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problems with recruiting professionals [1, 2 ], staff retention, creating healthy work environments [4, 5 ], and a growing demand for customer orientation pose challenges for nurse managers ' work. one essential area of nurse manager 's management skills is the use of different leadership styles. leadership styles can be seen as different combinations of tasks and transaction behaviours that influence people in achieving goals. earlier studies indicate that nurse manager 's effective leadership style is affiliated to staff retention, work unit climate, nurses ' job satisfaction [9, 10 ], nurses ' commitment, and patient satisfaction. transformational leadership style [5, 6, 13, 14 ] and transactional leadership help to respond to these issues. transformational leadership refers to the leader 's skills to influence others towards achieving goals by changing the followers ' beliefs, values, and needs. there are certain skills required from nurse managers so as to be able to use these effective leadership styles. the skills include the ability to create an organization culture that combines high - quality health care and patient / employee safety and highly developed collaborative and team - building skills. nurse managers also need to have the readiness to observe their own behaviour and its effects on the work unit ; as a result, employees can adjust to a better leadership style. these kinds of skills are related to manager 's emotional intelligence (ei). it has been defined as the ability to observe one 's own and others ' feelings and emotions, to discriminate among them and to use this information to direct one 's thinking and actions. self - awareness and self - management are reflections of personal competence, influencing the way the leader manages him / herself. social awareness and relationship management reflect social competence, which affects how the leader manages relationships with others. nurse managers with that skill can easily form relationships with others, read employees ' feelings and responses accurately, and lead successfully [1921 ]. have identified visionary, coaching, affiliate, and democratic styles as resonant, and pacesetting and commanding styles as dissonant leadership styles. the leadership styles of goleman. are applied as the basis of this study because earlier studies refer to the significance of these styles, especially that of ei in manager 's work. in addition, these leadership styles are one way of aiming to carry out transformational leadership. especially visionary, coaching, affiliate, and democratic styles include elements that promote transformational leadership. the finnish health care system is a strong institution where health care services are offered to all citizens and funded by taxes. it has widely recognized that health care services in finland are of high - quality despite recent concerns about equity issues, finns are in general very satisfied with their health care services.. consequently it is important to explore nurse managers ' leadership styles especially in this context. the intention of this study was to explore nurses ' and supervisors ' perceptions of nurse leaders ' leadership styles. the research questions were as follows : what kind of leadership styles do nurse managers use and what are the factors affected by their leadership styles. to achieve the aim of this study data were collected through open interviews. the majority of finnish nurse managers, nurses, and supervisors work in hospitals or long - term facilities. participants were selected paying attention to the fact they were of different ages, working in different wards and units (e.g., psychiatry, internal diseases, gerontology) in either hospitals or long - term facilities, and had worked with more than one nurse manager. the researcher contacted the participants and asked whether they were interested in taking part in the study. prior to the interviews each participant signed a form where they gave their consent to participate in the study. a total of 11 nurses and 10 supervisors, 20 women and one man, from eight finnish hospitals and five long - term care facilities participated in the study. the age of the nurses varied between 30 and 53 and their experience in health care between 7 and 25 years. the age of the supervisors varied between 38 and 59 and their experience as supervisors between 5 and 21 years. both nurses and supervisors had worked with many nurse leaders and they were interviewed about nurse managers in general. they thus had experience of different nurse managers on different wards and they were able to describe leadership styles from various aspects. semistructured interviews were used to gather data on the perceptions of nurse managers ' leadership styles and factors affected by leadership styles. they were subsequently asked about their perception of leadership styles and asked to describe the leadership styles used by their nurse managers. interviewing was continued until saturation of the data was achieved. because nurses and supervisors might have differed in their perceptions of leadership styles, the data were first analysed separately in two separate groups, following the same process for each group. content analysis was chosen because it is a research method for making valid inferences from data to the contexts of their use. the interview texts were read through multiple times, based on the author 's empirical and theoretical preunderstanding of the professional area of the participating nurses and nurse managers. a structured categorization matrix of leadership styles was developed based on the primal leadership model and research of vesterinen.. when using a structured matrix of analysis, an item of the data that does not fit the categorization frame is used to create its own concept, based on the principles of inductive content analysis. when both the data of nurses and superiors were analysed, the results were compared. the categories and subthemes were congruent and therefore the results are presented together, albeit paying attention to differences and similarities of the perceptions of nurses and superiors. a classification framework of the factors was formed inductively by defining categories and sub - themes. the criteria for allocating a unit to a category were formed by asking questions if the unit was suitable to the category. the sub - themes were named using descriptive concepts and classified as belonging to a particular category. the trustworthiness of this study has been ensured by confirming truth value, consistency, neutrality, and transferability of this study. when considering this study from the viewpoint of trustworthiness, there are some threats that should be taken into consideration. the researcher collected the data and performed the analysis alone and the interpretation could have been affected by her professional history. with interviews there is a risk that respondents try to please the interviewer by reporting things they assume s / he wants to hear. the researcher confirmed the truth value of the study by selecting participants in convenience sampling. the truth value of this study was also confirmed by analysing data as they emerged based on the interviews. to ensure the trustworthiness of the study quotes from interviews this gives a possibility to understand the limitations of the process of data collection and analysis. study the sample was small, consisting of finnish nurses and supervisors, and the results only reflect their perceptions of leadership styles. as a result, transferability of results is limited. however, when considering the main objective in this study, it was not transferability of research results, but it was to enhance understanding of leadership styles and use it for future studies. the data for this study were collected following approval from the administrations of the organizations. participants were asked to sign a form where they gave their consent to take part in the study. data analysis identified visionary, coaching, affiliate, democratic, commanding, and isolating leadership styles (figure 1). job satisfaction and commitment as well as operation and development work, cooperation and organizational climate in the work unit were the factors affected by leadership styles. visionary leadership stylesupervisors were of the opinion that today, nurse managers use a more visionary leadership style than previously. in the past, many organizations lacked a vision of their own and had fewer possibilities to engage in development for the future. even now, the skills of nurse managers to lead visionary development work varied. supervisors were of the opinion that today, nurse managers use a more visionary leadership style than previously. in the past, many organizations lacked a vision of their own and had fewer possibilities to engage in development for the future. even now, the skills of nurse managers to lead visionary development work varied. both nurses and supervisors reported that it was characteristic of the visionary nurse manager to emphasize and discuss the vision and provide information to employees. when establishing their vision, some nurse managers provided guidelines for attaining the work unit 's goals. these nurse managers had a systematic and purposeful leadership style, based on the knowledge of nursing science and practice. nurse managers had so - called performance development discussions with every employee once a year. during the discussion, the nurse manager explained and revised the goals and discussed the purpose of the employee 's work together with each employee. at the same time, they agreed on the goals of the employee for the next year. visionary nurse managers were described as being assertive and persistent in their attempts to get the work units to achieve their goals. nurse managers with more recent education were better equipped to search for information than nurse managers with older education. in addition, they often had a clear picture of the development needs in nursing practice. supervisors said that sometimes the fact that the organization did not have visions or direction for the future was an obstacle to a visionary leadership style. the managers were guided by various situations and there were no plans for the future.this manager had visions and we had long - term plans, but these plans often changed. this manager had visions and we had long - term plans, but these plans often changed. nurses emphasized the importance of making the vision understandable by giving information about current issues of the work unit. nurse manager 's skills to provide information objectively and positively influenced the way the personnel reacted to topical issues. coaching leadership stylenurses as well as supervisors felt that nurse managers with a coaching leadership style took into consideration both the professional development of the employees and delegation of work. the employees had resources and were seen as experts and the nurse manager delegated tasks to them. the skills of employees to work independently varied. some employees needed more coaching while others were satisfied with using their own professional skills independently. nurses as well as supervisors felt that nurse managers with a coaching leadership style took into consideration both the professional development of the employees and delegation of work. the employees had resources and were seen as experts and the nurse manager delegated tasks to them. some employees needed more coaching while others were satisfied with using their own professional skills independently. the nurse managers had a significant role in supporting the employees to cope with the problems at work. they were also responsible for coordinating and organizing work in the unit as a whole.a nurse manager draws plans for nursing practice so that there are these areas of responsibility