diagnostic procedures . the early detection of the bone metastases is of value in making decision regarding the treatment plan , which may vary extremely according to the bone status . the likelihood of the bone metastases at the first diagnosis varies with the histological score and serum level of prostate - specific antigen , and it is possible to some extent to estimate the degree of tumor spread in the light of the psa value . bone involvement is much less frequently involved with low psa level , so that the bone staging is not recommended with psa less than 10 ng / ml , except if there are known bone disorders which may later cause false positive findings [ 13 ] . however , in patients with psa relapse after primary therapy it is not easy to be guessed based on psa values whether the bone involvement or another involvement is behind a psa rising . in established skeletal involvement , there is a need of effective imaging method to monitor the status progress . bone scan is the widely used screening technique for assessing the bone status in the most malignancies , and it shows mostly high sensitivity in detecting the bone involvement . pet / ct with f - fluorodeoxyglucose ( fdg ) is known to have general limitation in prostate cancer . pet with radiolabeled choline such as f - fech - pet was found to be an effective method in diagnosis of primary and recurrent pca tumors as well [ 4 , 5 ] . two possible mechanisms have been proposed to explain the increased choline uptake in prostate cancer cells . the first is increased cell proliferation in tumors , because choline is a precursor for the biosynthesis of phosphatidylcholine and other phospholipids , the major components of the cell membrane . the second explanation is the overproduction of choline kinase in cancer cells , which was experimentally confirmed in human - derived prostate cancer [ 68 ] . although , f - fech - pet is being increasingly used primarily in patients with prostate cancer and for followup , its efficacy in detecting the bone involvement still needs further investigations in comparison with the established method ( bone scan ) . to our knowledge we aim in this study mainly to assess the value of f - fech - pet / ct in detecting the bone metastases and to compare the results with that of bone scan . 37 patients ( mean age 69 7 ) had been referred in our department for re - staging purposes due to biochemical recurrence , psa median 2.6 ng / dl ( range 0.321 ) . gleason 's score ranges among 3 and 9 : gs.7 ( n = 12 ) , gs.8 ( n = 8) , gs.9 ( n = 7 ) , gs . 3 ( n = 1 ) , and gs 5 ( n = 1 ) . the patients underwent a wide variety of initial therapies including radical prostatectomy ( n = 11 ) , radiotherapy ( n = 7 ) , and pure hormonotherapy in patients with locally advanced disease ( n = 3 ) . a number of patients underwent combined therapy due to previous slight psa increase such as radical prostatectomy followed by salvage radiotherapy ( n = 12 ) , radical prostatectomy followed by salvage antihormonal therapy ( n = 3 ) , or radical prostatectomy followed by hifu ( n = 1 ) . the study was approved by the ethics committee of the heidelberg university , and a written consent was obtained from all patients . bone scintigraphy was performed using a modern double - head gamma - camera ( ecam , siemens medical solution ) equipped with low - energy , high - resolution collimators . whole - body images were obtained 2 - 3 h after intravenous injection of 700 mbq ( 19 mci ) of tcm - mdp at the scan speed of 15 cm / min in the anterior and posterior projection ; additional imaging were acquired , when required . all pet studies were carried out using a biograph 6 pet / ct ( siemens / cti ) . imaging was started as dynamic imaging immediately parallel to administration of a standard of 250 mbq of f - ethylcholine for 10 min before proceeding to whole body imaging . for attenuation correction of the pet scan , a low - dose ct ( 130 kev , 30 mas ; care dose ) without contrast medium was done . static emission scans , corrected for dead time , scatter , and decay were acquired from the vertex to the proximal legs requiring eight bed positions , 4 minutes each . the images were iteratively reconstructed with the osem algorithm using four iterations with eight subsets and gauss filtering to an in - plane spatial resolution of 5 mm at full - width half - maximum . the delay between both modalities ranged between 2 and 45 days ( average 14 days ) . the images were evaluated visually by two skilled nuclear medicine physicians in presence of a skilled radiologist after viewing the images in different planes . the image assessment was achieved in combination with the clinical information and close correlation with other available imaging studies . the findings concerning the bone status were assessed as positive in presence of typical findings validated with followup and/or by correlation with other modality and as negative in absence of the pathological bone uptake ( there was no need to prove the negativity ) , or false positive / negative ( if proved the contrary ) . sensitivity , specificity , npv , and ppv are the statistical indicators utilized in assessing the universal outcome . in the patients with confirmed bone metastases ( n = 18 ) , the skeleton was divided into five regions ( skull , ribs , pelvis , vertebra , and extremities ) to simplify the comparison between the matching regions in both examinations . the metastases had been drawn as black points on a separate skeleton outline ( figure 1 ) . each imaging was evaluated separately , so that the interpretation would not be influenced . the anona test was used to compare the extent of metastatic spread in both examinations in the different regions . suvs were acquired by manually drawing a volume of interest over the pathologic bone lesions that had been identified on visual analysis of static emission images . by using attenuation - corrected pet data , suvs were calculated as the ratio of regional radioactivity concentration ( becquerel / milliliter ) divided by the injected amount of radioactivity ( becquerel ) normalized to body weight in gram . ct reading was based on characteristic patterns of morphological change ; all detectable lesions on ct were categorized by a radiologist as sclerotic , lytic , or mixed metastases . the quantitative radiodensity of sclerotic lesions was measured by means of the hounsfield unit ( hu ) . for quantitative assessment of mdp uptake , we considered an uptake score based on the visual assessment and graduated from 0 till 4 ( 0 no uptake , 1 decent uptake , 2 moderate uptake , 3 high uptake , 4 extremely severe uptake ) . so the intensity of choline uptake was once compared with the radio - density represented by hu and once with mdp uptake ( evaluated visually ) . lesions detected in ct and evaluated as high suspected were encompassed in this comparison , regardless whether they showed a positive choline uptake or not . some limitations should be kept in mind such as the absence of pathological verification of the bone metastases . thus , the patients were confirmed as having bone metastases just depending on clinical followups in correlation with other diagnostic tools , for example mri . the average clinical followup duration to confirm the final diagnosis was 1 year . the characteristics of the patients are summarized in table 1 . generally , 18 patients out 37 patients referred with biochemical relapse were confirmed to have bone involvement . since the pathological confirmation was not available , the followup and correlation with other modalities were adopted in our verification . ( followup was available in 11 patients ; in the remaining seven patients , the verification was based on a typical findings in pet , assessed by two nuclear medicine physicians matching with morphological images such as ct or mri . ) in patients with positive bone findings , the psa median was 4 ng / dl ( range 121 ) . by contrast , the patients with negative bone findings had a psa median value of 1 . 5 ng / dl ( range 0.319 ) . statistically there was a significant difference between both groups ( p = 0.02 ) ( figure 2 ) . in patients with negative bone findings the psa relapse was attributed to lymph node metastases ( n = 5 ) , local recurrence ( n = 2 ) , and lung metastases ( n = 1 ) . confirmed bone involvement cases , f - fech - pet / ct identified correctly the bone involvement in 15 cases with a sensitivity of 83.3% . no false positive was identified ; consequently its specificity was 100% , npp 86.3% , and ppv 100% . on the other hand , bone scan showed the presence of the metastases in 17 out of 18 confirmed cases with sensitivity of 94.4% . from 19 negative findings , the bone scan showed two false positive cases due to paget disease and trauma . as mentioned earlier , the skeleton had been divided into multiple regions to simplify the matching of findings . totally , the patients with positive findings have 122 lesions . f - fech - pet was capable to identify 101 lesions with a sensitivity 82.7% versus 109 identified using bs ( bone scan ) with sensitivity of 89.4% . the sensitivity of both modalities differs extremely with the anatomical region ( table 2 ) . indeed this superiority of bone scan was demonstrated in ribs ( 71% in f - fech - pet versus 94% in bs ) and cranium . in remaining regions , f - fech - pet was superior in identifying more metastases , in vertebrae ( 92% versus 85% ) and pelvis ( 91% versus 89% ) . in the extremities f - fech - pet shows also superiority over bone scan ( 90% versus 50% ) in spite of limited field of view ( fov ) figure 3 . statistically using anova test there was no significant difference between both modalities ( p > 0.1 ) in the number of detected metastases in all anatomical regions . degenerative changes : f - fech - pet / ct versus bs ( figure 4)whereas the bone scan demonstrates an intensive uptake in this degenerative change , the f - fech - pet / ct shows a negative choline uptake . whereas the bone scan demonstrates an intensive uptake in this degenerative change , the f - fech - pet / ct shows a negative choline uptake . trauma : f - fech - pet / ct versus bs ( figures 5 and 6)these images show a discrepancy between mdp uptake and choline uptake in a compression fracture and rib fractures . these images show a discrepancy between mdp uptake and choline uptake in a compression fracture and rib fractures . a total of 94 out of 122 with the following distribution : ( 3 in cervical spine , 10 in lumbar spine , 35 in pelvis , 7 in ribs , 4 in sternum , 15 in thoracic spine , 12 in the extremities and 7 in other regions ) were selected to make a further quantitative comparison between f - fech - pet and both ct and bone scan using the above mentioned quantitative parameters . 28 lesions were excluded from this analysis , for example , in ribs due to neighborhood to liver ( false high suv through partial volume effect ) or due to technical limitations . as previously mentioned , the quantitative assessment in bone scan was based on visual score escalating from 0 till 4 . based on the radiological portrayal in ct , lesions were divided into osteoblastic lesions ( n = 42 ) , osteolytic ( n = 19 ) , and mixed ( n = 16 ) . 16 lesions of positive choline uptake : ( 11 in the spine7 thoracic and 4 lumbar2 in the pelvis and 3 in the extremities ) were lacking morphological alterations . they were characterized with high choline uptake ( suv 5 , 95 1 , 5 ) and mostly with decent or absent mdp uptake . they were attributed to bone marrow involvement ( figure 7 ) 13 of sclerotic lesions ( 6 in pelvis , 3 in thoracic spine and 4 in ribs ) ( hu mean 739 216 ) were of negative choline uptake but mostly of intensive mdp uptake ( qmdp score 3 ) ( qmdp quantitative mdp evaluated visually ) . the decline or absence of choline uptake in sclerotic metastasis was noted in particular in patients receiving systemic therapy ( figures 8 and 11 ) . in contrast , the osteolytic metastases ( 4 in the pelvis and 1 in the humerus ) were characterized with high choline uptake ( 7 2 , nadir 5.5 ) and wide extent of mdp uptake ( 4 showed negative uptake , 4 showed intensive uptake and 2 showed moderate uptake ) . a significant negative correlation was demonstrated between tracer uptake suv(max ) and the density of sclerotic lesions assessed by hu ( r = 0.58 , p < 0.01 ) ( figure 9 ) . on the other hand , numerical score of mdp was correlated negatively with suv(max ) value ( r 0.01 ) and positively with hu ( r 0.34 ) . however , they were statistically not significant . in few mixed metastases it was demonstrated that the choline uptake was solely concentrated in the osteolytic part evading the sclerotic part ( figure 10(c ) ) . in pure sclerotic metastases it was shown that uptake concentrates at the rim of metastases , where there is a transitional region with a moderate sclerosis ( figures 10(a ) and 10(b ) ) . examples of the effects of ongoing systemic therapy on pet findingssee figures 11 and 12 . it is known that the skeleton is a favorable place for metastasis in prostate cancer . it is the second most common site of metastatic disease after lymph nodes and considered as the main cause of morbidity and mortality in prostate cancer patients and mostly related to poor prognosis . although bone scan is ranked first in assessing bone status in many malignancies including pca , it still lacks specificity . the comparison between pet modality and bone scan in detecting bone involvement was a concern of many studies , mostly involving f - fdg pet / ct [ 911 ] . cheng et al . showed , in their review of six studies , that f - fdg pet / ct has both higher sensitivity and specificity than bone scintigraphy . in patients with pca , tiwari et al . proved that f - fdg - pet / ct can play a complimentary role to the conventional skeletal scintigraphy , particularly in detection of bone marrow disease . whether or not f - fech - pet / ct is capable to assess the bone status in an adequate way or requires supplementary test is still blurred . according to our knowledge , there is lack of studies concerning the comparison of f - fech - pet / ct with standard bs . . showed that f - choline- pet / ct had lower sensitivity than f fluoride pet - ct for detection of bone metastases ( f - fluoride is bone seeking pet radiopharmaceutical ) . bone scan showed superiority over f - fech - pet / ct in patient - based results ( sensitivity 81.2% versus 93.7% ) . however , the high sensitivity of bone scan encounters a high likelihood of false positive findings . by contrast , f - fech - pet / ct appears to be less affected by such unspecific lesions . the benefit of associated diagnostic ct ( occasionally with contrast media ) should be kept in mind in minimizing the false positive cases . yet , ct is not the sole factor affecting the increase of the specificity of f - fech , mccarthy et al . suggested that f - fech - pet can separate benign conditions such as trauma and arthropathy from malignancy and may be useful as an assistant to bone scan in equivocal cases . basically , there is need to perform bone scan in case of negative f - fech - pet / ct finding if there is clinical suspicion , under suggestion that f - fech - pet is of less sensitivity . however , bone scan can be reserved and an unnecessary radiation exposure can be avoided , if proven otherwise . so whether or not bone scan can be abandoned as a complementary test to f - fech - pet / ct is of a high clinical value . we found that f - fech - pet / ct showed less sensitivity compared with bs ; however , two out of three patients demonstrating false negative pet findings were under systemic therapy , suggesting a potential impact of systemic therapy in minimizing the sensitivity . in our patient group , looking at the findings of both modalities in the various anatomical regions , we observed better lesion detection efficiency of bs in the ribs with sensitivity of 98% versus 73% . on the other hand , the f - fech - pet / ct was more effective in pelvis ( 93% versus 87% ) and in extremities in spite of the presence of few metastases outside the field of view . in vertebra the f - fech - pet / ct was also more accurate than bs . because of poor spatial resolution of bs , an uptake in the spine could be incorrectly attributed to degenerative changes that may explain the low sensitivity of bs in spine compared with f - fech - pet / ct . moreover spect ( single photon emission computed tomography ) can minimize the shortcoming of planar bs in the assessment of the spine ; however it is not routinely applied . in this regard , it should be mentioned that spinal metastases have typical locations , mostly in the posterior part of vertebrae due to the many short intraosseous arteries [ 16 , 17 ] , and they can be easily recognized and differentiated from degenerative changes in f - fech - pet / ct in the light of morphological view provided by ct component . in other anatomical regions f - fech - pet / ct showed advantage over bs ; this can be attributed to the superior spatial resolution . of course the associated ct contributed to a certain degree in increasing the sensitivity of f - fech - pet / ct through detecting the bone metastases of a negative choline uptake . in the same topic , we found that fech uptake decreases with increasing sclerosis ( negative correlation between hu and suv(max ) r = 0.58 , p < 0.01 ) . this issue emphasizes the importance of ct in compensating the deficiency of f - fech - pet in patients under aht . further benefit of ct is the validating the pet finding through excluding other benign changes that may share the same findings with the metastases . since the bone marrow is associated with minimal morphological changes and unusual to cause bone reaction , it remains undetectable or can be easily overlooked using morphological modalities or using bone seeking nuclides . kato and coworkers reported a case of bone marrow metastasis finally detected by marrow biopsy , without any abnormality on bone scintigraphy or computed tomography . generally , the bm aspiration and biopsy remain the procedures of choice to detect bm involvement . f - fdg pet is supposed to have special value in detecting bm involvement ; aydin et al . described a case of bone marrow metastasis detected by f - fdg pet and missed by bone scintigraphy in widespread melanoma . f - fech - pet / ct in prostate cancer patients shares this property , whereas there is no role of ct or bs . described a positive choline bone marrow metastasis in the thoracic spine without any ct abnormality . in our results , pet positive findings without morphological changes in spine , pelvis and proximal extremities were attributed to bone marrow involvement . bs was not of further benefit in identifying them ; they were either of negative or decent mdp uptakes . that raises the following question : what is the real clinical benefit of bs over f - fech - pet / ct , if the deficiency of choline - pet is mainly in osteoblastic metastases , which are simply to be visualized using associated ct ? in other words , if ct can adequately compensate the deficiency of f - fech - pet , still bone scan offers further diagnostic information ? indeed , in order to validate its further performance as complementary test , bone scan is required to show a superiority over f - fech - pet / ct both components pet and ct . just looking at lesion - based results , there is no real justification for additional bone scan , since the gain in detecting more lesions is not valuable in patients with multiple metastases , who should anyway undergo systemic therapy . however , in patient - based results we notice that associated ct failed in identifying three false negative cases in f - fech - pet / ct , which were ultimately detected using bs . more advantage of bone scan is emphasized in its availability and ease to repeat for followup . moreover , in multiple bone metastases if pain radiotherapy is indicated , bone scan is necessary because mdp distributes in a similar way as bone seeking therapy - tracer . in f - fech - pet / ct , choline uptake is supposed to be linked with presence of tumor cells which can transport and metabolize it . in contrast , bs displays the bone metastases indirectly through demonstrating the structural alterations . for this reason , any alteration concerning the tumor focus ( whether progress or regress ) is supposed to be first detected in choline - pet . this fact and the possibility to perform a quantitative assessing grant the f - fech - pet a unique value in therapy monitoring . subsequent to therapy onset , a drop in suv is an appropriate indicator for response . by contrast , in bone scan the therapy response may lead to transient increasing mdp uptake due to an amplified osteoblastic response in the so - called flare phenomenon . this can be mistaken with progress and typically lasts about 6 months after therapy [ 2123 ] . for this reason , the early response ct is also of negligible role in therapy monitoring , since the morphological alterations occur in delay . to understand the weakness of bs in therapy monitoring , we ought to return to the pathology of the bone metastases in prostate cancer . in this type of bone metastasis , intramembranous ossification takes place in regions of fibrous stroma and after therapy administration ; the ossification process stays for a certain time running in the manufactured stroma despite damaging the causing tumor cells and evoked mediators . in other words , the ongoing mineralization processes , thus mdp uptake , would not be inhibited after therapy onset even if the therapy is successful . compared with bs , as mentioned earlier , choline uptake in f - fech - pet / ct is supposed to reflect directly the tumor focus , and the reduction in suv early after initiation of therapy can distinguish responders from nonresponders . however , does uptake decline always indicate response ? in some cases , it was demonstrated that shortly after treatment onset , the f - fech - pet / ct turned to be negative just in osteoblastic metastases , while osteolytic metastases were preserved . this effect of aht in the sclerotic metastases was already described by beheshti et al . . it may also explain the lower total sensitivity of f - fech - pet / ct compared with bs in the area - based results ( 6 patients were under ongoing aht in our study ) . in another case of our study under aht , the followup shows a dramatic uptake decline in bone metastases therefore this uptake decline in bone metastases is likely to reflect a temporal diminishing of uptake rather to be an indicator for real response . the loss of choline uptake was also observed in osteoblastic metastases in patients undergoing no therapy . as mentioned earlier , choline uptake declines with increasing sclerosis , probably due to the drop in blood supply thus in nuclide availability . . found that a hu level above 825 is associated with an absence of metabolic activity . due to the lack of long - term followup , it is still unknown whether or not these metastases are still alive if reaching the stage of negative choline uptake . obviously , even if the metastasis center reaches this stage , the rim where there is a moderate sclerosis remains active . ultimately , the potential role of f - fech - pet / ct in therapy monitoring seems to be limited in case of bone involvement , because choline uptake is likely to be affected by local factors such as the level of sclerosis . no significant gain in sensitivity was achieved using bone scan compared with f - fech - pet / ct . in lesion - based results , the diagnostic potential of both modalities varies in the different anatomical regions and shows f - fech - pet / ct mostly of superior value . that was attributed to the higher spatial resolution and the additional benefit of accompanied ct except for its value in detecting the bone marrow involvement .