and everybody knows what is their area and they answer for that. a nurse manager draws plans for nursing practice so that there are these areas of responsibility and everybody knows what is their area and they answer for that. the nurse manager paid attention to employees ' professional skills and encouraged them to study further. both personnel 's competence and leaders ' skills to lead influenced the development work in the unit. it was useful to clarify what kind of needs the work unit and employees had for additional education and to draw up an education plan. the nurse manager encouraged the employees to collect information without prompting and to think independently. the employees and their best interests were the most important value to the nurse manager. they knew the rules and guidelines of the organization, but they considered the hopes and needs of employees in a flexible manner. the nurse manager had skills to understand the feelings of another person and supported him / her by listening sensitively. nurses reported that this encouraged the employees to discuss their personal concerns with the nurse manager. the way to act, pay attention to the employee, do you listen to her or not, that is the basic question. nurses as well as supervisors described an affiliate leadership style. the employees and their best interests were the most important value to the nurse manager. they knew the rules and guidelines of the organization, but they considered the hopes and needs of employees in a flexible manner. the nurse manager had skills to understand the feelings of another person and supported him / her by listening sensitively. nurses reported that this encouraged the employees to discuss their personal concerns with the nurse manager. the way to act, pay attention to the employee, do you listen to her or not, that is the basic question. the way to act, pay attention to the employee, do you listen to her or not, that is the basic question. on the other hand, supervisors reported that leading could be too solicitous, in a completely motherly way. the basis of the leadership style could be supporting the well - being and job satisfaction of the employees ; this might be more important than the development of nursing practise. the purpose a harmonious atmosphere without conflicts can be an obstacle to planned changes. when there are big changes in the work unit, nurse manager is present to the employees and listen [sic ] to them. she tries to support and say [sic ] : there is no problem and we manage of this.when a new employee begins to work, she leads in [sic ] more paternalistic way and takes care of them all the time. when there are big changes in the work unit, nurse manager is present to the employees and listen [sic ] to them. she tries to support and say [sic ] : there is no problem and we manage of this. when a new employee begins to work, she leads in [sic ] more paternalistic way and takes care of them all the time. both nurses and supervisors deemed it important that the nurse manager respects differences and personal characteristics of the employees, not forgetting employees ' equality. a nurse manager who respects and accepts the employees as individuals was easy to approach. on the other hand, nurse manager 's close friendship with employees could make it more difficult to examine the work unit and its functions objectively.there are managers who are very permissive and let the employees behave each in their own way, it is typical that new small managers rise beside them. there are managers who are very permissive and let the employees behave each in their own way, it is typical that new small managers rise beside them. according to the findings nurse managers sometimes behaved in a manner the employees felt to be unequal.it seems that if you are a strong - willed person, you are more likely to get what you want than a person who is adaptable. it seems that if you are a strong - willed person, you are more likely to get what you want than a person who is adaptable. democratic leadership styleboth nurses and supervisors reported that it was typical for the democratic nurse manager to emphasize teamwork and commitment to work. the employees had a possibility to voice their opinions and take part in problem - solving and decision - making. however, the nurse manager was ultimately expected to be a decision - maker. and find and make the decision by thinking together and listening to opinions of the employees and discussing together ; however, she is in some cases the final decision - maker. both nurses and supervisors reported that it was typical for the democratic nurse manager to emphasize teamwork and commitment to work. the employees had a possibility to voice their opinions and take part in problem - solving and decision - making. however, the nurse manager was ultimately expected to be a decision - maker. and find and make the decision by thinking together and listening to opinions of the employees and discussing together ; however, she is in some cases the final decision - maker. and find and make the decision by thinking together and listening to opinions of the employees and discussing together ; however, she is in some cases the final decision - maker. there were different perceptions of the nurse managers ' positions in this leadership style. on the one hand, they were deemed to be responsible for the work unit and to make reasonable decisions after discussing with the employees. on the other hand, some supervisors felt that some nurse managers did not stand out as managers, but as team members. this meant that the nurse manager 's own tasks could be of secondary importance. she is working a lot with us and she has difficulties performing her own duties as a nurse manager. she is working a lot with us and she has difficulties performing her own duties as a nurse manager. supervisors said that a nurse manager had an important role in cooperation and its development with the members of different professional groups and between work units. his / her skills to get the employees to commit to the common goals were deemed as significant. leadership style influences operation as a whole, for example, how a manager gets employees to commit to common decisions leadership style influences operation as a whole, for example, how a manager gets employees to commit to common decisions commanding leadership styleboth nurses and supervisors identified a commanding leadership style, characterized by an emphasis on compliancy and control. nurses as well as supervisors reported that it was important to the nurse managers with a commanding leadership style to follow clear directions and advice which they expected to get from others, for example, their own superiors. the nurse manager could ask employees ' opinions on how to find a solution to a problem in the work unit ; usually s / he had already made a decision and it was not changed by the opinions of the employees. the leadership style was described as authoritarian, hierarchical, and inflexible.nurse managers who do not have the latest knowledge of leadership, they demand that there should be clear rules and laws for everything and there is no flexibility. both nurses and supervisors identified a commanding leadership style, characterized by an emphasis on compliancy and control. nurses as well as supervisors reported that it was important to the nurse managers with a commanding leadership style to follow clear directions and advice which they expected to get from others, for example, their own superiors. the nurse manager could ask employees ' opinions on how to find a solution to a problem in the work unit ; usually s / he had already made a decision and it was not changed by the opinions of the employees. the leadership style was described as authoritarian, hierarchical, and inflexible.nurse managers who do not have the latest knowledge of leadership, they demand that there should be clear rules and laws for everything and there is no flexibility. nurse managers who do not have the latest knowledge of leadership, they demand that there should be clear rules and laws for everything and there is no flexibility. commanding leadership style was more common in the 1970s and 1980s and it was now considered traditional and out - of - date. it was, however, described as a convenient leadership style when employees are inexperienced or when there are big changes in the work unit. nurse managers were described as controlling the behaviour of the personnel, although observations of that kind have diminished considerably. isolating leadership styleboth nurses and supervisors described that nurse managers could isolate themselves from the work unit and retire to their own room where they worked alone without active communication with the employees. in that case neither the nurse manager nor the employees got the information they needed in their work. the nurse manager is quite isolated, she works alone in her room, we visit her when we have something to discuss with her. both nurses and supervisors described that nurse managers could isolate themselves from the work unit and retire to their own room where they worked alone without active communication with the employees. in that case neither the nurse manager nor the employees got the information they needed in their work. the nurse manager is quite isolated, she works alone in her room, we visit her when we have something to discuss with her. the nurse manager is quite isolated, she works alone in her room, we visit her when we have something to discuss with her. both nurses and supervisors reported that nurse manager 's leadership style affects employees ' job satisfaction and commitment to work. it is felt that nurse manager 's fairness and trust in the employees promotes their motivation and participation in work. leadership style contributes to job satisfaction when the nurse manager has skills to prevent and solve conflicts. all the participants reported that nurse manager 's skills to lead the work unit and motivate people affect the success of the work unit. it is important that there are enough trained employees and the employees know and are in charge of their areas of responsibilities. supervisors remarked on the influence of leadership style on efficiency and economy, because the fluency of operation has an impact on how much money is spent. it is important that the employees have a possibility to take part in development work as well. nurse manager 's leadership style can promote or hinder development in the work unit. supervisors emphasized that nurse managers have a significant role in cooperation within the work unit and outside it. some nurse managers want to work only inside their own unit, while others take a larger view of the matter. nurse manager 's leadership style has an influence on how externally orientated the staff are and whether they have connections outside the work unit. a nurse manager can promote the continuity of patient care by cooperation with other units. s / he is a role model in how to treat nurse students. both nurses and supervisors felt that problems in the organizational climate, such as conflicts between the employees or dissatisfaction with the nurse leader are reflected in patient care. the activity or passivity of the nurse manager affects the image of the work unit.if there is patient mistreatment, it is the nurse manager whose responsibility it is to decide how to react, for example, in our unit we treat patients well or we do not react at all to this complaint. if there is patient mistreatment, it is the nurse manager whose responsibility it is to decide how to react, for example, in our unit we treat patients well or all in all, organizational climate, personnel 's job satisfaction and commitment, work unit 's operation and development work, and cooperation influence the way patient care succeeds and how a patient experiences the care he / she gets. if the nurse manager 's basic value is good patient care, it influences in many ways his / her leadership style and how s / he organizes things in the work unit. an isolating leadership style was identified as distinct from the leadership styles that goleman presented, whereas pace - setting leadership style was not reported. the participants reported that nurse managers used many leadership styles, but normally they had one which they used more than others. nurses who worked for leaders with resonant leadership styles were more satisfied with supervision and their jobs. furthermore, visionary leadership style, coaching leadership style, affiliate leadership style, and democratic leadership style seem to promote transformational leadership because they motivate and involve staff. that is why nurse managers should develop themselves in the use of these leadership styles. nurse managers ' leadership style depends on many issues, such as organization, situation, and employees. reynolds and rogers argue that employees have variable levels of competence depending on the situation. it is important that nurse managers have skills to reflect on their own leadership style and receive feedback about it. health care is meeting ever - increasing new challenges where it has to react rapidly. it is important to the health care organizations to make long - term plans and prepare for the future by paying attention to the needs of inhabitants and the resources needed. having knowledge of nursing science and practice gives nurse managers the tools to use a visionary leadership style and make plans for the future. morjikian. argued that communication of future plans, goals, and strategies is important between the nurse manager and the employees. it is important to give information of the vision and explain it regularly to the employees, because sometimes the employees forget the purpose of their work and their working style is not appropriate. when nurse managers work like this they are also carrying out transformational leadership [4, 5 ]. in the future, securing skilled employees will be a big challenge in health care.. found that nurse managers with a coaching leadership style appreciated employees ' professional skills and encouraged them to study further. nurse manager 's consideration of employees ' profession and educational needs influenced nurse retention positively. kenmore argued that a coaching leadership style works when the employees are keen to develop and make use of possibilities to do so. although the nurse manager organizes the work unit as a whole and is responsible for the development work in the unit, his / her support has a significant role in helping employees to cope with the problems they meet in their work. this is also an important part in nurse managers ' role as emotionally intelligent leaders. as a consequence of globalization, an affiliate leadership style with acceptance of difference could be suited for the multicultural work unit. it is a challenge for the manager to listen sensitively and consider to employees ' personal needs individually and at the same time objectively, not forgetting employees ' equality. the basis of the leadership style could be supporting the well - being and job satisfaction of the employees. as kenmore argues, if a nurse manager is too concerned with creating harmony, it can lead to evasion of problems. because of shortage of employees, nurses have many possibilities to choose and change their workplaces. it is important that the employees can express their opinions and take part in decision - making. a commanding leadership style prevents the empowerment of the nurses, because they do not have possibilities to participate in work planning. however, there are situations where a commanding leadership style is appropriate. the majority of finnish nurses will retire in the next few years and there are many nurses with less work experience in the work units. employees with less work experience may need clear directions, for example, in acute situations when a patient 's life is in danger. according to huston, essential nurse manager competencies for the future include the ability to create an organization culture that combines high - quality health care and patient / employee safety and highly developed collaborative and team building skills. as a result of this study, an isolating leadership style was found : the nurse manager worked alone without active communication with the employees. the employees have to work without a leader, and that could cause anxiety for the employees who need support from their leader. a good question in this case if the leader does not show consideration towards the employees, it could affect their health and well - being negatively. leaders and their supervisors should be considered collectively to understand how leadership influences employee performance. a nurse manager has an important role in leading the work unit as a whole. a work unit is seen as a reflection of the nurse manager. according to rosengren., nurses reported that nursing leadership was considered being present and available in daily work, facilitating professional acknowledgement, supporting nursing practice and improving care both as a team and as individuals. a nurse manager with an emotionally intelligent leadership style creates a favourable work climate characterized by innovation, resilience, and change. nurse managers have to be flexible in the changes they have directly initiated or by which they have been indirectly affected. leadership style affects the organizational climate and the ways how information is given and communicated and how questions of the day are discussed. the nurse manager creates the basis for how different opinions are handled and problems solved in the work unit. it is perceived that nurse manager 's trust in the employees promotes their motivation and participation in work. way. found that trust and job satisfaction are strong links with greater commitment and intent to stay on at work. leader encourages the employees to develop goals and plan to achieve them. in this way their skills to build bonds and seek out mutually beneficial relationships affect cooperation in the work unit and around it. on the other hand, there is no one and only correct leadership style ; the same result can be achieved in many ways. a manager who has the ability to reflect on his / her own behaviour, that is, who has high ei, is better able to regulate and estimate his / her leadership style with different employees in different situations. a nurse manager has a significant role in using a leadership style that promotes good patient care. nurse managers had many leadership styles, but normally they had one that they used more than the others. the nurse managers should consider their leadership style from the point of view of employees, situation factors, and goals of the organization. leadership styles where employees are seen in a participative, active role have become more common. together with health care organizations, nursing education programmes should include education of nurse managers to improve their self reflection, through which they are better able to vary their leadership style.	nurse managers who can observe their own behaviour and its effects on employees can adjust to a better leadership style. the intention of this study was to explore nurses ' and supervisors ' perceptions of nurse managers ' leadership styles. open - ended interviews were conducted with 11 nurses and 10 superiors. the data were analysed by content analysis. in the study, six leadership styles were identified : visionary, coaching, affiliate, democratic, commanding, and isolating. job satisfaction and commitment as well as operation and development work, cooperation, and organizational climate in the work unit were the factors, affected by leadership styles. the nurse managers should consider their leadership style from the point of view of employees, situation factors, and goals of the organization. leadership styles where employees are seen in a participatory role have become more common.
the scdr was approved by the research ethics committee at karolinska institutet and the swedish data inspection board. this study is based on 14,721 incident cases of childhood - onset t1d occurring from january 1, 1978, to december 31, 2007, and recorded in the scdr. the scdr has recorded incident cases of childhood - onset t1d (014.9 years) since july 1, 1977, with a high level of coverage (9699% of cases) ascertained by internal revisions and matching to official population databases (7,8). similar methods of data collection and verification have been used since the start of the register. during 2 years (1999 and 2000), three pediatric hospitals did not deliver data prospectively, but this has been adjusted afterward. except for the yearly internal validation procedures as previously described (3) and the studies using external sources for validation, we have instituted a continuous validation with another source since 2003, i.e., the swedish quality assessment register, which covers age - groups 018 years. all children with newly diagnosed t1d in sweden are initially treated at pediatric clinics in a hospital setting. the clinics report their t1d cases to the scdr with date of diagnosis, birth date, and each patient s unique personal identification number. patients recorded july 1, 1977, to december 31, 1977, were excluded because it was a not a full year s contribution of cases. we excluded three patients with diabetes onset after 15 years of age who were accidently registered. age - standardized yearly incidence rates were extracted from the scdr and relevant population data from statistics sweden (9). mean annual incidence rates were calculated and described for the whole study population and stratified by sex and age - groups (04, 59, and 1014 years). a generalized additive model (gam) for a poisson response was used to investigate trends in incidence. gams are fitted for the poisson family of distributions with the log link function. smoothing terms are allowed in gams that permit flexible, nonlinear modeling of selected covariates. in the model, the impact of each calendar year at onset, age - group (04, 59, and 1014 years), sex, and interaction terms were tested. a nonparametric smoothing function for the time trend (year) is used by a penalized regression spline approach, with automatic smoothness selection. because the response variable is a rate rather than a count, as custom for the poisson model, we include the population size in the respective age sex group as an offset for each of the models. to analyze possible trend shifts for the latest birth cohorts, we fit a linear regression curve with the cumulative incidence as a dependent variable and age at onset as a predictor. in the regression analysis, standard methods are used to test differences in slopes between the different birth cohorts using the cohort 2000 as the reference cohort. a total of 14,721 swedish children (7,769 boys and 6,952 girls) were registered with t1d before 15 years of age during the study period. when stratified by age at onset groups (04, 59, and 1014 years), the mean age - specific annual incidence rates over the full observation period were 20.5, 33.9, and 37.6/100,000, respectively. the average increase in incidence rate for the entire cohort was highest between the periods 19992001 and 20022004, whereas the increase seems to level off during 20052007. age- and sex - specific rates of t1d in sweden 19782007 : incidence per 100,000 children per year in 3-year periods (95% ci) using linear models for time trends constitutes a great restriction ; therefore, the more flexible gams were used. table 2 shows that although calendar year and age at onset both significantly add to the model fit, sex alone gives a nonsignificant contribution. addition of an age year interaction term contributes