18f - fech - pet / ct has been proved to be an imaging agent for prostate carcinoma . however , its role in detecting the bone metastases is still blurred owing to the lack of related studies . the purpose of our study was to assess the efficacy of pet with 18f - ethylcholine in assessing the bone status and to compare the results with that of conventional bone scan findings . for this purpose , we selected 37 patients ( mean age 69 7 ) , who had been referred for restaging purposes due to biochemical recurrences and underwent both 18f - fech - pet / ct and bone scan in a short interval . generally 18 patients out 37 patients referred with biochemical relapse were confirmed to have bone involvement . from 18 confirmed bone involvement cases , 18f - fech - pet / ct identified correctly the bone involvement in 15 cases with overall sensitivity of 83.3% . on the other hand , bone scan identified 17 out of 18 confirmed cases with overall sensitivity of 94.4% . the lesion - related results show that the sensitivity of each investigation differs with the anatomical regions , and by comparing both results , 18f - fech - pet / ct was mostly superior to bone scan ; however , without a statistical significance ( p > 0.1 ) . in conclusion , no significant gain in sensitivity was achieved using bone scan compared with 18f - fech - pet / ct .

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

PMC3306921

further improvements in sequencing techniques have allowed sequences to be produced on a large scale and inserted into a computer . appropriate programs , capable of processing data , have been developed to facilitate access and make all of the information available throughout the process ( morozova and marra , 2008 ) . after dna sequencing is necessary to re - build the complete genome from the fragments obtained . genome assembly uses programs that read the sequences , in some cases find redundancies between them and merge the sequences , forming larger continuous consensus sequences . multiple rounds of assembly are sometimes required for find the best genome assembly sets . in the end of assembly process is necessary ordering the contigs between them or by anchoring in a reference genome to obtain the genome scaffold ( fagin et al . , 1992 ) . this entire process requires a large processing capacity , due to enormous amount of sequences generated by modern equipments . even a simple organism requires many hours of processing and memory for its complete assembly . the increasing availability of computers with massive computational power connected to high - speed networks has enabled the aggregation of geographically dispersed resources for the execution of large - scale applications . grid computing is a model that proposes the use of computing resources of several machines located in various places , even on separate continents , to solve problems that require massive computational power , such as data mining , weather forecasting , computational biology , and medical image processing ( foster , 2001 ; foster et al . , 2002 ; berman et al . , 2003 ) grids are currently being used as an alternative for clusters for achieving large - scale processing capacity . grid computing differs from cluster computing because of the heterogeneity of their resources ( which may consist of computers with different architectures , operating systems , and processing capabilities ) and due to the dynamic character of the same . cluster computing is defined as the sharing of resources working cooperatively and managed by a single global system synchronized and centralized . in grids , on other hand , each node has its own manager and resource allocation policy , which is not so visible ( foster et al . , 2008 ) . programs are composed of small pieces with specific responsibilities and clearly defined , called tasks . each task has a set of attributes , among them the priority that should be assigned according to their importance . each task is performed independently , but they need to interact with each other so that the system meets its objectives . in order for rules to be applied to a set of tasks that may use information from the various computers , it is necessary to adopt appropriate scheduling policies for each application . applications of the type bag - of - tasks ( bot ) facilitates scaling because they are composed of independent tasks , allowing the use of policies based on data from only a few systems . they do not require information on grid infrastructure , such as bandwidth , network topology , and network latency . policies can be called static or dynamic , depending on how the schedule is set ( foster and kesselman , 2004 ) . in spite of the bot applications are simple , it is not easy job to make the scheduling in a heterogeneous and dynamic environment such as grid computing . the scheduling of independents tasks in grid is still difficult due to the use of resources that are shared and due restraint created by other applications , which are running simultaneously . to obtain a good performance in this type of situation requires the use of good information to make scheduling more efficient . in order to achieve this purpose , we developed a dynamic algorithm for bot applications on grid environments because it has a better match and do not require detailed information ( cirne et al . the employment of an efficient algorithm for managing resources is crucial to reduce the time needed to finish tasks in a grid . here , we propose a task scheduling algorithm that takes grid characteristics into account and can be implemented within abyss , software used for parallelized de novo genome assembly , in order to optimize the performance of the genome assembly stage and consequently improve the efficiency of this process as a whole ( bittencourt and madeira , 2006 ; simpson et al . , 2009 ) . the main goal of our experiments was to evaluate the performance of abyss using the scheduling algorithm that we developed compared to the default scheduler bundled with abyss . the abyss is a software used for the de novo genome assembly and can be used in grids by using message passing interface ( mpi ) for the communication between nodes ( pacheco , 1996 ) . reads are distributed among the nodes to form a distributed graph , such that each node knows where the rest of the graph is . the proposed algorithm acts only at this stage of assembly of the distributed graph ( simpson et al . , 2009 ) . these experiments allowed us to evaluate the influence of the heterogeneity of networks ( different speeds ) , heterogeneity of the tasks ( size variation ) , and the granularity of tasks ( number of tasks per machine ) . we used genomic data available from the institute of biological sciences , federal university of para , consisting in a set of 33 millions short reads with fixed length ( 25 bp ) and 110 coverage of corynebacterium pseudotuberculosis i19 genome . this is a structured set of functions implemented in c language for the simulation driven to events , using an extendend mackup language ( xml ) file as input to define the network topology and the characteristics of resources and responsibilities . in these simulations , the network transfer times are negligible , because the focus of this analysis to verify the processing efficiency of the cpus using the proposed task scheduler ( casanova , 2001 ) . in order to run a simulation in simgrid , it is necessary to perform the following steps ( legrand et al . , 2003 ) : model the file of deployment of application , where is specified the location of the creation and allocation of agents . the experiments were conducted with six heterogeneous machines , i.e. , with different operating systems , hardware , and processing power . only the k - mer ( length hash ) parameter was changed to 17 , such value has proved satisfactory . the proposed scheduler does not change the mode of action of abyss , just the way as the tasks are distributed among the nodes . thus , the final assembly was not altered , because the main goal is not to improve the quality of the assembly using the abyss , but minimize the processing time that this step demands . the scheduler developed in the c language provides abyss with the capacity to distribute his tasks according to the rules above . all the files ( the scheduler itself , the configuration files for simgrid , the dataset used , and the detailed instructions ) are available on http://sourceforge.net/p/abyssgrid . the trend , which can be observed in figure 1 , is that in situations with a greater number of tasks per machine and less granularity , performance improves when there are many tasks . the situation changes only at the end of the execution due to load imbalance , which degrades performance . another point to consider is the execution time of tasks , which is relatively long because of the large computational load in the case of genomic data . figure 2 shows the impact of the heterogeneity of the machines in the grid . each point shows the levels of heterogeneity of tasks . we can conclude that the scheduler responds effectively in terms of processing time , even when machines are very heterogeneous . as shown in figure 2 , we can observe that processor 3 failed to perform well , because of the considerable heterogeneity of the machines . the possibility of a slow machine performing a large task is considerable , though it has only a small impact on the execution time of tasks , as was the case for processors 4 and 5 . figure 3 shows the impact of heterogeneity on the performance of the tasks scheduling algorithm . we can conclude that the heterogeneity of the tasks did not have a significant impact on the performance of the task scheduler that we proposed , demonstrating its efficiency in this context figure 4 show that using abyss without our algorithm spent more time spent to execute all the tasks in the same environment . this is due to the fact that the abyss uses , to distribute its tasks , the first in , first out ( fifo ) algorithm , which does not take into account the differences between the available resources in the environment or its load . grid computing has greatly advanced because of the demand for scientific applications that require processing , storage , and manipulation of large amounts of information . fields that require such processing power include molecular biology , astronomy , and earthquake prediction . genome sequencing techniques are also advancing and generating very large amounts of data that must be processed using resources with great computational power . computational grids can be used to address this demand for execution of parallel or distributed applications . though computers have become much more powerful , the genome assembly stage continues to confront limitations because of the type and volume of data generated by next - generation sequencers . increasing amounts of data exponentially rise the processing time , which can overwhelm the assembly process , so that it becomes necessary to implement techniques that will minimize these limitations . future work will be based on improvements in the scheduler so the distribution of tasks is made by first checking if the processors available have enough computing power to speed up processing . based on the experiments , the obtained results were considered satisfactory , since the assembly can be completed , and when such results are compared to the assembly without the use of the scheduler developed , they proved to be faster . the fact of the machines has used different configurations , and the tasks have different sizes , such fact did not affect significantly the completion and assembly performance . the simulations also showed that the scheduling algorithm submitted to testing can be considered scalable in its entirety , since it reached a minimum efficiency , and for any tested task , there was a minimum number of processors available or imminently available to be perform the processing . we proposed a scheduling algorithm for computer grids , using the de novo assembly software abyss . the algorithm proved being very effective in tests with heterogeneous tasks , and there was no impact on processor performance . however , when granularity increased , we identified a small imbalance , causing decreased processor performance . the runtime of the tasks did not affect processor performance in the tests that we run . when the tests were performed with a heterogeneous group of machines , the scheduler responded effectively to the processing challenge . even when there was deterioration in performance due to a slow machine that was allocated a large task , other processes ended up compensating for the slight loss of time in the task . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

improvements in genome sequencing techniques have resulted in generation of huge volumes of data . as a consequence of this progress , the genome assembly stage demands even more computational power , since the incoming sequence files contain large amounts of data . to speed up the process , it is often necessary to distribute the workload among a group of machines . however , this requires hardware and software solutions specially configured for this purpose . grid computing try to simplify this process of aggregate resources , but do not always offer the best performance possible due to heterogeneity and decentralized management of its resources . thus , it is necessary to develop software that takes into account these peculiarities . in order to achieve this purpose , we developed an algorithm aimed to optimize the functionality of de novo assembly software abyss in order to optimize its operation in grids . we run abyss with and without the algorithm we developed in the grid simulator simgrid . tests showed that our algorithm is viable , flexible , and scalable even on a heterogeneous environment , which improved the genome assembly time in computational grids without changing its quality .