significantly, indicating that the time trend differs by age at onset. addition of an interaction term for year sex did not contribute significantly to the model, but the interaction term for age at onset sex significantly contributes, illustrating the known difference in age - specific incidence peak in the different sexes that coincides with puberty onset. the best fit model (model 7) explains 91% of the variation in incidence, i.e., the estimated and recorded cases are virtually the same. generalized additive models for the poisson family with a log link the models are evaluated with the response variable being the incidence of t1d. a smooth term is estimated for the time trend and linear coefficients for age, sex, and interaction terms. a : incidence of childhood diabetes by calendar year for boys and girls, respectively. observed and estimated data, gam ; model 4. b : incidence of childhood diabetes in three ages at onset groups by calendar year. observed and estimated data, gam ; model 7. figure 1a illustrates the observed and estimated (gam model 4) incidence rates for the entire cohort and study period by sex. a modest increase by time is seen the first 67 years of registration followed by a transient decrease between 1985 and 1990. in the 1990s, a steep increase is seen, followed by a leveling off during the last 7 years of the study period. the trend in incidence differed among the three age - groups over time and is illustrated in table 1 and fig. 1b. from the beginning to the end of the 1990s, the two youngest age - groups (04 and 59 years) had the highest relative increase. during the last decade, the 59- and 1014-year age - groups had the highest rates of relative increase. the highest incidence is now seen in the 1014-year age - group, as in the years before 2000, indicating birth cohort effects of risk exposures. although the average incidence for the entire cohort has increased by every consecutive 3-year period, the youngest age - groups (04 and 59 years) have had a decreasing average incidence during the last 3-year period compared with the previous period. when studying the birth year cohorts for the entire study period in 5-year periods, there is a clear shift to younger age at onset in birth cohorts 19781999, but the birth years 19992002 and 20032006 display a change in this trend (fig. 2b, which shows the decline in cumulative incidence appearing in each consecutive birth year cohort for the years 20002006. this illustrates a possible trend break in incidence increase with older age at onset. a : cumulative incidence of childhood t1d in five calendar year groups. b : cumulative incidence of childhood t1d for yearly birth cohorts 20002006. to analyze the cumulative incidence trends in the cohorts of year 2000 and thereafter all cohorts subsequent to year 2000 show a significant decrease in cumulative incidence at the 0.01 level. for the birth cohort of year 2006, the difference from the cohort of year 2000 is the largest. here, the decrease in cumulative incidence is 30.12 for each year increase in age at onset for the 2006 cohort compared with the 2000 cohort (p = 0.007). a similar analysis of the birth cohorts 19781987 showed no clear trend that could explain the transient decrease in mean incidence seen between 1985 and 1990. a total of 14,721 swedish children (7,769 boys and 6,952 girls) were registered with t1d before 15 years of age during the study period. when stratified by age at onset groups (04, 59, and 1014 years), the mean age - specific annual incidence rates over the full observation period were 20.5, 33.9, and 37.6/100,000, respectively. the average increase in incidence rate for the entire cohort was highest between the periods 19992001 and 20022004, whereas the increase seems to level off during 20052007. age- and sex - specific rates of t1d in sweden 19782007 : incidence per 100,000 children per year in 3-year periods (95% ci) using linear models for time trends constitutes a great restriction ; therefore, the more flexible gams were used. table 2 shows that although calendar year and age at onset both significantly add to the model fit, sex alone gives a nonsignificant contribution. addition of an age year interaction term contributes significantly, indicating that the time trend differs by age at onset. addition of an interaction term for year sex did not contribute significantly to the model, but the interaction term for age at onset sex significantly contributes, illustrating the known difference in age - specific incidence peak in the different sexes that coincides with puberty onset. the best fit model (model 7) explains 91% of the variation in incidence, i.e., the estimated and recorded cases are virtually the same. generalized additive models for the poisson family with a log link the models are evaluated with the response variable being the incidence of t1d. a smooth term is estimated for the time trend and linear coefficients for age, sex, and interaction terms. a : incidence of childhood diabetes by calendar year for boys and girls, respectively. observed and estimated data, gam ; model 4. b : incidence of childhood diabetes in three ages at onset groups by calendar year. observed and estimated data, gam ; model 7. figure 1a illustrates the observed and estimated (gam model 4) incidence rates for the entire cohort and study period by sex. a modest increase by time is seen the first 67 years of registration followed by a transient decrease between 1985 and 1990. in the 1990s, a steep increase is seen, followed by a leveling off during the last 7 years of the study period. the trend in incidence differed among the three age - groups over time and is illustrated in table 1 and fig. 1b. from the beginning to the end of the 1990s, the two youngest age - groups (04 and 59 years) had the highest relative increase. during the last decade, the 59- and 1014-year age - groups had the highest rates of relative increase. the highest incidence is now seen in the 1014-year age - group, as in the years before 2000, indicating birth cohort effects of risk exposures. although the average incidence for the entire cohort has increased by every consecutive 3-year period, the youngest age - groups (04 and 59 years) have had a decreasing average incidence during the last 3-year period compared with the previous period. when studying the birth year cohorts for the entire study period in 5-year periods, there is a clear shift to younger age at onset in birth cohorts 19781999, but the birth years 19992002 and 20032006 display a change in this trend (fig. 2b, which shows the decline in cumulative incidence appearing in each consecutive birth year cohort for the years 20002006. this illustrates a possible trend break in incidence increase with older age at onset. a : cumulative incidence of childhood t1d in five calendar year groups. b : cumulative incidence of childhood t1d for yearly birth cohorts 20002006. to analyze the cumulative incidence trends in the cohorts of year 2000 and thereafter, all cohorts subsequent to year 2000 show a significant decrease in cumulative incidence at the 0.01 level. for the birth cohort of year 2006, the difference from the cohort of year 2000 is the largest. here, the decrease in cumulative incidence is 30.12 for each year increase in age at onset for the 2006 cohort compared with the 2000 cohort (p = 0.007). a similar analysis of the birth cohorts 19781987 showed no clear trend that could explain the transient decrease in mean incidence seen between 1985 and 1990. during the past few decades, a large number of studies from single centers, nationwide registers, and multicenter studies covering many parts of the world have shown a dramatic increase in the incidence of childhood - onset t1d (13,10,11). these studies clearly indicate the strong impact of lifestyle - related risk factors in childhood - onset t1d. in most reports, the incidence increase is steepest among the youngest children, and taken together with the notions of a stable or even decreasing incidence rate in young adults (1215), these findings indicate an accelerated preclinical autoimmune destruction of the -cell to younger age at onset suggested to cause at least part of the increase over time (6,16,17). the current study indicates for the first time a reversed pattern with a shift to older age at onset by birth cohort among children born after the year 2000. the change in incidence by time may be transient as reported from norway (11) and seen in the swedish data 19851990, but the shift to older age at onset by birth cohort in the last 7-year period is of clear interest because it indicates a more specific change in a risk exposure that affects the youngest children. a major strength of the scdr is that we were able to follow prospectively, with a high level of ascertainment of cases, nationwide incidence rate over a long time. thus, this study allows us to consider shifts in time trends in large datasets. our rich data combined with precise statistical methods, i.e., using a flexible model approach (gam), increase the accuracy of the statistical analysis. a weakness in analyzing cumulative incidences in children born after 1993 in our study is obviously that the birth year cohorts are not followed for the full age span. thus, an unknown number of potential new cases may change the future picture, highlighting the need to continue monitoring incidence trends, performing analysis by birth cohort, and including incidence data from older age - groups (12). population - based case - control studies confirm that high calorie intake (18,19) and rapid growth and weight development are risk markers for childhood - onset diabetes (2022). during the past decade, efforts have been made to decrease child obesity through preventive health care programs at child health care centers and schools through guidelines from the swedish national institute of public health (23). recent studies after the implementation of these guidelines have shown a declining prevalence of overweight and obese 4-year - old swedish children (24). certainly other environmental risk exposures that specifically affect young children may contribute to the pattern of change in trend now seen in sweden, but the ecological relationship with a change in overweight in sweden is striking. some studies have tried to predict future incidence and prevalence of childhood diabetes using different models of time trend data (2,10). obviously, such predictions are dependent on the stability of trends and a large enough dataset. a prediction based on our own 30-year data and gam models would imply a grave picture, suggesting an increase of new t1d cases by > 60% in the coming decade (data not shown). however, such a forecast may be an overestimate in case the trend break now seen is not transient. in conclusion, our data point at a break in the worrying trend of increase in childhood - onset t1d in sweden, indicating that prevention by changes in lifestyle habits is possible. still, adequate health care resources must be allocated to meet children s needs in both promoting better lifestyle habits for potential prevention of t1d and type 2 diabetes and taking care of the many children worldwide in whom diabetes will develop.	objectiveduring the past few decades, a rapidly increasing incidence of childhood type 1 diabetes (t1d) has been reported from many parts of the world. the change over time has been partly explained by changes in lifestyle causing rapid early growth and weight development. the current study models and analyzes the time trend by age, sex, and birth cohort in an exceptionally large study group.research design and methodsthe present analysis involved 14,721 incident cases of t1d with an onset of 014.9 years that were recorded in the nationwide swedish childhood diabetes registry from 1978 to 2007. data were analyzed using generalized additive models.resultsage- and sex - specific incidence rates varied from 21.6 (95% ci 19.423.9) during 19781980 to 43.9 (95% ci 40.747.3) during 20052007. cumulative incidence by birth cohort shifted to a younger age at onset during the first 22 years, but from the birth year 2000 a statistically significant reversed trend (p < 0.01) was seen.conclusionschildhood t1d increased dramatically and shifted to a younger age at onset the first 22 years of the study period. we report a reversed trend, starting in 2000, indicating a change in nongenetic risk factors affecting specifically young children.
there is a complex mechanism in cells that controls which genes are expressed. transcription factors, which are a special type of protein and capable of regulating the expression of other genes by binding to their upstream regions, play a crucial role in this mechanism. transcriptional regulatory relationships between transcription factors and their targets can be represented by a network, called a transcription regulatory network, where each vertex denotes a gene and each edge denotes a regulatory relationship. identifying transcription regulatory networks is critical, because it facilitates understanding biological processes in cells. gene expression data, which measure mrna levels of genes, are widely used for inferring transcription regulatory networks. bayesian networks, a type of probabilistic graphical model, have been proposed to infer transcription regulatory networks from gene expression data [3, 4 ]. despite their success in learning regulatory networks, models inferred by the bayesian networks tend to overfit the data because, in a gene expression dataset, the number of variables is normally large compared to the number of samples. to cope with this problem, segal. designed the module network method, which is a special type of bayesian network algorithm. in this method, each module represents a set of variables that share (1) a single variable or a set of variables as their parents and (2) local distributions. compared to standard bayesian network algorithms, this design significantly reduces the number of parameters to be learned and consequently leads to more accurate inferences. the module network method has yielded promising results in learning regulatory networks [57 ]. given a gene expression dataset and a list of candidate transcription factors, the process of learning module networks consists of two tasks : clustering genes into modules and inferring the regulation program (transcription factors) of each module. segal. designed an expectation - maximization - based learning algorithm that alternates between these two tasks. moreover, joshi. separated the two tasks, where they grouped genes into modules before learning the regulation program of each module. experimental results showed that the separation improves the performance of the module network method in inferring regulatory networks. many techniques have been applied to infer the regulation program of a given gene module, that is, the second task in learning module networks, such as logistic regression, moderated t - statistics from limma, gibbs sampling, and linear regression. a common characteristic of these methods is that they are able to calculate the confidence (i.e., regulatory score) for the assignment of a transcription factor to a gene module, which is referred to as a regulator - module interaction. consequently, their results can be sorted into an ordered list of regulator - module interactions according to their regulatory scores. the higher the ranking of a regulator - module interaction in the ordered list given by a method, the more confidence this method assigns to the interaction. in addition, since these methods resort to distinct techniques, they show very different biases in detecting regulatory relationships. for example, in the nitrogen utilization module in yeast, lemone favors regulatory relationships where transcription factors and genes are globally coexpressed, while the limma - based method favors regulatory relationships where transcription factors and genes are locally coexpressed. this suggests that integrating results from different regulation program learning algorithms can be a promising direction in better inferring regulatory networks. in this work, we extend our previous work by integrating its results with those given by two other learning algorithms [9, 13 ]. to the best of our knowledge, experimental results indicate that the union and weighted rank aggregation methods produce more accurate predictions than those given by individual algorithms, whereas the intersection method does not yield any improvement in the accuracy of predictions. the rest of this paper is organized as follows : section 2 describes the dataset, integration methods, and regulation program learning algorithms studied in this work. the yeast stress dataset has been used as a benchmark to validate the performance of module network learning algorithms [5, 9 ]. this dataset consists of 173 arrays and measures the budding yeast 's response to a panel of diverse environmental stresses. the conditions covered by the dataset consist of temperature shocks, amino acid starvation, nitrogen source depletion, and so on. in previous work [5, 17 ], 2355 differentially expressed genes in the dataset were selected and these genes were clustered into 69 gene modules. genes in the modules show functional enrichment. this module consists of 47 genes mostly involved in two pathways : the methionine pathway (regulated by met28 and met32), and the nitrogen catabolite repression (ncr) system (regulated by gln3, gzf3, dal80, and gat1). both pathways relate to the process by which the budding yeast uses the best available nitrogen source in the environment [18, 19 ]. in this work, we apply three algorithms [9, 10, 13 ] to infer regulators of these modules using a list of 321 transcription factors prepared by segal. as candidate transcription factors. then, we integrate the results of these algorithms by methods described in section 2.2. the regulatory relationships recorded in yeastract (released on april 27, 2009) are used as the reference database to validate results given by individual algorithms and our integration methods. we apply union, intersection, and weighted rank aggregation integration methods to integrate results from different regulation program learning algorithms. the former determines the ranking of a regulator - module interaction using the highest ranking given by all candidate learning algorithms. in contrast, the latter determines the ranking of an interaction using the lowest ranking. for example, given a regulator - module interaction, which is the 1st, 3rd, and 5th items in rankings given by three individual learning algorithms, respectively, the union method assigns the 1st as its ranking, while the intersection method assigns the 5th as its ranking. after determining the ranking of each interaction, these methods can each produce an ordered list of regulator - module interactions by sorting interactions by their rankings. in comparison, the weighted rank aggregation method is much more computationally intensive than the union and intersection methods. given a set of learning algorithms m, this integration algorithm searches for an ordered list, that is, simultaneously as close as possible to the list produced by each algorithm in m. let lm = (a1, a2,, ak) represent an ordered list of k regulator - module interactions produced by the algorithm m. let r(a) denote the rank of the interaction a under m. finally, let m(i) (i = 1,2,, k) denote the p value (weight) that algorithm m assigns to the interaction ranked at the ith position in the ordered list. this can be represented by the following minimization problem : (1)=arg min (), where (2)()=mmd(,lm) represents the sum of the distances between an ordered list and the lists from all algorithms. the distance between and lm is determined by the weighted spearman 's footrule distance : (3)d(,lm)=alm\|m(r(a))m(rm(a))\| \|r(a)rm(a)\|. to determine, we apply the cross - entropy monte carlo algorithm. we select lemone, inferelator, and the limma - based method, as candidate regulation program learning algorithms. in this section, in addition, in order to apply the weighted rank aggregation to integrate their results, for each algorithm, we also define how to calculate the p value for the assignment of a regulator to a module. for each gene module in the yeast stress dataset, lemone sampled 10 regression trees and then calculated regulatory scores for assigning transcription factors to this module based on these trees. regulatory scores of all regulator - module interactions were downloaded from the supplementary website of. we calculate the lemone - based p value for the assignment of a regulator r to a module as follows. first, given a regression tree t of this module, we define the p value of the split with r and a splitting value z at an internal node t in t (i.e., p value(t)(r, z)) as the probability of observing a split with a higher average prediction probability than this split at the node t. the average prediction probability of a split is defined as in equation (4) of. then, the p value for assigning r to t is defined as : (4)p value(t)(r)=wt\|z\|zzp value(t)(r, z), where wt is the number of experimental conditions in t divided by the total number of conditions in the data, and z represents the set of possible splitting values for r in t. furthermore, given a set of regression trees f, the lemone - based p value for assigning r to this module can be calculated as : (5)p value(r)=1\|f\|tf ttp value(t)(r). inferelator uses linear regression and variable selection to identify transcription factors of gene modules. in each gene module in the yeast stress dataset, we fit a linear model to the mean of the module 's genes in each condition using the 321 candidate transcription factors as predictor variables. the regulatory score for assigning a regulator to the module is decided by the absolute value of the regulator 's regression coefficient in the fitted model. the inferelator - based p value for the assignment of a regulator to a module is defined as follows. first, we permute the values of the expression value matrix from the row direction (gene). third, we fit the distribution of nonzero coefficients obtained from the permuted dataset by the weibull distribution defined as : (6)pdf(x)=k(x)k1e(x/)k, with k = 0.889 and = 0.015 (figure 1). last, we define the inferelator - based p value for a regulator - module interaction with a regulatory score s as the probability of observing a value more than s from the weibull distribution (6). in our previous work, moderated t - statistics proposed in limma were applied to infer transcription factors of gene modules. for each gene module in the yeast dataset, ten condition clusterings were sampled by a two - way clustering algorithm. then the regulatory score for assigning a transcription factor to this module was calculated by summing the transcription factor 's standardized moderated t - statistics based on the sampled condition clusterings. we next describe how to define the method 's p value for the assignment of a transcription factor to a module. first, we randomly generate ten condition clusterings, each of which consisted of two clusters. we then calculate the regulatory score for each candidate transcription factor based on these randomly generated clusterings. last, the probability density function of these randomly generated scores is approximated by the stretched exponentials defined as : (7)pdf(x)={hmaxexp[br(xxmax)cr],for xxmax,hmaxexp[bl(xmaxx)cl],for x < xmax, with hmax = 0.127, br = 0.024, bl = 0.083, cr = 2.45, cl = 1.70, and xmax = 0.050. as shown in figure 2, the approximated fit is very close to the empirical distribution of the randomly generated regulatory scores, so the p value for a regulator - module interaction with a regulatory score s can be defined as the probability of observing