Introduction Materials and Methods Results Discussion Conclusion Conflict of Interest Statement

PMC3302993

rheumatoid arthritis ( ra ) and other connective tissue disorders are frequently associated with certain types of corneal or / and scleral pathology such as peripheral ulcerative keratitis , dry eye syndrome , and scleritis . it is also known that these conditions may lead to perforation of the eye by compromising the integrity of its outermost layers . keratoconus ( kc ) is generally regarded as an isolated condition with no apparent ophthalmic or systemic associations at the time of diagnosis . however , elevated levels of corneal proinflammatory cytokines such as interleukin ( il)-1 have been linked to corneal thinning , implying the involvement of the immune system in the pathogenesis of kc . only a small number of autoimmune diseases ( e.g. , ra , asthma , inflammatory bowel disease ) have been related to kc , while others have not ( e.g. , systemic lupus , crohn s disease , myasthenia gravis , and multiple sclerosis ) . cell - mediated mechanisms are suggested to play a pivotal role in the pathogenesis of ectatic corneal disorders and kc itself . further , confocal microscopy has revealed that in patients with ra , the central corneal and stromal thicknesses are significantly lower compared with controls , and these patients have significantly higher numbers of hyperreflective stromal cells . the activation of these keratocytes is mediated by proinflammatory cytokines , such as il-1 and il-6 . we report a case of paracentral corneal perforation in a patient with keratoconus and a previously undiagnosed connective tissue disorder . a 53-year - old man presented in the emergency room complaining of severe ocular pain with sudden onset and a significant decrease in visual acuity in his left eye . regarding his ocular history , the patient noted that his ophthalmologist had been modifying his spectacle prescription annually over the last 13 years because of the increasing myopic shift . the patient reported no systemic drug intake except for analgesics for constant lumbar back pain . the patient s best corrected visual acuity was measured cf at 10 cm for the left and 20/60 for his right eye . his spectacle prescription at the time of the examination was : 1.00/5.5 100 os and 4.5/4.5 70 od . slit lamp biomicroscopy revealed a paracentral corneal perforation , 1 2 mm , accompanied by iris prolapse , corneal thinning , and a markedly shallow anterior chamber in the left eye . the right eye displayed clinical features of moderate keratoconus , which was confirmed by corneal topography ( magellan mapper , nidek ; figs . 1 and 2).fig . steep corneal curvature on biomicroscopy raised suspicions of a coexistent keratoconus topography image demonstrating significant keratoconus on the right eye the patient was immediately prepared for surgical intervention in order to preserve the integrity of the affected eye . amniotic membrane ( am ) was primarily available and transplanted in an attempt to seal the perforation and promote corneal healing ( fig . cryopreserved am was attached over the perforation in multiple layers and covered with an epithelial - side - up , limbal - sutured sheet . the surgical technique involved scraping of residual corneal epithelium and 360 limbal peritomy of the conjunctiva prior to am transplantation . the outermost of the multiple layers was sutured with 8/0 vicryl on the cornea whereas the overlaid transplant was sutured at the limbus with a running 10/0 nylon . a bandage contact lens was applied at the end of the operation in order to promote reepithelialization of am and to prevent mechanical rubbing of the transplant on blinking.fig . 3photo of left eye on second post - op day after amniotic membrane transplantation in multiple layers in an attempt to maintain the eye s anatomical integrity photo of left eye on second post - op day after amniotic membrane transplantation in multiple layers in an attempt to maintain the eye s anatomical integrity further blood tests revealed a significantly elevated rheumatoid factor at 2,170 iu / ml ( reference value < 20 iu / ml ) . specific blood tests in addition to detailed clinical examination carried out by an orthopedic surgeon and immunologist confirmed the diagnosis of ra , and the patient was referred to rheumatologists for further assessment . the operated on eye displayed a well - formed anterior chamber , without any leakage , over the 3 post - op weeks . visual acuity was slightly improved to cf at 50 cm . however , reepithelialization failed to cover am completely whilst subsequent tissue thinning resulted in re - leakage at week 4 postoperatively . one month after corneal grafting and while on systemic immunosuppressants , the patient developed endophthalmitis with unfavorable outcome . kc is a noninflammatory corneal disorder in which stromal thinning and ectasia result in an atypical corneal shape . although the etiology is not completely understood , there is evidence suggesting that kc probably represents a multifactorial process . kc with acute hydrops can be complicated with spontaneous corneal perforation ; however , this has been reported mainly in cases with advanced kc . nevertheless , spontaneous perforation in mild to moderate kc cases has also been reported recently . ophthalmic involvement in autoimmune disorders such as ra , systemic lupus erythematosus , and sjgren syndrome has been extensively described . however , many patients experience ocular sequelae during the course of undiagnosed immunological disorders . reduced tear secretion , chronic inflammation of the ocular surface , as well as ultrastructural abnormalities are believed to play a major part in decompensating the ocular surface . this condition may lead to spontaneous perforation in sporadic cases . in our case , am transplantation was used as a first - line solution since previous studies showed that urgent penetrating keratoplasties in perforated eyes displayed higher rates of graft failure compared to those performed several weeks to months following the perforation [ 10 , 11 ] . multilayer am transplantation has been reported to promote epithelial healing and reduce inflammation , neovascularization , and scarring in a diversity of external eye disorders . am - related local anti - inflammatory effects contribute to faster and more efficient reepithelialization . furthermore , am serves as a biological scaffolding in cases where normal tissue is lost and promotes reinforcement or even sealing of small perforations . am has been used in patients with ra - related epithelial defects as a temporizing solution with the aim for delayed reconstruction . however , as solomon and colleagues reported , am transplantation showed poorer outcomes in patients with systemically undertreated ra compared to other autoimmune conditions like ocular pemphigoid or even sjogren s . other authors have proposed cyanoacrylate and fibrin glue adhesives as an efficacious solution in cases where corneal perforations occur [ 13 , 14 ] . in our case , am transplantation was preferred over other treatment modalities mainly due to severe inflammation of the ocular surface and the small size of the perforation . besides , the diagnosis of ra had not yet been established . furthermore , penetrating keratoplasty does not represent a first - choice operation in our country due to the relatively restricted number of centers authorized to perform corneal transplantations and to limited availability of corneal grafts . in our patient , although the am transplant initially provided a sufficient corneal sealing , it failed as a long - term solution due to poor reepithelialization and the progressive tissue degradation . recurrent aqueous humor leak necessitated an emergent penetrating keratoplasty , which took place in another center . there are quite a few studies that extensively describe the relationship between ra and ocular surface disorders that may permanently affect vision [ 15 , 16 ] . kc is another thoroughly studied condition that , apart from major refractive alterations , may occasionally cause corneal perforation when undiagnosed or / and untreated . to the best of our knowledge , this is the first report of a case with corneal perforation arising from coexistent kc and ra . it seems possible that the impact of these conditions is more severe when they interact . we should therefore raise awareness and either rule out or efficiently treat underlying connective tissue disorders in patients with kc . this article is distributed under the terms of the creative commons attribution license which permits any use , distribution and reproduction in any medium , provided the original author(s ) and source are credited .

purposethe purpose of this study is to report a case of corneal perforation in a patient with undiagnosed keratoconus and underlying rheumatoid arthritis.methodsthis is a retrospective case study based on the patient s medical records and followed by a brief review of the literature.resultsa 53-year - old patient was referred to our department for acute pain and sudden decrease of visual acuity in his left eye . corneal perforation was detected and attributed to a previously undiagnosed and untreated keratoconus . additional laboratory work - up and clinical examination revealed a coexisting rheumatoid arthritis . amniotic membrane was originally transplanted in order to maintain the structural integrity and promote healing of the perforated eye . subsequently , the patient underwent a penetrating keratoplasty , though with unfavorable results due to postoperative endophthalmitis.conclusionsrheumatoid arthritis may be associated with higher risk of corneal perforation in patients with ectatic degenerative diseases such as keratoconus .