a value more than s from the approximated fit. note that the assignment of a regulator to a module is associated with a p value for each regulation program learning algorithm, and the p value is required by the weighted rank aggregation method to integrate results from different learning algorithms. in contrast, the assignment is also associated with a p value based on records in the reference database, yeastract. this p value is calculated by the hypergeometric distribution and is used to evaluate the performance of individual learning algorithms and integration methods. we applied each regulation program learning algorithm described in section 2.3 to calculate the regulatory score for assigning a regulator to a module. then we sorted all of its regulatory scores between 321 candidate transcription factors and 69 modules in descending order. this led to an ordered list of 22,149 regulator - module interactions for each method. in addition, for each regulator - module interaction, we used the hypergeometric distribution to calculate the p value of this interaction, using regulatory relationships in yeastract as the reference database. this p value is based on the number of genes regulated by the regulator in the dataset, the number of genes regulated by the regulator in the module, and the number of genes in the module, and is used to determine if the regulator - module interactions is a true positive. moreover, for a given ordered list of regulator - module interactions, we define the precision of the top i items in this ordered list as : (8)p(i)=t(i)i, where t(i) denotes the number of interactions with p values less than 0.05 in the top i items (i.e., the number of true positives among these i interactions). in figure 3, we show the precision of the top i regulator - module interactions (i = 1,2,, 100) in the ordered lists obtained by inferelator, lemone, and the limma - based method. when less than 20 interactions are selected, the limma - based method outperforms the other two methods. most true positives given by limma - based method are for the module of nitrogen utilization. however, inferelator and lemone outperform the limma - based method when the number of selected interactions is in the range of 20 and 50. in addition, when more than 50 interactions are selected, the three methods show similar performance in the yeast dataset. the weighted rank aggregation method searches for a synthesized list that is simultaneously as close as possible to the ordered lists from lemone, inferelator, and the limma - based method. however, it is not feasible to directly apply this integration method on a list with 22,149 interactions due to the extensive computational workload. hence, we resort to a tradeoff by integrating the top k (k 22,149) interactions in the ordered lists given by these algorithms. this is, for a given ordered list and k, interactions ranked lower than k (i.e., k + 1, k + 2,, 22,149), are associated with a same weight (p value) of one. the larger k, the closer the list produced by the rank aggregation is to the lists given by the three candidate algorithms, but the rank aggregation costs more computation time. for example, it takes 12 hours and 48 hours for k = 75 and k = 100, respectively, on a hp rackmount server with amd opteron processors (86, 64 bit, dual core) and 16 gb memory. in order to select a proper value for k in the yeast dataset for each k, this led to 10 ordered lists, and we calculated the average of the precision of the top i (i = 1,2,, k) interactions in these ten lists. as shown in figure 4, when k increases from 25 to 50 and then to 75, the precisions obtained by the rank aggregation method are improved, but the precisions at k = 75 and k = 100 are about the same. this indicates that after k reaches 75, considering more interactions from the ordered lists of the candidate algorithms can no longer improve the performance of the rank aggregation method. hence, k is set to 100 in our tests using the yeast dataset. in this section, we compare the performance of integration methods with individual algorithms. in order to make the comparison clear, for a given i (i = 1,2,, 100), we define the baseline precision as that obtained by selecting the maximum of the precisions of the top i interactions given by all individual algorithms. that is, given a set of individual learning algorithms m, it is determined as : (9)p(i)=maxmm(pm(i)), where pm(i) denotes the precision of top i interactions in the ordered list given by algorithm m (8). note that baseline precision represents an upper optimistic bound that can not be achieved by individual algorithms as we can only use one of them at a time. hence, even if the precision obtained by an integration method is only comparable to baseline precision, it still shows that this integration method yields a better overall performance than those of individual algorithms. we compare the precision of the top 100 predictions from the three integration methods with baseline precisions (figure 5). the union and rank aggregation methods generate better or similar results compared to the baseline precision. in addition, somewhat surprisingly, the union method, which has a lower computational cost than rank aggregation, achieves comparable results as given by rank aggregation. the first twenty interactions from union and rank aggregation are shown in tables 1 and 2, respectively. on the other hand, we observe that baseline precisions are generally better than precisions given by the intersection method. the intersection method sorts interactions by their lowest rankings from all candidate algorithms, so it tends to assign interactions with moderate confidences from all algorithms with high ranks. for example, as shown in table 3, its first 20 interactions include several not highly ranked by any algorithm, such as the twelfth interaction (rds2 to module 16) ranked 219th, 125th, and 273rd by the limma - based method, lemone, and inferelator, respectively ; and the eighteenth interaction (dal81 to module 58) ranked as 199th, 310th, and 190th by the limma - based method, lemone, and inferelator, respectively. we also compare areas under precision curves for the top 100 predictions given by the integration methods and individual learning algorithms (table 4). the union and weighted rank aggregation methods achieve better results than those from the individual learning algorithms, but the intersection method only yields a comparable result with the individual learning algorithms. these results indicate that we should be cautious to apply the intersection method to integrate results from algorithms of different natures. a metalearner approach was applied to infer transcription factors of coexpressed gene modules in a yeast stress dataset, with the regulatory relationships recorded in yeastract as the gold standard. we integrated the predictions of three existing inference techniques [9, 10, 13 ] by three different methods : union, intersection, and weighted rank aggregation. experimental results show that integrated predictions based on union or rank aggregation have higher precision than any of the individual methods. the justification of this work is that the results generated by different algorithms are not identical and often have clearly different influences from the datasets used. the experiments confirm our expectation that integrating the output of several algorithms results in higher quality predictions. to the best of our knowledge, an interesting extension of this work is to investigate if integrating results from more algorithms can lead to even better performance. in particular, we expect that when more algorithms are combined, we may see significant difference between the union and weighted rank aggregation methods. the experiments in this work are conducted on a yeast dataset, and results are validated by the regulatory relationships recorded in yeastract, which does not represent a complete reference database of the regulatory network in the yeast. hence, another direction for future work is to perform experiments on expression data from other species (e.g., e. coli) to verify if results are consistent with those we obtained in the yeast dataset. in addition, we are interested in performing experiments on synthetic datasets (e.g., dream), where complete reference networks are available.	the module network method, a special type of bayesian network algorithms, has been proposed to infer transcription regulatory networks from gene expression data. in this method, a module represents a set of genes, which have similar expression profiles and are regulated by same transcription factors. the process of learning module networks consists of two steps : first clustering genes into modules and then inferring the regulation program (transcription factors) of each module. many algorithms have been designed to infer the regulation program of a given gene module, and these algorithms show very different biases in detecting regulatory relationships. in this work, we explore the possibility of integrating results from different algorithms. the integration methods we select are union, intersection, and weighted rank aggregation. experiments in a yeast dataset show that the union and weighted rank aggregation methods produce more accurate predictions than those given by individual algorithms, whereas the intersection method does not yield any improvement in the accuracy of predictions. in addition, somewhat surprisingly, the union method, which has a lower computational cost than rank aggregation, achieves comparable results as given by rank aggregation.
menopause is defined as the permanent experience of long - lasting endocrinal, somatic and psychological changes. during these periods, women experience some symptoms which begin with vasomotor signs (like flushing, night sweat, etc.), changes in menstruation cycle, vaginal dryness, itching and dyspareunia and continue with temper changes, memory reduction, disorders of sexual arousal reduction, stress urinary incontinence and complaint from musculo - eskeletal pains. even though some of the complications subside during the time, the symptoms of vasomotor, vaginal dryness and dyspareunia which are connected to disorder in sexual function related to lack of sexual hormones (especially estrogen) irrespective of treatment will progress markedly and unfortunately will not be solved without treatment. following the subsidence or discontinuity of this hormone, women are affected by symptomatic vaginal atrophy and basic changes will occur in their genitor - urinary mucous. these changes include vaginal dryness, irritation, itching, post - coital bleeding, vaginal discharge and dyspareunia and in the urinary system, urine frequency and urinary incontinence appear. as a whole, it is estimated that 10.0 - 40.0% of women experience the symptoms connected with atrophy and on the other hand about 16 million women (500 thousand new cases) show such symptoms every year. in confirmation to the prevalence of this problem, crandall c et - al. (2010) considered this matter and reported that the vaginal dryness was observed from 23.4% pre - menopause to 61.5% post - menopause among the women under the study. (2009) and mehta and bachman also showed that 10.0 - 40.0% of women at the post - menopause stage face inconvenience and problems related to vulva and vaginal atrophy that requires treatment but only 25.0% of them refer for treatment. two hormonal and non - hormonal methods are usually used in treatment of such problems. in the studies which applied non - hormonal method, materials like lubricants and vaginal moistures, vitamin e oil and improving lifestyle like stopping cigarette smoking have been mentioned. for hormonal methods also the conjugated estrogen in two forms of systemic (oral and parenteral) and topical are prescribed. the systemic method is useful for those women who are suffering from flushing and sleep disorder related to vaginal atrophy. on the other hand, the contraindication of this method for tumors sensitive to estrogen, liver failure and having thromboembolization history related to estrogen should also be considered. also, attention should be paid to their side effects like breast sensitivity, nausea and vomiting, vaginal bleeding, mild increase in the risk of affecting the neoplasms dependent on estrogen and in lesser amount the pain in the perineal area. topical treatment in the form of cream, tablet and ring (conjugated estrogen 0.625) which has been confirmed by fda (food and drug association) with the objective of preparing sufficient estrogen for reducing the symptoms of atrophy and relief of its resultant complications is applied. in this regard, researches show that topical drugs have similar effect and even though the probability of general absorption is there, as to the influence and improvement of the symptoms to the rate of 80.0 - 90.0%, which is expected, they are similar. topical hormones are also not without complication ; the results of a study (2004) showed that they have similar effects in the incidence rate of hyperplasia and endometrial thickening. creams are probably accompanied with more side - effects compared with ring or tablet which may be due to the application of a dose more than recommended. considering the aforesaid points, for those who do not select estrogen - therapy due to the medical prohibitions or having side - effects, the non - hormonal interventions which are mostly neglected for the sexual problems are propounded. in these methods, applying lubricants, moisture creams and using dilators are recommended. it was reported in a study in the year 2010 that their use will reduce the complications of vaginal atrophy. also, in this connection, materials which could be applied as gel in the form of the extract of some plants like vitex agnus - castus and compounds like hyaluronic acid alone or in combination with vitamin e for the treatment of sexual disorders and vaginal atrophy are mentioned. regarding hyaluronic acid which is a natural polysaccharide, it can be mentioned that, it forms an important part of extra - cellular matrix of the skin and cartilage. this substance is able to conserve a large amount of water molecules and due to the properties like formation and conservation of extra - cellular inflation, skin moistening in the case of inflammation and preservation of water equilibrium has a key role. also, it is effective widely in treatment of skin diseases due to preservation of tissue consistency, facilitating the cellular emigration in the cases of inflammation and also the process of improvement and regeneration of the tissues. various studies carried out regarding hyaluronic acid have shown that this compound has been tolerated well without side - effects among patients and the complications have been observed only when applied in the form of parenteral jelly by creating susceptibility at injection sites as mild inconveniences, redness, edema and cyanosis. it should be notified that this medicine in the form of suppositories or tablets has rarely been used in iran for treatment of atrophy of genitor - urinary system. the present study aimed at achieving an appropriate and uncomplicated treatment, which is accepted by those who have contraindication for hormonal drug and or desire to use non - hormonal methods. therefore, we tried to compare the effects of conjugated estrogen cream 0.625 mg (hormonal) and hyaluronic acid cream (non - hormonal) for the treatment of atrophy and its complications with the aim to promote the health of menopause women. in spite of the importance of the matter and effect of vaginal atrophy symptoms on women s life, they are mostly not reported and do not go under treatment subsequently therefore, to remove such problems, beginning of treatment and taking care of them by physical evaluation, talking about their sexual problems and the qualitative problems of their life seem necessary. this study was approved by ethics committee under number : ct-92 - 6681 on oct 2013 and was carried out in multi - stages as a simple randomized controlled clinical trial on 56 menopause women referred to shahid motahari clinic during 6 months (september to march 2013 - 2014). they were selected on the basis of the aim and by sample size determination with error of 5%, confidence of 95%, a power of 80%, an effect size of 6.0 (mean=4 and standard deviation=6), and correlation of 70% using the formula : to consider the possibility of loss of 15% and the longitudinal nature of the study and sizes repeated, we used the formula : finally, the sample size in each group was determined 28. married and menopause women, existence of moderate to severe dryness at the vaginal region, endometrial thickening with vaginal sonography maximum 5 mm were among the inclusion criteria. smoking cigarette, and using anti - coagulate drugs (heparin), topical hormonal and nonhormonal drugs one month before the study, existence of vaginal infection requiring treatment in the primary examination for pup smear, sensitivity (such as rash, erythema, inflammation) to drug or its compounds, existence of doubtful or known history of hormone relative diseases (such as breast cancer, unknown cases of vaginal bleeding, severe thrombophlebitis or thromboebolism disorders related to estrogen), existence of chronic diseases (such as cardiac diseases, hypertension, diabetes). at first, multi - stage sampling was done with no blinding eligible individuals. in this way, the researcher selected the existing centers in proportion to the number of referrals from 30 to 50 percent through simple sampling method. consort flow diagram of participants then, the numbers from 1 to 80 were written on the same card and then put in a bag covered ; then we assigned the odd numbers to one group (group a) and even numbers to the other group (group b). groups of a and b (both groups had inclusion criteria) filled in the informed consent form. group a received conjugated estrogen 0.625 mg cream (production by aborayhan pharmaceutical company) and group b received hyaluronic acid vaginal cream (containing 5 mg sodium salt), which had been prepared from shiraz pharmacy college. group a applied one applicator of drug (0.058 mg) every night before sleep for a period of two weeks and two times a week for the next six weeks and group b used one applicator (5 mg) every night before sleep for a period of 8 weeks. the manner of putting cream inside vagina, proper place and using at a specified time were explained for both groups and follow - up through telephone calls. the study of the rate of vaginal atrophy symptoms at zero week (before treatment) and eighth week (after treatment) was carried out for both groups with a compound scale including vaginal dryness and itching, dyspareunia and urinary incontinence. stress urinary incontinence (the urinary incontinence followed by increasing the intra - abdominal pressure at the time of sneezing, coughing, etc.) and urgency (sudden and severe feeling in urinary urgency) were considered in this study. the severity of each sign of atrophy was evaluated by vas (visual analog signals) before and after the intervention and on the basis of four points scale in which zero = asymptomatic, one = mild (score 1 to 3), two = moderate (score 4 to 6) and three = severe (score 7 to 10) were propounded, respectively. the signs were evaluated by the researcher with attending the interview sessions. to recognize the cellular maturation (before and after intervention) also by carrying out vaginal and cervical pap smear, the available rate and type of cells (para - basal, medial and surface) were determined. the smear samples were colored with ethyl alcohol 90.0%, studied as uni - blind by a cytologist (unaware of the type of treatment) and the maturation degree was calculated according to the formula [(percentage of surface cells x1)+(percentage of medial cells x0.5)+(percentage of para - basal cellsx0)=maturation degree ] with index : lack 0 - 25, low estrogenic effect=26 - 49, moderate estrogenic effect=50 - 75, sevear estrogenic effect=76 - 100. the vaginal ph was studied before and after the intervention using ph indicator strip inserted into the vagina ; its rate was divided into 4 degrees as zero (ph6.49) (9). it is necessary to mention that the cytologist, sample taker, the type of ph marker band and the laboratory were fixed throughout the study. finally, the collected data were analyzed through spss 20 software, using descriptive statistics, chi - square, paired and independent t - tests, and retest by wilcoxon and mann - whitney tests at the confidence interval of 95.0%. married and menopause women, existence of moderate to severe dryness at the vaginal region, endometrial thickening with vaginal sonography maximum 5 mm were among the inclusion criteria. smoking cigarette, and using anti - coagulate drugs (heparin), topical hormonal and nonhormonal drugs one month before the study, existence of vaginal infection requiring treatment in the primary examination for pup smear, sensitivity (such as rash, erythema, inflammation) to drug or its compounds, existence of doubtful or known history of hormone relative diseases (such as breast cancer, unknown cases of vaginal bleeding, severe thrombophlebitis or thromboebolism disorders related to estrogen), existence of chronic diseases (such as cardiac diseases, hypertension, diabetes). at first,, the researcher selected the existing centers in proportion to the number of referrals from 30 to 50 percent through simple sampling method. consort flow diagram of participants then, the numbers from 1 to 80 were written on the same card and then put in a bag covered ; then we assigned the odd numbers to one group (group a) and even numbers to the other group (group b). groups of a and b (both groups had inclusion criteria) filled in the informed consent form. group a received conjugated estrogen 0.625 mg cream (production by aborayhan pharmaceutical company) and group b received hyaluronic acid vaginal cream (containing 5 mg sodium salt), which had been prepared from shiraz pharmacy college. group a applied one applicator of drug (0.058 mg) every night before sleep for a period of two weeks and two times a week for the next six weeks and group b used one applicator (5 mg) every night before sleep for a period of 8 weeks. the manner of putting cream inside vagina, proper place and using at a specified time were explained for both groups and follow - up through telephone calls. the study of the rate of vaginal atrophy symptoms at zero week (before treatment) and eighth week (after treatment) was carried out for both groups with a compound scale including vaginal dryness and itching, dyspareunia and urinary incontinence. stress urinary incontinence (the urinary incontinence followed by increasing the intra - abdominal pressure at the time of sneezing, coughing, etc.) and urgency (sudden and severe feeling in urinary urgency) were considered in this study. the severity of each sign of atrophy was evaluated by vas (visual analog signals) before and after the intervention and on the basis of four points scale in which zero = asymptomatic, one = mild (score 1 to 3), two = moderate (score 4 to 6) and three = severe (score 7 to 10) were propounded, respectively. the signs were evaluated by the researcher with attending the interview sessions. to recognize the cellular maturation (before and after intervention) also by carrying out vaginal and cervical pap smear, the available rate and type of cells (para - basal, medial and surface) were determined. the smear samples were colored with ethyl alcohol 90.0%, studied as uni - blind by a cytologist (unaware of the type of treatment) and the maturation degree was calculated according to the formula [(percentage of surface cells x1)+(percentage of medial cells x0.5)+(percentage of para - basal