Introduction Case report Discussion Conclusion Conflict of interest Open Access

PMC5065320

specificity is one of the foundational components of training success . in sport performance training , one of the initial steps in the design of an exercise regimen is the identification of movements specific to the needs of the individual , which may include joint angles , and muscle actions and recruitment ( 1 , 4 ) . movement analysis is an integral step in the selection of exercises that mimic sport - specific actions and transfer to competition . furthermore , the intensity and movement velocity ( explosiveness ) utilized in the performance of an exercise can be a vitally important component of specificity ( 1 , 4 ) . sprinting , and the acceleration phase in particular , is characterized by the explosive extension of the hip , knee , and ankle joints ( 3 ) , with a greater overall contribution of the hip joint particularly as speed increases ( 2 ) . the kettlebell swing ( kbs ) is similarly characterized by activation of the posterior chain muscles , particularly in relation to the hip , in the horizontal plane that occurs during the sprint motion ( 3 , 8 , 11 , 16 ) thereby indicating movement specificity . additionally , the rapid concentric phase and ballistic nature of the kbs ( 8 , 10 ) may align well with sprint performance , which also requires rapid force production . comparisons between the kbs and a traditional one - repetition maximum back squat resulted in a greater impulse demand in the kbs , pointing towards the potential for a large rate of force development despite differences in load , and demonstrating the importance of the velocity of this exercise ( 8) . thus , it is likely that this velocity specificity would also align well with sprinting . despite the popularity and potential applicability of kettlebell training in multiple domains ( 7 , 10 , 13 ) , the sport performance community has not responded with an appropriate depth of rigorous scientific studies into how kettlebell training may transfer into sprinting . in 2012 , lake and lauder reported six weeks of two - handed kbs exercise progressing to 60% of maximal loads , improved maximal and explosive strength ( 7 ) . conversely , results from a comparison study suggested that kbs at 60% of maximal loads are not sufficient to develop lower body maximal and explosive strength , and concluded that the kbs may be best used as adjuvant training during a strength and conditioning program ( 8) . additionally , otto et al . reported that six weeks of traditional weightlifting induced significantly greater improvements in strength compared with kbs training , however , both were effective in increasing muscular strength and power in recreationally - active men ( 13 ) . regardless of training modality , it is understood that volume plays an important role in the degree of muscular strength and power improvement . it is plausible that different training volumes used in the aforementioned studies may have resulted in ambiguous conclusions about the efficacy of kbs on power performance . moreover , though hypothesized , it has yet to be determined whether kbs training can have positive effects on sprint performance ( 7 ) . therefore , the present study examined the effect of an eight - week kbs program versus a program of equal intensity , volume , and movement specificity using the stiff - leg deadlift ( sdl ) on sprint performance . the sdl was chosen as it utilizes very similar muscle recruitment as the kbs , while allowing a slower movement velocity ( 1 , 4 ) . in this fashion , it is possible to utilize similar movement specificity and volume , while addressing the additional velocity specificity of the kbs . therefore , it was hypothesized that the eight - week kbs program would improve sprint performance to a greater degree than an eight - week sdl program . twenty ( n=20 ) healthy college - age female students ( 1825 yrs ) were recruited for the current investigation . approval by the slippery rock university institutional review board was acquired and all participants completed informed consent . participants were instructed to maintain their current aerobic exercise and dietary programs throughout the duration of the study . no participants had extensive experience with kettlebell exercise or completed sprint or resistance training in the previous 6 months as a means to limit extraneous variables from prior training history . study investigators , however , were trained in the safe and effective implementation of resistance exercise , and completed specific sessions on coaching the kbs and sdl . participants were excluded from the present study if they were not cleared by their physician for vigorous exercise , were currently utilizing a resistance training program , were outside of the age range ( 1825 ) , or were unable attend the scheduled sessions . participants were assigned into one of two groups ( kbs or sdl ) based on their initial sprint performance , to ensure that there were participants of similar ability in each cohort . following the group assignment all participants participated in one instructional session on their assigned exercise prior to data collection . technique mastery was not necessary during the single instructional session as the participants were observed ( i.e. appropriate cueing ) throughout the duration of the training intervention for both safety and effectiveness . each group underwent an 8-week ( twice - weekly ) exercise intervention consisting of sixteen training sessions . quite simply , this schedule was chosen as it fulfilled training frequency guidelines for beginners ( i.e. 23 sessions per week ) , and fit within the time constraints of a university academic semester . each training session began with a 5 min light ( <3 mets ) aerobic warm - up on a treadmill followed by two 15 meter striders with a 30 second rest . all training sessions were performed using a tabata interval timer ( 20 seconds of exercise ; 10 seconds of rest ; 8 rounds ) based on previous work demonstrating a large anaerobic component to this training approach ( 5 , 15 ) . the participants were required to elect two non - consecutive days each week to exercise under the supervision of a project coordinator . a hardstyle kettlebell swing emphasizing maximal hip recruitment and minimal knee flexion was utilized in this study ( 7 ) . training started with a consistent prescribed weight ( ~9.1 kg ) with the potential for a ~2.3 kg ( next kettlebell weight increment ) increase following week three , and another ~2.3 kg increase at week six , if the participant maintained proper form throughout each session . form was monitored by trained instructors throughout the sessions , and included emphasis on maintaining an explosive concentric phase during maximal hip recruitment . training started with a consistent barbell weight ( ~27.3 kg ) , with the potential for a ~4.5 kg increase following week 3 , and another ~4.5 kg increase at week 6 , if the participant maintained proper form throughout each session . form checks for the sdl group included the maintenance of a two - second up / two - second down exercise tempo and maximal hip recruitment . each participant completed both a 30-meter sprint test and countermovement vertical jump test on a consistent , indoor track . identical post - testing was completed between 48 and 72 hours of the final exercise session . if a participant performed the vertical jump test before the sprint test in pre - testing , they were asked to perform the sprint test first for post - testing . each participant performed the same warm - up during the pre- and post - training testing . this warm - up consisted of a 400-meter jog followed by a 40-meter interval of the following drills : high knees , butt kicks , walking knee - to - chest stretch , walking quad stretch , toe touches , hamstring swings , carioca , and striders . participants were given the option to add additional warm - up striders before performing their maximal sprint ability . thirty meter sprint performances were timed using a tc - photogate ( brower timing systems , draper , ut ) . participants used a standing position and began the maximal sprint individually without a formal start . the tc - photogate recorded when the participants crossed the start and the finish line . each participant performed two sprint trials with a two - minute rest in between attempts , with the fastest sprint recorded . a vertical jump measurement device ( vertec , jump usa , sunnyvale , ca ) was used to measure maximal vertical jump height . participants reached to maximum vertical height on the vertec with their dominant arm while their feet were flat on the ground . each participant performed two countermovement jumps with arm swing and displacement of the highest vane was determined . to calculate maximum vertical jump height , all analyses were performed using a standard statistical software program ( ibm spss statistics for windows , version 21 , 2012 ) . pre- and post - testing differences among groups were assessed using an independent t - test . a repeated measures anova was utilized to assess potential differences in pre- and post - testing differences across groups . an a - priori -significance level of 0.05 was accepted as a reflection of differences in the mean . twenty ( n=20 ) healthy college - age female students ( 1825 yrs ) were recruited for the current investigation . approval by the slippery rock university institutional review board was acquired and all participants completed informed consent . participants were instructed to maintain their current aerobic exercise and dietary programs throughout the duration of the study . no participants had extensive experience with kettlebell exercise or completed sprint or resistance training in the previous 6 months as a means to limit extraneous variables from prior training history . study investigators , however , were trained in the safe and effective implementation of resistance exercise , and completed specific sessions on coaching the kbs and sdl . participants were excluded from the present study if they were not cleared by their physician for vigorous exercise , were currently utilizing a resistance training program , were outside of the age range ( 1825 ) , or were unable attend the scheduled sessions . participants were assigned into one of two groups ( kbs or sdl ) based on their initial sprint performance , to ensure that there were participants of similar ability in each cohort . following the group assignment , all participants participated in one instructional session on their assigned exercise prior to data collection . technique mastery was not necessary during the single instructional session as the participants were observed ( i.e. appropriate cueing ) throughout the duration of the training intervention for both safety and effectiveness . each group underwent an 8-week ( twice - weekly ) exercise intervention consisting of sixteen training sessions . quite simply , this schedule was chosen as it fulfilled training frequency guidelines for beginners ( i.e. 23 sessions per week ) , and fit within the time constraints of a university academic semester . each training session began with a 5 min light ( <3 mets ) aerobic warm - up on a treadmill followed by two 15 meter striders with a 30 second rest . all training sessions were performed using a tabata interval timer ( 20 seconds of exercise ; 10 seconds of rest ; 8 rounds ) based on previous work demonstrating a large anaerobic component to this training approach ( 5 , 15 ) . the participants were required to elect two non - consecutive days each week to exercise under the supervision of a project coordinator . a hardstyle kettlebell swing emphasizing maximal hip recruitment and minimal knee flexion was utilized in this study ( 7 ) . training started with a consistent prescribed weight ( ~9.1 kg ) with the potential for a ~2.3 kg ( next kettlebell weight increment ) increase following week three , and another ~2.3 kg increase at week six , if the participant maintained proper form throughout each session . form was monitored by trained instructors throughout the sessions , and included emphasis on maintaining an explosive concentric phase during maximal hip recruitment . training started with a consistent barbell weight ( ~27.3 kg ) , with the potential for a ~4.5 kg increase following week 3 , and another ~4.5 kg increase at week 6 , if the participant maintained proper form throughout each session . form checks for the sdl group included the maintenance of a two - second up / two - second down exercise tempo and maximal hip recruitment . each participant completed both a 30-meter sprint test and countermovement vertical jump test on a consistent , indoor track . identical post - testing was completed between 48 and 72 hours of the final exercise session . if a participant performed the vertical jump test before the sprint test in pre - testing , they were asked to perform the sprint test first for post - testing . each participant performed the same warm - up during the pre- and post - training testing . this warm - up consisted of a 400-meter jog followed by a 40-meter interval of the following drills : high knees , butt kicks , walking knee - to - chest stretch , walking quad stretch , toe touches , hamstring swings , carioca , and striders . participants were given the option to add additional warm - up striders before performing their maximal sprint ability . thirty meter sprint performances were timed using a tc - photogate ( brower timing systems , draper , ut ) . participants used a standing position and began the maximal sprint individually without a formal start . the tc - photogate recorded when the participants crossed the start and the finish line . each participant performed two sprint trials with a two - minute rest in between attempts , with the fastest sprint recorded . a vertical jump measurement device ( vertec , jump usa , sunnyvale , ca ) was used to measure maximal vertical jump height . participants reached to maximum vertical height on the vertec with their dominant arm while their feet were flat on the ground . each participant performed two countermovement jumps with arm swing and displacement of the highest vane was determined . to calculate maximum vertical jump height , all analyses were performed using a standard statistical software program ( ibm spss statistics for windows , version 21 , 2012 ) . pre- and post - testing differences among groups were assessed using an independent t - test . a repeated measures anova was utilized to assess potential differences in pre- and post - testing differences across groups . an a - priori -significance level of 0.05 was accepted as a reflection of differences in the mean . one individual was removed due to an unrelated ankle injury ; and following the eighth week of training , another individual withdrew due to an appendectomy . pre- and post - testing data were recorded for eight kbs participants , and ten sdl participants . kbs had 95% adherence to the training intervention , with a total training volume of 15,850 swings . the average number of repetitions performed was 124 , with an average training session volume - load of ~1378.2 kg . however , only 6,746 sdls were completed ( due to the intentionally slower repetition velocity ) , with the average number of repetitions per training session at 43 . despite lower repetitions , there were no significant differences noted in the pre - training 30 m sprint times between the kbs and sdl cohorts ( table 1 , p>0.05 ) . likewise , there were no significant differences in the post - training 30 m sprint times between the kbs and sdl groups ( table 1 , p>0.05 ) . in kbs or sdl pre- to post - testing sprint times were not significantly different ( table 1 , p>0.05 ) . there were no significant differences noted in the pre - training vertical jump performances between the kbs and sdl groups ( table 1 , p>0.05 ) . likewise , there were no significant differences in the post - training vertical jump performance between the kbs and sdl groups ( table 1 , p>0.05 ) . however , across groups , pre- to post - testing vertical jump performances improved by approximately 4% ( table 1 , p<0.05 ) , with no effect of training group . to the knowledge of the authors , this is the first study to determine the effect of an 8-week kbs program versus a program of similar volume - load , and movement specificity using sdl on sprint and counter movement vertical jump performance . the primary finding in the current investigation was kbs training ( 8 week ; 16 sessions ) did not significantly increase sprinting performance when compared to sdl . additionally , vertical jump performance was improved with training . due to the design of the study , no differences existed in pre - intervention sprint times or vertical jump heights between groups . the current investigation measured recreationally - active females who did not partake in resistance training activities . it is known that training status and modality can influence determinants of power ( e.g. sprinting ) . our recreationally - active cohort was not unusual in that they demonstrated similar average 30 meter sprint times for females when compared to those studied by mangine and colleagues ( 5.20.2 v. 5.50.5 seconds ) ( 9 ) . interestingly , no differences in the post - intervention sprint performances between kbs and sdl were observed . while both cohorts increased training loads and volumes over the course of the eight - week intervention , likely indicating adaptation , kbs training provided no additional transfer to the actions of sprinting , though a modest increase in countermovement vertical jump performance was observed . to our knowledge , few investigators have attempted to quantify the efficacy of kettlebell exercise with equivocal findings . kbs has been reported to improve strength and power ( 7 , 10 ) , while another study has reported no additional benefit over traditional training ( 13 ) . it is likely that improvements with kbs may be due to the cyclical , eccentric loading phase of the exercise combined with the rapid reversal of force necessary to transition to the concentric phase . furthermore , the mechanical demand of the kbs is dictated by both a vertical and horizontal mechanical output ( 8) , with a ballistic component provided by the long lever arm and variable center of mass of the participant / kettlebell system . therefore , considering the movement- and velocity - specificity between kbs and sprinting , additional explanations for a lack of observed transfer in our findings must be considered . the multifaceted nature of sprint performance must be considered first and foremost in the discussion of the current findings . based on the duration of the training intervention , neural adaptations would likely account for a majority of the participants ability to adapt to the training regimen and increase training volume ( 1 , 12 ) . neural adaptations , while important , may not account for the spectrum of underlying changes necessary for improved sprint performance . it is likely that structural adaptations including muscle hypertrophy ( 3 ) , increases in stride frequency and length , and enhanced sprint technique ( 12 ) are critical components of sprint performance , but were not addressed in this intervention . the results of this study suggest that kbs training alone may not provide a sufficient stimulus to improve sprint performance in recreationally - active females even with a focus on movement and velocity specificity . it is likely that the present study may not have supplied a great enough training volume - load and intensity for improved sprint performance . in comparison to a study that demonstrated increases in explosive strength ( vertical jump ) in athletes using kbs ( 7 ) , our training volume was likely inadequate . assuming similar kbs movement and velocity specificity , when extrapolating an average training session from our study ( < 70 kg subject ; ~9.1 kg kettlebell ; 15 swings/20 seconds ; eight work intervals ) to training parameters of similar work in athletes ( < 70 kg subject ; 12 kg kettlebell ; ~22 swings/30 seconds ; twelve work intervals ) , our trainees received only one - third of the average training session volume ( 7 ) . additionally , it is likely that the work to rest ratio ( 2:1 ) of the tabata protocol resulted in muscular fatigue which limited participants from generating maximum power outputs throughout the exercise sessions . thus , the drop in power output and relative intensity could limit the transferability of the intervention to sprint performance , which utilizes maximum power output . it is likely that an altered work to rest ratio ( e.g. 1:4 or 1:5 ) would preserve movement quality and explosiveness to a greater degree than the tabata protocol . while athletes may respond differently to training , and pose greater challenges than their recreationally - active counterparts ( 12 ) , it is probable that the volumes and intensities used in this investigation were underestimated . a conservative starting load was chosen for the kbs group , as the best current guidelines for power programming highlight the need for rapid force development ( 5 ) . consistent load assignment was deemed for this experiment in order to ensure similar training volume across participant groups , though it is likely that some means of individual strength or power testing in order to more accurately assign participant loads may have improved measured outcomes . currently , however , specific load , intensity and volume recommendations for the various potential applications of the kbs are not readily available . with the effectiveness of kbs training on vertical jump performance documented ( 7 , 13 ) , vertical jump was used as a comparable benchmark to gauge participant responsiveness to training . as countermovement vertical jump height changed significantly in both training cohorts ( approximately 4% improvement ) it is more likely that a lack of emphasis on sprint mechanics and technique may have had a larger impact on the lack of transfer to sprint performance than a simple lack of training volume . though correct form and adherence to the hardstyle kettlebell swing and stiff - legged were coached across all 16 training sessions , nonconsecutive training days per week was chosen purely out of a need for freedom in scheduling between the participants and investigators , a more rigorous schedule format may have altered the findings of the study . it is plausible that both sprint and jump performances are sensitive to diurnal variation ( 14 ) . in the current study , scheduling conflicts resulted in a difference between pre- and post - testing time of day ( 12:002:00 pm and 7:009:00 am , respectively ) . furthermore , changes in the time of testing may have also impacted variables related to food consumption , hydration , body temperature , motivation , and sleep patterns . in conclusion , an eight - week kbs program was not shown to improve measures of sprint performance when compared to an sdl program . training volumes are an important consideration for effectiveness in interventions designed to improve sprint outcomes , however , an equal consideration must be placed on mechanics and technique . due to movement- and velocity - specificity , the kbs should not be excluded from resistance training programs designed to accompany sprint training , especially given the limited volume of literature currently available . recently , it has been reported that a two - handed kbs is appropriate for the training of ballistic or explosive outcomes ( 6 ) . it is plausible that an appropriate kbs prescription as part of a comprehensive training program ( e.g. teaching sprint technique ) can improve sprint performance . however , data from the current investigation do not confirm or support the efficacious use of kbs in sprinting . therefore , future research is still needed to determine the value of kbs in sprint - training programs and provide volume and intensity recommendations .

the kettlebell swing ( kbs ) , emphasizing cyclical , explosive hip extension in the horizontal plane , aligns with movement- and velocity - specificity of sprinting . the present study examined the effect of an eight - week kbs intervention on sprinting in recreationally - active females , in comparison to an eight - week intervention using the stiff - legged deadlift ( sdl ) . following a pre - testing session measuring 30 meter sprint and countermovement vertical jump performance , participants were divided evenly by sprint time into kbs ( n=8 ) and sdl ( n=10 ) cohorts . following familiarization with the exercises , kbs met twice weekly to perform swings using the tabata interval ( 20s work , 10s rest , 8 rounds ) , stressing a rapid , explosive tempo . in contrast , the sdl group performed their tabata stiff - legged deadlifts at a conventional resistance training tempo ( 2 seconds concentric , 2 seconds eccentric ) . following eight weeks and greater than 95% training adherence , the sdl group only had a slightly greater average training volume ( ~3% ) than kbs . no significant differences in pre - test values , or changes were noted in sprint performance from pre- to post - intervention in either group . an improvement in vertical jump performance was noted across groups . potential explanations for the lack of sprint improvement compared to previous studies include differences between recreationally - active and athletic females , and low exercise volume ( ~46% of a comparable study with improvements in vertical jump ) . future studies should seek to determine the appropriate volume and intensity for kbs components of sprint programming .

INTRODUCTION METHODS Participants Protocol Statistical Analysis RESULTS DISCUSSION

PMC4436771

a core principle of both financial investment and biology is that diversity leads to strength . in biology , hybrid vigor produces healthy organisms , because each parental genome contributes complementary strengths and balances the other 's weaknesses . in investing , diversity spreads risk , maximizing the chances of finding some big winners while reducing the chances of collapse based on sudden declines in a few companies ' fortunes . diversity at all levels from the kinds of science to the regions in which it is conducted to the backgrounds of the people conducting it strengthens the institute 's research portfolio and should lead to the best returns on the taxpayers ' investments . it is impossible to know where or when the next big advances will arise , and history tells us that they frequently spring from unexpected sources . it is also impossible to know what threads of foundational knowledge will be woven together to produce a new breakthrough . supporting a wide variety of lines of inquiry will improve the chances of important discoveries being made . this includes studying a diversity of organisms , because important and useful processes almost certainly remain to be discovered in areas of biology we have not yet explored . scientists ' past or current experiences have a significant impact on the problems they choose to study and on the ideas they have for approaching these problems . health burdens from specific diseases differ from state to state and population to population , and these variances can drive the kinds of questions researchers ask . in addition to variations in regional health burdens , other environmental factors such as local plants and animals can influence an investigator 's research directions . for example , baldomero olivera , now a prominent researcher at the university of utah , began studying deadly cone snail venom while he was working in the philippines , where the animals are endemic and people routinely died of snail stings ( telis , 2014 ) . olivera 's work on cone snail toxins has transformed neuroscience and has already led to one food and drug administration approved drug and several more in clinical trials . we also need to consider the identification and development of scientific talent when planning how we invest in biomedical research . for example , if cutting - edge biomedical research were only being conducted in 25 states , it would mean that high school and college students in the other 25 states could get research experience only if they were willing and able to move . the loss of talent to science in the united states caused by this research experience gap would be severe . in addition to the value of supporting a diverse research portfolio , it is important to recognize that the best ideas come from investigators themselves . although there are times when management and top - down direction can help break through systemic barriers or open up bottlenecks particularly when the development of new technologies is required fundamental research works best when investigators are following their noses and setting their own directions . during the national institutes of health ( nih ) budget - doubling period ( 19982003 ) , the fraction of the nigms portfolio dedicated to investigator - initiated research declined from 99 to 80% because of increasing use of programmatic initiatives ( figure 1 ) ; that is , funding targeted at specific scientific areas . with the budget doubling more than a decade behind us , it is time to return the institute 's focus to investigator - initiated research , to ensure that new scientific territory is opened for exploration by adventurous investigators . the fraction of the nigms portfolio committed to investigator - initiated research has declined over the past two decades . the blue bars ( left axis ) show the funds nigms committed to funding opportunity announcements ( foas ) targeted at specific areas of research in each fiscal year shown . the red line with triangles shows the change in the percentage of the nigms portfolio dedicated to investigator - initiated research . the analysis does not include fellowship , career development , and training awards ; programs transferred to nigms from the former national center for research resources ; and some other programs . a criticism of nigms ' renewed emphasis on investigator - initiated research has been that it will disadvantage team science and collaboration . but the dichotomy is not between investigator - initiated research and team science ; it is between investigator - initiated research and targeted or top - down research , in which funds are earmarked for specific scientific areas . team science aimed at studying fundamental biomedical problems can and usually should be investigator - initiated . interdisciplinary team science is undoubtedly extremely important and will become increasingly so as we delve deeper into the complexities of biological systems . one way to support team science is for independently funded principal investigators ( pis ) to form collaborations organically based on common interests and complementary skills , a method that , for fundamental research , seems likely to yield better results than collaboration borne of incentives such as targeted funding nih also has several mechanisms dedicated to supporting investigator - initiated team science , including multi - pi r01s and program project grants ( p01s ) . the centers of biomedical research excellence within the nigms institutional development award program also support team science , with a focus on developing the careers of junior investigators ( nigms , 2015 ) . moving forward , we plan to try to understand what kinds of teams benefit the most from unified grant mechanisms such as multi - pi r01s and p01s . press coverage , prizes , and renown all revolve around the concepts of discoveries and breakthroughs . eureka moments in which a paradigm shift occurs . and yet most major advances in science actually happen when a series of small steps coalesce into an important new understanding . for example , the discovery of restriction enzymes , a truly transformative advance that propelled biomedical research in the 1970s into the age of molecular biology and launched the biotechnology industry , began in the 1950s with the description of bacterial resistance to phage infection . dozens of papers published in various journals led to the insights that brought the nobel prize to warner arber , daniel nathans , and hamilton smith in 1978 ( loenen et al . , 2014 ) . discoveries arise from a complex web of knowledge , and without the network created by the steady progress of many researchers , they would not occur . as discussed earlier , it is impossible to know in advance where in this web the next big breakthroughs will arise or which strands of knowledge will be required to make them . the original observation of crispr sequences , for example , was made at the end of a 1987 journal of bacteriology paper that was otherwise devoted to reporting the sequence of the gene for the escherichia coli alkaline phosphatase isozyme converting protein iap ( ishino et al . describing the mysterious repeat sequences , the paper ends with the sentence , so far no sequence homologous to these has been found elsewhere in procaryotes , and the biological significance is not known . slowly , through years of careful characterization of the crispr pathway , our understanding of what this initial observation meant fueled the development of a novel technology that has dramatically improved our ability to replace genes in living cells , paving the way for advances in medicine and biotechnology . this and many other instances like it show how important it is for us to support as wide a web of research as possible . in 1985 , bruce alberts wrote a prescient commentary in which he laid out the inefficiencies created in the basic biomedical research enterprise when labs become too large ( alberts , 1985 ) . there are a number of reasons for these inefficiencies , but they mostly come down to bandwidth . for example , the more people a pi has in his or her lab , the less time he or she can devote to training and supervising each one . in addition , the bigger a lab gets , the more time the pi must spend on the grant - writing and administrative tasks needed to keep the operation running and the less time he or she has for actually doing research . for these reasons , alberts argued that the per capita output of a lab would generally diminish above a certain size and that nih and other agencies should cap pi funding , thereby limiting lab size and optimizing scientific productivity and quality . in 2010 , nigms conducted an analysis of the productivity and scientific impact of the research the institute funds as a function of each nigms investigator 's total direct - cost support from nih ( figure 2 ) . this study showed that , on average , these metrics increase only shallowly with funding above a moderate level ( $250,000300,000 ) and then actually decrease above $750,000 ( berg , 2010a , b ) . although a few investigators beat the averages and increase their productivity at a proportional or better rate as their funding increases , other well - funded investigators perform even worse than the averages . similar results have recently been published for chemical and biological research supported by other agencies ( fortin and currie , 2013 ; gallo et al . , 2014 ) and by the nih ( berg , 2015 ; danthi et al . , 2015 a 2010 analysis of researchers funded by nigms showed that , on average , productivity did not scale proportionally with funding beyond a relatively moderate direct - cost threshold . ( top panel ) average number of publications associated with nih grants of nigms - funded investigators as a function of their total nih direct costs ( red line with circles ) and the average impact factor of the journals in which each set of investigators published ( blue line with squares ) . ( bottom panel ) the unbinned data used to generate the averages shown in the top panel . jeremy berg , paul sheehy , and matt eblen ( nigms ) performed the data analysis . thinking about these data from the perspective of an investor of taxpayer funds , one can do a simple back - of - the - envelope calculation to determine what the best investment strategy for fundamental research should be . the first is from a talented established investigator and is for a new r01 , which would be his third , bringing him from $ 400,000 to $ 600,000 in total direct funding . in the first case , the additional $ 200,000 would buy taxpayers , on average , approximately one more paper during the funding period over what the established pi would have produced with $ 400,000 ( figure 2 ) . in the second case , the new pi would produce five papers on average in the funding period if she were awarded the grant . thus the choice seems obvious : taxpayers net four more papers by funding the new pi than by giving the established pi a third grant . , some biomedical research simply costs more money than the average , and this needs to be considered when grant budgets are set . and a difficulty here is that recent analyses have indicated that the peer - review process does not have sufficient resolution to accurately distinguish among the most promising applications , with at best modest correlations between score and productivity or impact for funded applications ( berg , 2011 , 2013 ; gallo et al . , 2014 ; so , for example , if the established pi 's application scored in the fifth percentile , and the new pi 's scored in the 15th percentile , could we really be confident that the former is likely to produce more important work than the latter ? along these same lines , as mentioned earlier , although we fund some pis who beat the average productivity versus funding curve , we also fund some who are below it , again indicating that picking the high performers is not easy , at least with the current process . these considerations suggest that we should be very selective in allowing pis to accumulate high funding levels and that , in general , funding more investigators at a moderate level rather than a few at a high level will yield the best payoffs . a question that at first glance may seem trivial but is , i believe , a significant one is whether our key metric for how we use the funds we invest in biomedical research should be the number of grants we award or the number of investigators we support . for example , we report to the scientific community and congress on the success rate for grant funding : how many grants we awarded in a year divided by the number of grant applications we received . in theory , focusing on grants tells us how many projects we are funding . however , because pis can have more than one nigms research grant , this focus distances our funding decisions from the key question of how many investigators are in our portfolio . if we instead used the number of pis we support as the key parameter to drive our funding and programmatic decisions , it would reduce the number of variables , allowing us to focus on the most important ones . for example , once we decided on the optimal number of nigms - supported investigators , we could then set a mean and median direct - cost target per investigator based on our total research budget . once we had established the total number of investigators who should be supported by the institute , we could determine how many new pis should enter and how many established pis should exit the system each year . we would also be better able to ensure the diversity and breadth of the institute 's research portfolio . overall this seemingly simple shift in how we view our mission could be a useful catalyst for reequilibrating the biomedical research enterprise to maximize its effectiveness , efficiency , and sustainability . promoting this shift is something we are working on at nigms , both as part of our new strategic plan and through a new funding mechanism , described in the following section . reequilibrating the biomedical research enterprise to make it more efficient and sustainable will require major changes in every part of the system . because we do not know a priori which changes will work best and because there is always a risk of unintended negative consequences , the soundest approach will be to experiment and to expand initiatives that succeed and abandon those that do not . this model requires us to define in advance the outcomes we hope to achieve and to collect the necessary data to measure them as the experiments progress . one such experiment is the nigms maximizing investigators ' research award ( mira ; preusch , 2015 ) . the mira program aims to transform how we support fundamental biomedical research , shifting away from the current paradigm of funding specific , predefined projects to one in which we focus on supporting the overall research program in each investigator 's lab . the goals of the mira pilot are to 1 ) increase funding stability for investigators to enhance their ability to take on ambitious research and approach problems creatively ; 2 ) increase flexibility for investigators to follow new directions as ideas and opportunities arise , which should help maximize the chances for breakthroughs ; 3 ) improve the distribution of funding , allowing the institute to support an optimally broad and diverse portfolio of investigators ; and 4 ) reduce the amount of time spent writing and reviewing grant applications , freeing up time to focus on research , training , and mentoring . each of these goals aims to help maximize the scientific returns on taxpayers ' investments . much discussion of the problems facing the biomedical research enterprise ( e.g. , ioannidis , 2011 ; bourne , 2013b , c ; alberts et al . , 2014 ) . among the many challenges , perhaps the most worrying is that junior scientists are becoming increasingly discouraged about their career prospects ( bourne , 2013a ; polka and krukenberg , 2014 ) , a growing crisis that could leave a serious deficit in the nation 's scientific capacity for years to come . but despite the many challenges we face , there are reasons to be optimistic about the future . scientifically , our deepening and expanding knowledge is opening up incredible new frontiers in research , and developments in technology are allowing us to address questions that a decade ago seemed completely inaccessible . although i have not discussed it here , renewed focus on improving scientific education , training , and career development seems likely to lead to an even more skilled , productive , and efficient workforce in the coming years . there is a growing recognition that we owe it to the nation and future generations to reoptimize the biomedical research enterprise ( lorsch , 2015 ) , and momentum is building in many sectors to make the changes this reoptimization will require . for example , the nih ( rockey , 2012 , 2015 ; maas , 2015 ) , members of congress ( harris and young , 2014 ; hearing on the fy 2016 national institutes of health budget request , 2015 ) , professional societies ( federation of the american societies for experimental biology , 2015 ) , and academic leaders ( daniels , 2015 ) are focusing attention on the challenges facing junior scientists and are trying to develop strategies to help this critically important group . if these trends continue , and all of the stakeholders take on their share of the responsibility , i am confident we can develop a more efficient and sustainable biomedical research enterprise and , in the process , accelerate advances in human health and prosperity .

the national institute of general medical sciences ( nigms ) at the u.s . national institutes of health has an annual budget of more than $ 2.3 billion . the institute uses these funds to support fundamental biomedical research and training at universities , medical schools , and other institutions across the country . my job as director of nigms is to work to maximize the scientific returns on the taxpayers ' investments . i describe how we are optimizing our investment strategies and funding mechanisms , and how , in the process , we hope to create a more efficient and sustainable biomedical research enterprise .

A diverse investment portfolio is a strong portfolio Emphasis on investigator-initiated research The value of team science Building the foundation for breakthroughs: the value of steady progress The optimal lab size Changing our funding metric A funding experiment: supporting research programs instead of specific projects Creating a bright future for biomedical research

PMC5364737

while the benefits of digital pathology in routine clinical use continue to inspire initiatives around the world , some european countries are arguably ahead of the curve . there are now a number of sites using digital pathology for primary diagnostic review , for example , in sweden , the netherlands , finland , lithuania , and denmark . the nordic symposium on digital pathology ( ndp ) was created in 2013 to promote exchange of state - of - the - art knowledge . the pioneering experiences in sweden have been a sounding board for the care providers moving in the same direction . in contrast to research use of digital techniques , ndp specifically targets the clinical adoption of whole - slide imaging ( wsi ) and other digital technologies . while there appears to be broad consensus on the opportunities that lie ahead , a foundational motivation for the symposium is that effective progress is dependent on tight collaboration between health care , industry , and academia . judging from the attendees evaluations from this and previous years , this type of venue with ample room for interactive discussions is highly appreciated and much needed . here , we provide a summary of the ndp 2016 , in the form of a meeting overview , results from the symposium 's integrated diagnostics workshop , speaker contributions , and finally brief conclusions . the 2016 ndp symposium took place on november 89 , 2016 , in linkping , sweden . a total of 170 attendees gathered , of which 47% listed health care as the primary affiliation , 35% industry , and 18% academia . pathologists constituted the lion share of health care representatives , with managers , laboratory technologists , and information technology ( it ) staff also being well - represented categories . the participants represented 13 different countries across europe , asia , and north america , with the nordic attendees being a large majority ( 88% ) . a large part of the program was devoted to invited talks and a workshop on integrated diagnostics . the contents of these sessions will be outlined in the sections below . in the science and innovation session , 12 posters were presented , with contributions from groups in linkping , bremen , tampere , uppsala , lund , and norrkping . the ndp also included an industrial exhibition with 12 vendors , showing a wide range of digital pathology products and services . figure 1 shows a snapshot from the symposium and the program details are available at the ndp website www.ndp2016.se . a central component of this year 's ndp was a workshop discussing collaboration between radiology and pathology and how digital tools relate to developments in this area . it is important to note that the survey respondents represent a biased selection among the pathology community . since only ndp participants were asked , this means that respondents are likely to be among the most positive to digital pathology and also among the most experienced . there is also strong geographical dominance from the nordics and , in particular , sweden . it is likely that the questions that require deep knowledge of clinical practice were dominated by pathologists as those questions to a substantial extent were skipped or received role distribution of survey respondents the survey first asked , today , to what degree do you use digital images of histology slides in your practice ? ( in percentage of all histology cases ) . the results are shown in figure 3 , for instance that a majority of respondents report substantial use of digital imaging in primary diagnostics ( another bias to note for these questions is that several people from the same institution may have responded ) . current use of digital pathology among survey respondents the main theme of the survey was integrated diagnostics , i.e. , the idea of closer collaborative work practices between pathology and radiology . such proposals have been voiced over the years and appear to be gaining traction in the respective communities , likely fueled by the increasing possibilities arising when pathologists also work in a primarily digital environment . to the end of forming a baseline , the survey asked pathologists , how often do you have direct interaction with a radiologist regarding a patient case ? figure 4 shows the results , painting the picture that multidisciplinary team meetings are where the interaction primarily happens but also that the direct interaction overall is low . one could argue that computer chat would be a very convenient and efficient way of interdisciplinary communication as it has proven to be within radiology settings . the low use may , however , merely be a sign that appropriate it tools are currently missing . interaction frequency between pathologist and radiologist , across different communication types the respondents were also asked to imagine well - crafted future collaboration support in the form of new it tools and workflows , and in that environment assess the potential : what impact will pathology - radiology collaboration have ? the impact within different areas is presented in figure 5 . overall , the ndp attendees were very positive about the potential , in particular , about the possibility for deeper discussion on diagnosis discordance . assessed impact of deepened pathology - radiology collaboration of different types , assuming well - crafted information technology tools and workflows are available . what impact will pathology - radiology collaboration have , in the area of the survey results were presented as a segway into a panel discussion . jeroen van der laak ( radboud university , nijmegen , the netherlands ) , and prof . some of the main points brought forward were as follows : the underlying technology is there , now we need to focus on integrating them into workflows and good user experiences ( prof . hamilton ) ; it is important to step out of the back office mindset and actively contribute to shaping the best possible care chain for the patient ( dr . devlin ) ; integrated diagnostics should also be seen in the broader perspective of regional / national integration ( prof . farstad ) ; it is vital to avoid misdirected diagnostic activities the diagnosticians should challenge any request they believe to be unnecessary ( dr . devlin ) ; an attractive concept is to have a matre d for diagnostics , a new role guiding diagnostic paths and providing effective single - point communication to referrers ( prof . the overall conclusion from the panel and the discussion with the ndp attendees is that closer collaboration between pathology and radiology is a positive and important development track going forward . levenson presented the latest achievements with slide - free microscopy technology based on ultraviolet ( uv ) excitation . the technology , dubbed microscopy with uv surface excitation , allows direct depiction of unsectioned specimens at full microscopic resolution . levenson demonstrated many case examples also including image features not represented in traditional staining and microscopy , including three - dimensional tissue information , features whose diagnostic value is now being explored . along another vein , levenson shared insights on using trained pigeons as unbiased proxies for human visual pattern recognition in pathology , work which was both humorous and thought - provoking . the second keynote speaker , associate prof . jeroen van der laak , radboud university , provided ample insight into state of the art in image analysis for wsi . among other topics , van der laak shared result details from the recent grand challenge , called camelyon16 , in lymph node metastasis detection . the research challenge encompassed a whole - slide task , in this sense closer to the clinical reality than previous challenges , and leading results demonstrated levels of accuracy in line with human assessment . of interest , however , was that machines were being compared with the ultimate human effort ( i.e. , the painstaking detailed microscopic review of every part of the lymph node , something not always feasible in clinical practice ) . in tandem with those promising results , van der laak emphasized the demanding efforts still needed to bring the algorithms into the everyday clinical context . johan hartman , karolinska institutet , presented work concluding that computational analysis outperformed manual assessment for breast cancer subtyping . for example , the image analysis algorithm showed higher sensitivity and specificity for the luminal b subtype , being considered a very challenging distinction . arvydas laurinavicius , vilnius university , spoke to the distinct prognostic value of bimodality of ki67 expression - an analysis requiring computational methods . instead of restricting proliferation measures to a hotspot , laurinavicius showed results where heterogeneity across the slide proved to be a more powerful survival indicator in breast cancer . he also shared his group 's experiences on running routine primary review using telepathology , with several colleagues reporting routine clinical work remotely . peter van ooijen , university medical center groningen , described the challenges an informatician will meet , ranging from overarching strategic efforts to mundane pragmatic problem - solving . a key takeaway was the importance of adopting and enforcing standards when building an effective and robust digital system . devlin , altnagelvin hospital , presented the journey to a fully digitized national radiology system in northern ireland . apart from advice on handling technology and workflow change , devlin emphasized the need to establish direct support from stakeholders in top leadership layers . bethany williams , leeds university , contributed to the ongoing efforts in wsi validation by presenting a systematic analysis of the type of cases where discordance with microscopic review has occurred and what can be learned from them . among the most challenging areas were diagnosis of dysplasia and finding small objects ( e.g. , micrometastases ) . tieto sweden had invited theo papaioannou from the vstra gtaland region , sweden , to present their ongoing large - scale digitization project . from philips , dr . hamilton presented computational imaging solutions and insights on remaining challenges for digital pathology in the context of workforce shortages and the need to collaborate . jesper molin from sectra presented key takeaways from his phd thesis , devoted to new opportunities for pathologist interaction in digital image review . finally , dr . per hertz from eizo nordic discussed the importance of high and controlled display performance in the context of diagnostic quality . finally , a seminar on implementing digital microscopy in medical education was hosted by clinicum , linkping university . the topics covered have evolved from entry - level concerns about how to scan slides properly , or whether complete digitization is possible , to mature discussions on how to design systems and workflows to accomplish quality and productivity gains gains that are undisputed . as in previous years , the attendees expressed the great value of this experience sharing across organizations , disciplines , and sectors . darren treanor is a member of the aperio / leica advisory board and the sectra advisory board . darren treanor is a member of the aperio / leica advisory board and the sectra advisory board .

the nordic symposium on digital pathology ( ndp ) was created to promote knowledge exchange across stakeholders in health care , industry , and academia . in 2016 , the 4th ndp installment took place in linkping , sweden , promoting development and collaboration in digital pathology for the benefit of routine care advances . this article summarizes the symposium , gathering 170 attendees from 13 countries . this summary also contains results from a survey on integrated diagnostics aspects , in particular radiology - pathology collaboration .

I M W S C Financial support and sponsorship Conflicts of interest

PMC2646651

the children with idiopathic speech and language disorders are very heterogeneous by phenotype . the diagnosis of specific language impairment ( sli ) is generally made by applying exclusion and inclusion criteria according to icd 10 ( 1992 ) . sli is diagnosed , if the speech language development is significantly delayed , deviated or unclear and if the problem does not result from defective sense organs , any known neurological or neuropsychiatric disease , anomaly of oral apparatus or mental retardation ( icd 10 1992 ; barry et al 2007 ) . shriberg and colleagues ( 2005 ) have examined diagnostic and phenotype markers for genetically transmitted speech delay . . it is also difficult to evaluate the effect of recurrent early middle ear infections , particularly those with effusion , on speech and language development . it is generally thought that lengthened periods of otitis media with effusion impair acoustic - phonetic perception and persistent brain imprinting of speech sounds ( ptok and eysholdt 2005 ) . it is difficult to assess the prevalence and incidence of hereditary speech - language disorders because of the lack of clear criteria . the prevalence of familiar sli is reported to be about 20%80% in speech disordered children ( bishop and edmundson 1986 ; tallal et al 1989 ; barry et al 2007 ) . in typically developing children the respective estimation has been about 3%8% ( bishop and edmundson 1986 ; tomblin et al 1997 ; barry et al 2007 ) . the suggestions of causes resulting in idiopathic speech - language disorders have varied during decades and affected the terminology and diagnostic criteria . consistent articulation errors have been thought to originate from speech motor problems , whereas the label dysphonology has been attached to variable and unstable speech sound errors with no detectable physiologic or physical association . it has been argued that motor impairment can not explain that a child is capable of producing a specific sound in one condition , but not in another . the inconsistency of speech sound errors has been taken as a linguistic disorder and a deficiency to use the sounds as a part of the language system ( gibbon 1999 ) . because of the difficulty in recognizing the pathophysiology of the speech sound errors the term articulation / phonology disorder ( apd ) has been used . the omissions and substitutions of speech sounds have been suggested to represent phonological , ie , linguistic , disorders , while distortions indicate articulatory problems , ie , consistent dysfunction of articulating speech organs . these errors probably origin from undifferentiated lingual gestures , and thus reflect delayed or deviant control of functionally independent regions of the tongue . the lack of differentiation of articulators has been documented by electropalatography ( gibbon 1999 ) . the lack of differentiation of the tongue tip and blade from the movements of posterior part of the tongue , as well as the difficulty in controlling the lateral parts of the tongue in sagittal groove formation , is typical in apd . apd children also tend to lift the posterior part of the tongue , when only anterior lift is required . undifferentiated tongue movement reflects insufficient spatial acuity of the articulators that is typical in immature articulation . however , the motor variability in producing speech sounds may also result in linguistic impairment in terms of phonological inconsistency of the speech sound system of the language ( gibbon 1999 ) . considering developmental speech sound errors the term speech sound disorder ( ssd ) is currently preferred because this fairly common developmental disorder may have antecedents in both articulatory ( sensorimotor ) and phonological ( cognitive - linguistic ) domains ( lewis et al 2006 ; mcgrath et al 2007 ) . it is considered to be clinically useful to subtype the children with ssd into those that present with concomitant language disorder and to those without it ( lewis et al 2006 ) . the phonetico - phonological symptoms of apd and ssd share common speech characteristics with developmental verbal dyspraxia ( dvd ) , childhood apraxia of speech ( cas ) , or developmental apraxia of speech ( das ) . das and cas have been described as disorders of speech motor programming and control . in them , the behavioral characteristics are numerous , including inconsistent speech sound errors and dysprosody ( forrest 2003 ; nijland et al 2003 ; jacks et al 2006 ) . the later developing consonants ( eg , /l/ , /r/ , /s/ ) are typically omitted or substituted by early developing ones . the initial and final consonants are often omitted , the overall consonant accuracy is low and the production of consonant clusters and complex mono- and polysyllables is typically difficult ( jacks et al 2006 ) . the vowels may also be distorted , but the distortion is not always eliminated by advancing age ( davis et al 2005 ) . children with cas ( das / dvd ) often present with severe and long lasting unintelligibility of speech , and the treatment so far lacks generally accepted effective methods ( davis et al 1998 ; jacks et al 2006 ) . in summary , the developmental disorders apd , ssd , dvd , cas , and das share common characteristics and the terms seem to be used somewhat synonymously ( cf . gibbon 1999 ; forrest 2003 ; nijland et al 2003 ; jacks et al 2006 ; mcgrath et al 2007 ) . apd , ssd , dvd , cas , and das are idiopathic expressive speech disorders that may be included in developmental language impairment ( dli ) or specific language impairment ( sli ) . even though there is so far no consensus on the diagnostic inclusion or exclusion criteria and definition of dvd ( forrest 2003 ) , a consensus has been obtained about the definition of general developmental dyspraxia ( sanger et al 2006 ) . it is typical that the child has not yet achieved the skills to perform complex motor functions ( in the absence of muscular weakness , involuntary muscular movements , or deficient selective control of muscular functions or ataxic disability to activate correct motor patterns ) appropriate for age ( sanger et al 2006 ) . this definition fits also well in dvd , which may be taken as narrow band disorder among developmental dyspraxias . disorders of motor development associated with dli and sli are common , widely recognized and reported ( bishop et al 1987 ; robinson 1991 ; rintala et al 1998 ; hill 1998 ; webster et al 2006 ) . motor problems are found to be common also in association with reading disability , even in 60% of cases it is generally thought that the children start walking without support at the average age of 12 months . according to the who multicenter study ( 2006 ) , 50% of the children have started to walk unassisted by the age of 12 months , about 25% by the age of 11 months , and 3%10% by the age of 910 months . very little attention , if at all , has been paid on speedy motor development in association with speech - language disorders . even though speedy walking babies are less common in general population , they seem to be common among speech disordered children ( haapanen 2007 ) . according to clinical observations , early walking seems to be familial also among those speech - language disordered children , particularly in children with expressive disorders that have started walking at typical age . haapanen ( 2007 ) has described a case series of 10 children with a particular developmental behavioral triad . the children have started to walk unassisted earlier than typical ( at the latest by the age of 11 months ) , and they experience with expressive speech disorders resembling das and tongue movement dysfunction ( lingual dysfunction with undifferentiated lingual movements resulting in dentoalveolar ( ie , linguoalveolar ) sound disorder . to name and describe them shortly , these children are here called speedy babies ( vauhtiveikot in finnish ; haapanen 2007 ) with spee referring to speech and dy to developmental dyspraxia or speech - language disorder such as developmental dysphasia , and dysfunction of the tongue . the aim of the present case series that describes four children with unbalanced psychomotor development ( speedy babies ) is to draw attention to children who walk early and have speech - language disorders , so that professionals may recognize the problem without delay . the coexistence of particular psychomotor characteristics is discussed in terms of interpreting them as a syndrome . in addition , a rough preliminary analysis of relevant demographic and clinical data in a series of 60 consecutive cases was carried out in order to make a preliminary estimation of the frequency of early independent walkers among speech disordered children and to explore the distribution of the age at unassisted walking between two diagnostic groups , ie , children with mainly expressive and mainly receptive verbal problems . the children were referred to special medical consultation because of unclear speech , which caused social or emotional problems . all these four children were followed up and evaluated by a speech therapist several times each . the speech - language disorder was diagnosed on the basis of multidisciplinary evaluations , which included clinical examination performed by the medical speech pathologist and also individually administered , normative , and/or orienting tests and series of tasks that were performed by the speech therapist and the neuropsychologist . also , a series of 60 consecutive cases was preliminary examined . the data gathered included the diagnostic type of speech - language disorder ( ie , whether mainly expressive vs receptive ) , the age of the occurrence of walking without support and the familial occurrence of speedy walking and speech / language / reading disabilities . the children with intelligence deficiency , abnormality of orofacial structures ( except a minimal and nonsignificantly tongue tie , ie , short lingual frenulum ) , sense organ deficiency , psychiatric diagnosis or any recognizable neurological disease were excluded , as were also the children with a mother tongue some other than finnish , or with the age of more than 6 years . the data were analyzed by mann - whitney u - test and fisher s exact test . no sucking problems or dysphagia were ever reported . according to the parents reports and the carefully made baby clinic registrations , he had begun to walk without support at the age of 10 months , he had about five verbal symbols by the age of 1518 months , and he spoke short sentences by the age of 22.5 years . although the first words occurred at a normal age , his active vocabulary grew up slowly . the child was further referred to medical speech pathologist , ie , phoniatrician , at the age of 6 years for diagnosis and a comprehensive treatment plan . speech therapeutic intervention had been started at the age of 3 years because of unclear speech . speech and language development was then assessed during 16 sessions by the speech therapist . the child presented with unintelligible words , sound substitutions , assimilations and other articulatory / phonological disorders typical of verbal dyspraxia . when re - evaluated and tested at the age of 6 years , he performed within mean age level in the reynell speech comprehension test ( finnish version ; kortesmaa et al 2001 ) , but achieved results that were slightly below mean age level in a finnish normative sentence comprehension test ( korpilahti 1996 ) . psychological evaluations carried out at the age of 6 years were based on wppsi - r ( 1995 ) , the sentence and functional order comprehension items of the nepsy test ( korkman et al 1997 ) , ro ( rorschach test 1948 ) , free play observation and as well a parent questionnaire and kindergarten report analysis . he performed better in the performance items than in the language ones , albeit no significant discrepancy between them was found . he had some conceptual difficulties , particularly related to time , and some naming difficulty , albeit on the other hand he could describe some concepts better than what was typical for a child at his age . his attention tended to impair slightly in complicated tasks . by 6 years of age , many consonants were substituted by a sound auditively resembling a velar plosive /k/. he was still actively using the posterior part of the tongue in articulation . he also tended to speak with tongue tip and blade fixed to the bottom of the mouth . however , initial consonant deletions , consistent substitutions , omissions or reversals were no more detected , except some consonant cluster assimilations . yet , inconsistent sound substitutions and reversals typical of verbal dyspraxia were observed in semi sense sentences and nonsense word repetition tasks ( such as pa - ta - ka ) , implicating the presence of latent dyspraxia . the nonverbal tongue movements were considerable slow and groping , but he was able to protrude the tongue out of the mouth , and bend it on the upper lip . the child was found active and skilled in rough motor functions such as running , climbing , bicycling , and swimming . in kindergarten he was found impulsive and not very concentrated on issues that he was not interested in . but , no attentional problems were registered in association with issues and activities that he was interested in . according to kindergarten reports he was active , helpful , talkative , but he experienced some social problems with verbal communication because of unclear speech . no suspicion of attention - deficit hyperactivity disorder ( adhd ) was ever roused even though the child was lively . the child had had only one middle ear infection so far , and no allergies were recognized . his younger sister had begun to walk without support at the age of 9 months . the parents had not suffered from speech , reading , or other learning disabilities . for rehabilitative purposes , the child was recommended to be supported by parents and kindergarten stuff in social interaction and concentration . the speech therapist set the targets of individual intervention to improve abstract and basic concepts , as well as the clarity of the speech . the possibility of dyslexia could not be ruled out , and therefore the school stuff was recommended to organize the possibility of pedagogic rehabilitation by special teacher before hand . the follow up examinations of the child s development were scheduled to communal basic health care . a physically healthy male child was born after normal pregnancy and delivery . according to parents report and health care registrations he begun to walk without support at the age of 7 months , but spoke the first words not earlier than by the age of 2.5 years . the first sentences consisting of two words occurred at the age of 3 years . at the age of 5 years , the boy was referred to a local health care speech therapist because of unclear speech . he received 10 assessment and intervention sessions by the therapist that found the child talkative , yet presenting with the short sentences and inaccurate narrating . object naming assessed by the boston test ( kaplan et al 1997 ) was not appropriate for age . the child was referred to medical speech pathologist at the age of 6 years because of speech production difficulties , and for diagnosis and further rehabilitation plan . he was then still found to have articulatory errors and dysphonology typical of developmental verbal dyspraxia . sound was still palatalized in continuous speech , but no more in single words or in controlled speech production situations . assimilations of alveolar sounds /s/ and /r/ occurred when produced in association with other consonants . the child had difficulties in protruding the tongue and bending it up to the upper lip . movement of the tongue was not fully differentiated from the movement of the lower jaw . no velopharyngeal or labial dysfunction was recognized . in neuropsychological testing by wpps1-r ( 1995 ) and the items 3 , 7 , 15 , 16 , 30 of the nepsy test ( korkman et al 1997 ) carried out at the age of 6 years , his nonverbal performance was in the average level for the age . verbal performance was poorer compared to the nonverbal performance , and it was lower than the age average for his age . auditive short term memory span was more limited than normal as tested by digits and sentences . understanding of complicated sentences and instructed narrating of a heard story was age - appropriate , even though he scored a level of low normal . it was also found important to focus on phonological awareness to improve readiness for reading . according to health care recordings and information given by parents , he walked without support by the age of 1011 months . the first words occurred before the age of 12 months , but the speech development was slow and remained limited . he was examined by a local health care psychologist and assessed and rehabilitated by a speech therapist for 25 times . the following tests and assessment were carried out : auditive reasoning , understanding , and short term memory items of the itpa - test ( kuusinen and blfield 1974 ) , a finnish articulation test ( remes 1975 ) , a finnish test for active vocabulary ( odell 1953 ) , the reynell language comprehension items ( kortesmaa et al 2001 ) and a finnish sentence comprehension test ( korpilahti 1996 ) , wppsi - r ( 1995 ) , and nepsy ( linguistic items ) ( korkman et al 1997 ) , boehm test for basic concepts ( boehm 1993 ) , drawing tasks , and play observation . speech comprehension was below normal according to the reynell test at the age of 4 years , but had become age average at the age of 5 years when re - tested . at that age , he had no problems in color naming or in basic concepts , but the speech was still severely unclear including instable sound substitutions , assimilations , and other errors typical of verbal dyspraxia . narrating was somewhat limited . the child was referred to special health care at the age of 5 years and 4 months for diagnosis and treatment design . the tongue tip was a little deformed in a little heart - like form due somewhat short but a thin and flexible frenulum of the tongue . the very mild tongue tie was not regarded to explain the phonological errors or stable lingual dysfunction resulting in dentoalveolar sound distortions , substitution , omission or assimilations . visual perception was strongly above normal , albeit fine motor hand movements , such as using a pencil or binding the bands of the shoes were regarded clumsy . his parents regarded him skilled in different rough motor functions such as climbing and running , he had no allergies and only few otitis media . individual speech therapy recommended and assisting it with intraoral removable oral - plate treatment was considered . the kindergarten personnel was involved in rehabilitation of the child s speech and language development , and an assisting nurse was introduced . his first words occurred at the age of 18 months and he formed sentences by the age of 2 years and 5 months . the speech was already from the beginning very unclear , and he had received speech therapy for three years from the age of 2 years . in the beginning his active vocabulary was below age level , but grew to be average for at the age of 4 years , when also the reynell iii test ( finnish version ; kortesmaa et al 2001 ) results indicated normal speech comprehension . according to kindergarten reports , the boy was very active , lively , and daring . he was regarded to be skilled in rough motor functions . although the child spoke a lot and had long sentences , his speech was still unintelligible and he suffered from it . he was referred to medical speech pathologist at the age of 5 years , when neuropsychological testing was also carried out . both nonverbal performance and verbal performance was at the age - appropriate level , and no significant discrepancy was found between them . , /s/ , and /l/ sounds and he was not able to produce low vowels . a physically healthy male child was born after normal pregnancy and delivery . no sucking problems or dysphagia according to the parents reports and the carefully made baby clinic registrations , he had begun to walk without support at the age of 10 months , he had about five verbal symbols by the age of 1518 months , and he spoke short sentences by the age of 22.5 years . although the first words occurred at a normal age , his active vocabulary grew up slowly . the child was further referred to medical speech pathologist , ie , phoniatrician , at the age of 6 years for diagnosis and a comprehensive treatment plan . speech therapeutic intervention had been started at the age of 3 years because of unclear speech . speech and language development was then assessed during 16 sessions by the speech therapist . the child presented with unintelligible words , sound substitutions , assimilations and other articulatory / phonological disorders typical of verbal dyspraxia . when re - evaluated and tested at the age of 6 years , he performed within mean age level in the reynell speech comprehension test ( finnish version ; kortesmaa et al 2001 ) , but achieved results that were slightly below mean age level in a finnish normative sentence comprehension test ( korpilahti 1996 ) . psychological evaluations carried out at the age of 6 years were based on wppsi - r ( 1995 ) , the sentence and functional order comprehension items of the nepsy test ( korkman et al 1997 ) , ro ( rorschach test 1948 ) , free play observation and as well a parent questionnaire and kindergarten report analysis . he performed better in the performance items than in the language ones , albeit no significant discrepancy between them was found . he had some conceptual difficulties , particularly related to time , and some naming difficulty , albeit on the other hand he could describe some concepts better than what was typical for a child at his age . his attention tended to impair slightly in complicated tasks . by 6 years of age , his spontaneous speech was still unclear and he presented with some unintelligible words . many consonants were substituted by a sound auditively resembling a velar plosive /k/. he was still actively using the posterior part of the tongue in articulation . he also tended to speak with tongue tip and blade fixed to the bottom of the mouth . however , initial consonant deletions , consistent substitutions , omissions or reversals were no more detected , except some consonant cluster assimilations . yet , inconsistent sound substitutions and reversals typical of verbal dyspraxia were observed in semi sense sentences and nonsense word repetition tasks ( such as pa - ta - ka ) , implicating the presence of latent dyspraxia . the nonverbal tongue movements were considerable slow and groping , but he was able to protrude the tongue out of the mouth , and bend it on the upper lip . the child was found active and skilled in rough motor functions such as running , climbing , bicycling , and swimming . in kindergarten he was found impulsive and not very concentrated on issues that he was not interested in . but , no attentional problems were registered in association with issues and activities that he was interested in . according to kindergarten reports he was active , helpful , talkative , but he experienced some social problems with verbal communication because of unclear speech . no suspicion of attention - deficit hyperactivity disorder ( adhd ) was ever roused even though the child was lively . the child had had only one middle ear infection so far , and no allergies were recognized . his younger sister had begun to walk without support at the age of 9 months . for rehabilitative purposes , the child was recommended to be supported by parents and kindergarten stuff in social interaction and concentration . the speech therapist set the targets of individual intervention to improve abstract and basic concepts , as well as the clarity of the speech . the possibility of dyslexia could not be ruled out , and therefore the school stuff was recommended to organize the possibility of pedagogic rehabilitation by special teacher before hand . the follow up examinations of the child s development were scheduled to communal basic health care . a physically healthy male child was born after normal pregnancy and delivery . according to parents report and health care registrations he begun to walk without support at the age of 7 months , but spoke the first words not earlier than by the age of 2.5 years . the first sentences consisting of two words occurred at the age of 3 years . at the age of 5 years , the boy was referred to a local health care speech therapist because of unclear speech . he received 10 assessment and intervention sessions by the therapist that found the child talkative , yet presenting with the short sentences and inaccurate narrating . object naming assessed by the boston test ( kaplan et al 1997 ) was not appropriate for age . the child was referred to medical speech pathologist at the age of 6 years because of speech production difficulties , and for diagnosis and further rehabilitation plan . he was then still found to have articulatory errors and dysphonology typical of developmental verbal dyspraxia . sound was still palatalized in continuous speech , but no more in single words or in controlled speech production situations . assimilations of alveolar sounds /s/ and /r/ occurred when produced in association with other consonants . the child had difficulties in protruding the tongue and bending it up to the upper lip . movement of the tongue was not fully differentiated from the movement of the lower jaw . no velopharyngeal or labial dysfunction was recognized . in neuropsychological testing by wpps1-r ( 1995 ) and the items 3 , 7 , 15 , 16 , 30 of the nepsy test ( korkman et al 1997 ) carried out at the age of 6 years verbal performance was poorer compared to the nonverbal performance , and it was lower than the age average for his age . auditive short term memory span was more limited than normal as tested by digits and sentences . understanding of complicated sentences and instructed narrating of a heard story was age - appropriate , even though he scored a level of low normal . it was also found important to focus on phonological awareness to improve readiness for reading . a male child was born after normal pregnancy and delivery with normal physical health . according to health care recordings and information given by parents the first words occurred before the age of 12 months , but the speech development was slow and remained limited . he was examined by a local health care psychologist and assessed and rehabilitated by a speech therapist for 25 times . the following tests and assessment were carried out : auditive reasoning , understanding , and short term memory items of the itpa - test ( kuusinen and blfield 1974 ) , a finnish articulation test ( remes 1975 ) , a finnish test for active vocabulary ( odell 1953 ) , the reynell language comprehension items ( kortesmaa et al 2001 ) and a finnish sentence comprehension test ( korpilahti 1996 ) , wppsi - r ( 1995 ) , and nepsy ( linguistic items ) ( korkman et al 1997 ) , boehm test for basic concepts ( boehm 1993 ) , drawing tasks , and play observation . speech comprehension was below normal according to the reynell test at the age of 4 years , but had become age average at the age of 5 years when re - tested . at that age , he had no problems in color naming or in basic concepts , but the speech was still severely unclear including instable sound substitutions , assimilations , and other errors typical of verbal dyspraxia . narrating was somewhat limited . the child was referred to special health care at the age of 5 years and 4 months for diagnosis and treatment design . the tongue tip was a little deformed in a little heart - like form due somewhat short but a thin and flexible frenulum of the tongue . the very mild tongue tie was not regarded to explain the phonological errors or stable lingual dysfunction resulting in dentoalveolar sound distortions , substitution , omission or assimilations . visual perception was strongly above normal , albeit fine motor hand movements , such as using a pencil or binding the bands of the shoes were regarded clumsy . his parents regarded him skilled in different rough motor functions such as climbing and running , he had no allergies and only few otitis media . individual speech therapy recommended and assisting it with intraoral removable oral - plate treatment was considered . the kindergarten personnel was involved in rehabilitation of the child s speech and language development , and an assisting nurse was introduced . his first words occurred at the age of 18 months and he formed sentences by the age of 2 years and 5 months . the speech was already from the beginning very unclear , and he had received speech therapy for three years from the age of 2 years . in the beginning his active vocabulary was below age level , but grew to be average for at the age of 4 years , when also the reynell iii test ( finnish version ; kortesmaa et al 2001 ) results indicated normal speech comprehension . according to kindergarten reports , the boy was very active , lively , and daring . he was regarded to be skilled in rough motor functions . although the child spoke a lot and had long sentences , his speech was still unintelligible and he suffered from it . he was referred to medical speech pathologist at the age of 5 years , when neuropsychological testing was also carried out . both nonverbal performance and verbal performance was at the age - appropriate level , and no significant discrepancy was found between them . , /s/ , and /l/ sounds and he was not able to produce low vowels . table 1 shows the summary data of the distribution of the age at unassisted walking and speech data among 60 consecutive speech and/or language disordered children studied preliminarily . of those 60 children 23 ( 38% ) began to walk unassisted at the age of 11 months or earlier ( group a ) , and 37 ( 62% ) walked at the age of 12 months or later ( group b ) . sli ( with or without symptoms of verbal dyspraxia ) was diagnosed in a total of 49 children , out of which 12/49 ( 24% ) suffered from receptive / mixed type of sli . a total of 21/49 ( 43% ) children with sli belonged to group a. more precisely , 18/37 ( 48% ) of children with expressive type of sli and only 3/12 ( 25% ) of children with receptive or mixed type of sli begun to walk unassisted at the age of 11 months or earlier ( group a ) . the age of walking without support was not evenly distributed between the two diagnostic groups . expressive sli was significantly associated with earlier walking age according to mann - whitney u test ( u = 126.00 , p = 0.022 ) . in the total group of 60 children , the ones with expressive speech production disorders ( including children with mild ankyloglossia and impaired speech without diagnosed sli ) walked significantly earlier than the ones with speech comprehension problems ( mann - whitney u test , u = 195.5 , p = 0.013 ) . counting out the children who began walking at the age of 11 or 12 months , 10/16 ( 63 % ) children were left , who had expressive speech problems and walked without support before the age of 11 months . also 6/16 ( 37% ) children were left who had expressive speech impairment , and who walked at the age of 13 months or later . none of those children that had receptive or mixed sli belonged to the earlier walking group . the difference was statistically significant ( fisher s exact test , p = 0.035 ) . the children with expressive sli had significantly more often relatives with speedy walking than the children with mainly receptive sli ( fisher s exact test , p = 0.044 ) . the groups did not differ from each in respect to familial occurrence of speech / language or reading disabilities . multidisciplinary speech evaluation and longitudinal follow ups were carried out in all the four cases described in this study . it appeared that the children demonstrated characteristics typical of apd , ssd , dvd / das , or expressive sli ( dsm iv , icd 10 ) . the milestones of gross motor development are reported in terms of starting to walk without support . data of the age at which the first unassisted steps were made and when the occurrence of the first words and sentences occurred were obtained by the parents reports and the carefully made baby clinic recordings . however , parents typically note those milestones that are often emotionally charged so that they write them into baby books that are common in finnish families . parents tend to remember the developmental milestones of walking very well , even when the child is 3-years - old , and still fairly well , when the child is 5-years - old . however , recall of first words is less exact ( majnemer and rosenblatt 1994 ) . in these children , parents reports of developmental milestones although the children were reported skilled in many rough motor functions , such as climbing , running , bicycling , and various sports , data were not collected because of the difficulties of obtaining reliable data . a behavioral pattern of unbalanced motor and speech development is typical to the present children and similar to those described earlier by haapanen ( 2007 ) . the children presented with a triad of speedy unsupported walking ability associated with expressive speech impairment including inconsistent error characteristics typical of developmental verbal dyspraxia , as well as stable misarticulations . the consistent speech sound production errors were found to result from lingual dysfunction , in terms of the inability to lift and broaden the tip of the tongue on the upper lip or alveolar ridge and the deficiency in the control of lateral borders of the tongue . the present misarticulations resemble those due to undifferentiated lingual gestures that were described by gibbon ( 1999 ) . misarticulations of dentoalveolars and low vowels probably indicate difficulty in lowering the body and back of the tongue . typically consistent misarticulations occurred in the late developing alveolar , nonnasal continuous consonants ( /s/ , /l/ , /r/ ) . they were consistently distorted , but they also underwent inconsistent dysphonological processes such as omissions , substitutions , assimilations or reversals . no labial or velopharyngeal dysfunction occurred even though labials of velar stop consonants were inconsistently correctly produced / omitted / substituted or changed by other processes typical of verbal dyspraxia . in the case of two children , the neuropsychological discrepancy criteria that is used when diagnosing sli ( eg , dsm iv , icd 10 ) was not fulfilled , which is not , however , necessary in diagnosing verbal dyspraxia ( cf . it may not be possible to differentiate between dvd and ssd , that may occur with or without coexisting language disorder ( lewis et al 2006 ) . consistent lingual dysfunction that occurred in the present children and in those described by haapanen ( 2007 ) has not so far been reported as typical of verbal dyspraxia . however , the lingual dysfunction found in speedy babies may be similar to tongue motor difficulties , of which kent ( 1992 ) has stated that speech motor variability resulting in phonetic variability of speech sounds can be so great that it results in an apparent variability in phonology , ie , in dysphonology . murdoch and colleagues ( 1995 ) have found that children with dvd may have weaker lingual musculature and exhibit significantly reduced tongue strength endurance than the controls . even though the present children experienced with only a few consistent misarticulations , their speech disorder can be regarded as moderate or severe because of particularly inconsistent sound substitutions , omissions and assimilations . /s/ , and /l/ sounds are sounds that develop late and are often misarticulated in children even at the preschool age ( luotonen 1995 ) . the finnish /r/ sound is generally found very difficult to produce because of anterior tongue tip vibration necessary for normative /r/. the persisting /r/ sound disorders in finnish children have been taken as a marker of verbal dyspraxia ( qvarnstrm et al 1993 ) . the difference between the present speedy babies and typically developing children is , however , their dysphonology which is rare in the common population ( tomblin et al 1997 ) . an interesting finding is that the present speedy babies , as well as those described by haapanen ( 2007 ) seem to be physically healthy . they also can be taken as troublemakers because of their motor activity that results in demands for continuous attention to their physical safety , particularly in early infancy . since speedy rough motor development seems to be clustered in some children with idiopathic expressive speech impairment , particularly with speech characteristics typical of developmental verbal dyspraxia and consistent lingual dysfunction , it seems justifiable to consider those behavioral phenomena occurring as a syndrome . it is obvious that early walking does not explain the speech disorder , but there may be an inherited link between those developmental characteristics . interestingly , according to our preliminary observations , children that start walking early independently seem to be fairly common among speech disordered children , particularly among those with expressive speech - language disorders . in the present preliminary series of 60 consecutive cases , none of the children who had receptive sli walked before the age of 11 months . on the contrary , speedy motor development seems to be more often associated with delayed achievement of expressive speech considered normal by articulation or phonolgy . an interesting question arises : does biodiversity among human beings also appear in terms of bipolarity of the achievement of behavioral developmental milestones ? speedy walking development also seems to be common in the family members of both speedy babies and of those speech - language disordered children , who have started to walk at typical age . the children that start walking at typical age , but present with expressive sli seem to have significantly more often relatives with speedy walking than the children with receptive sli . this has not so far evoked any attention . instead , much attention has been paid on familial co - occurrence of speech / language and reading / writing disorders ( stormswold 1998 ; barry et al 2007 ) , which also are common in speedy babies , both in the present children and in those presented by haapanen ( 2007 ) . however , speech or reading and writing disorders do not seem to be significantly associated with either of the sli subtypes , whether expressive or receptive . it seems that dyslexia may not be as seemingly associated with early independent walking as expressive speech disorders seem to be . it is obvious that the association between the timetable of independent walking and speech and learning development warrants further studies . albeit developmental disorders of motor functions have been studied much ( hill 1998 ; webster et al 2006 ) , hardly any attention is paid on atypically speedy motor development in association with speech - language disorders . an unclear phenotypic diagnostics obscures the study populations and slow down accumulation of knowledge of this population ( sli consortium 2004 ) . in order to gather phenotypic and genotypic information and to design recognition and rehabilitation strategies of individuals with communication disorders , it is important to try to search for particular developmental and behavioral characteristics that seem to cluster . to delineate and sharpen the clinical picture of speedy babies , it is worth further studies to eg , examine how common speedy unassisted walking is among speech - language disordered population , how do the behavioral characteristics vary in different ages , how variable is their cognitive profile , what are their fine motor functions like . speedy rough motor and postural control development does not guarantee skilled fine motor functions , such as eg , eye - hand motor functions ( viholainen et al 2006 ) . it is noteworthy that the present children were fairly old , ie , 5- or 6-years - old , before they were referred to a medical specialist for further studies and arrangement of the financial recourses for their rehabilitation . it is emphasized that early recognition of the problems speedy babies is important , so that treatment strategies may be developed . early intervention on speech problems and appreciation of early gross motor skills will aid in sustaining appropriate self confidence in these often lively and active children . speedy babies require health care that recognizes and respects their motor skills and supports their needs for motor activities .

even though difficulties in motor development in children with speech and language disorders are widely known , hardly any attention is paid to the association between atypically rapidly occurring unassisted walking and delayed speech development . the four children described here presented with a developmental behavioral triad : 1 ) atypically speedy motor development , 2 ) impaired expressive speech , and 3 ) tongue carriage dysfunction resulting in related misarticulations . those characteristics might be phenotypically or genetically clustered . these children did nt have impaired cognition , neurological or mental disease , defective sense organs , craniofacial dysmorphology or susceptibility to upper respiratory infections , particularly recurrent otitis media . attention should be paid on discordant and unbalanced achievement of developmental milestones . present children are termed speedy babies , where speedy refers to rapid independent walking , spee and dy to dyspractic or dysfunctional speech development and lingual dysfunction resulting in linguoalveolar misarticulations . speedy babies require health care that recognizes and respects their motor skills and supports their needs for motor activities and on the other hand include treatment for impaired speech . the parents may need advice and support with these children .

Introduction Methods Case 1 Case 2 Case 3 Case 4 Preliminary data of 60 consecutive cases Discussion