cellsx0)=maturation degree ] with index : lack 0 - 25, low estrogenic effect=26 - 49, moderate estrogenic effect=50 - 75, sevear estrogenic effect=76 - 100. the vaginal ph was studied before and after the intervention using ph indicator strip inserted into the vagina ; its rate was divided into 4 degrees as zero (ph6.49) (9). it is necessary to mention that the cytologist, sample taker, the type of ph marker band and the laboratory were fixed throughout the study. finally, the collected data were analyzed through spss 20 software, using descriptive statistics, chi - square, paired and independent t - tests, and retest by wilcoxon and mann - whitney tests at the confidence interval of 95.0%. the results showed that from the view point of age, menarche, age of menopause beginning, number of pregnancies, occupation, education, disease history and drug consumption both therapeutic groups were similar and the chi - square test did not show any statistical significant difference (p>0.05) (table 1). demographic characteristics of the samples in the study groups frequency meansd chi - square as to the atrophy symptoms before and after the intervention, it was specified that vaginal dryness and itching and also dyspareunia were significantly improved after the intervention among the groups (p0.05) (table 4). comparison of the frequency of vaginal ph in the groups treated before and after the intervention paired and independent t - test retest by wilcoxon and mann - whitney tests the results showed that the women in both groups did not have any difference and were similar with respect to some of the demographic characteristics (p>0.05). in a study by ziagham et - al. (2012) which compared the effect of hyaluronic acid vaginal suppository with vitamin e in the treatment of vaginal atrophy among menopause women, both groups were similar as to the age, menopause duration, occupation, educational level and economical status and had no statistical difference. in a study, the effect of hyaluronic acid vaginal tablet and estradiol was compared on the vaginal atrophy for a period of 8 weeks ; no significant difference was observed between the mean age and the age of menopause beginning among both groups. in another study, the researchers compared the effect of genestine with hyaluronic acid on vaginal atrophy also no significant difference was observed between age, menopause age and their effects on vaginal symptoms. the results of the research also indicated that both hyaluronic acid and permarine improved the vaginal atrophy symptoms, cellular maturation increase and reduced ph ; this improvement was sometimes more among the hyaluronic acid group. but many researchers studied separately the effect of two aforesaid drugs on atrophy and their results were similar to those of the present study in selection of the objective group and effect of drug but there were some differences in terms of drug form and duration of the intervention. in this respect, we reviewed some studies as castelo - branco c (2005) to study the management of post - menopausal vaginal atrophy and atrophic vaginitis with focuses on the changes involved in vaginal aging. it was shown that estrogen increased the content of the skin collagen, and hyaluronic acid to improve the skin moisture and genitourinary symptoms. another researcher studied the effect of conjugated estrogen cream in treatment of atrophy which was consumed daily or two times a week for a period of 12 weeks. the results indicated that applying both methods of using the drug caused more improvement in symptoms of atrophy, maturation index and vaginal ph significantly compared with the placebo group. in another study, the conjugated estrogen vaginal cream was used twice a week for a period of 12 weeks for the treatment of vaginal atrophy. the results showed that estrogen cream caused a reduction in the vaginal dryness, dyspareunia and ph and an increase in the vaginal cells maturation index. the findings of our study is similar to the results of the above studies in terms of improving atrophy symptoms after taking estrogen, but does not agree in the amount of consumption, the duration of intervention and use of a single drug without comparison with other drugs. the effect of hyaluronic acid on treatment of vaginal atrophy was studied in other researc-, the results f which were similar to those of the present study as to improving the symptoms of atrophy in the genito - urinary system. especially, the results of evaluating the effect of hyaluronic acid suppository of another study in iran on the severity of the symptoms of vaginal atrophy 2, 4 and 8 weeks after treatment became significant and more effect was observed in improvement of symptoms compared with the group receiving vitamin e. our findings are similar to the mentioned study in terms of reduction of symptoms and duration of the use drugs but and does nt match in terms of drug form and comparison with vitamin e. another researcher studied the effectiveness of three drugs, i.e. hyluronic acid, vitamin e and a in the form of suppository for a period of one month firstly in continuous days and then every other day on menopaused women and the results showed that using hyaluronic acid caused a significant reduction in signs and symptoms of vaginal atrophy. the results of the present study showed a significant reduction in symptoms with using hyluronic acid that is similar to the results obtained by castantino and guaraldi s study. but is there was a difference in terms of comparison of vitamin a and e and drug form. it is also reported that the effect of the gel of hyaluronic acid on the symptoms of vaginal atrophy was due to the chemo - therapy started from the first week. our results are in the same line with tea.s study in terms of improving the symptoms using hyaloronic acid and is different in terms of drug form, target - group and comparison with other drugs. another study on the effect of prescribing genestine vaginal suppository compared with hyaluronic acid suppository on the epithelium atrophy showed that using drugs for 15 continuous days in each month for a period of 3 months improved the symptoms of vaginal dryness and itching, dyspareunia, colposcopy specialties and the rate of vaginal cellular maturation. the results of our study were similar to those of le donne et-al.s survey in term of drug effects on atrophy and drug type, but they were different as to the drug form and duration of the intervention. to the best of our knowledge, no study was previously done in iran that used hyaloronic acid cream for vaginal atrophy treatment. the variety of admitted patients to the clinic and finding the menopause women from different department were the limitations of this study. this study showed the better and more relief of the symptoms of urinary incontinence, cellular maturation and vaginal dryness in menopause women in the hyaluronic acid cream therapeutic group compared with estrogen - therapy group. therefore, the hyaluronic acid could be a suitable alternative for those women who suffer from the complications of atrophy of the genital system and those with medical contraindications or negative experience in using hormonal drugs.	background : vaginal atrophy is a common complication in menopause which does not improve with time and, if untreated, can affect the quality of life for women. the aim of this study was to compare the effectiveness of the vaginal cream of hyaluronic acid and conjugated estrogen (premarin) in treatment of vaginal atrophy.methods:this study was a randomized controlled clinical trial on 56 menopausal women with symptoms of vaginal atrophy ; they were randomly allocated to two groups (recipient conjugated estrogen and hyaluronic acid). the severity of each sign of atrophy was evaluated by visual analog signals (vas) and on the basis of a four point scale. also to recognize the cellular maturation with pap smear and the maturation degree were calculated according to the formula and scores 0 - 100. as to the vaginal ph, we used ph marker band, the rate of which was divided into 4 degrees. data were analyzed using spss, version 20, and p0.05 was considered as significant.results:the results of this study showed that the symptoms of vaginal atrophy compared with the baseline level were relieved significantly in both groups. dryness, itching, maturation index, ph and composite score of the vaginal symptoms were relieved significantly in both groups (p<0.001). dyspareunia in premarin (p<0.05) and hyaluronic acid (p<0.001) decreased compared with pre - treatment. urinary incontinence only showed improvement in the hyaluronic acid group (p<0.05). improvement in urinary incontinence, dryness, maturation index (p<0.05) and composite score of vaginal symptoms (p<0.001) in the hyaluronic acid group was better than those in the premarin group.conclusion:according to the results of the present study, hyaluronic acid and conjugated estrogen improved the symptoms of vaginal atrophy. but hyaluronic acid was more effective and this drug is suggested for those who do not want to or can not take local hormone treatment.trial registration number : irct2013022712644n1
thomas fuller, md, gnomologia (1732) scoring systems are beloved by some intensivists. they can provide a means by which patients may be compared between facilities, therefore enabling sensible trials to be conducted. however, the holy grail for many enthusiasts remains their potential use as prognostic tools among the critically ill in an attempt to predict the future. one would hope that clinical acumen also plays a role in determining treatment and the study by rocker and coworkers is somewhat reassuring in this respect. that prospective study, which included some 851 mechanically ventilated patients, was performed to evaluate the predictive ability of, and outcomes associated with, daily clinician estimates of a probability of intensive care unit (icu) survival under 10%. the usual baseline characteristics were recorded, together with daily acute physiology and chornic health evaluation ii score and multiple organ dysfunction score. after morning ward rounds the attending physician and each bedside nurse were asked to predict the clinical probability of icu survival as one of the following : under 10%, 1040%, 4160%, 6190%, or over 90%. of those patients deemed to have a greater than 10% chance of surviving icu, 87.8% survived. of those with an expected survival chance of under 10%, 29% did actually survive their icu stay, although no data are given regarding whether they survived their hospital stay. the physicians tended to have a bleaker outlook than the nursing staff, but when both observations were combined this was, unsurprisingly, a more powerful predictor. indeed, the clinical prediction was more powerful than illness severity, use of inotropes and vasopressors, or organ dysfunction. however, the group thought to have a poor outlook was also more likely to have some form of life support withdrawn. therefore, it appears from this study that clinical assessments of prognosis remain strongly influential in determining outcome. a study conducted by ewig and coworkers takes prediction a step further (or back ?) onto the medical wards. in a 3-year prospective study of 696 sequential patients (after exclusions) with community - acquired pneumonia, these included the original and modified american thoracic society guidelines, the original and modified british thoracic society guidelines, the pneumonia severity index and the less cumbersome curb (confusion, urea, respiratory rate, blood pressure) index based on recent studies reported by lim and coworkers. the modified american thoracic society guidelines were superior to the other tools in predicting the severity of community - acquired pneumonia, as judged by need for icu care. fulfilling two out of three minor criteria (systolic blood pressure 2 lobes ] involvement ; and arterial o2 tension [in mmhg]/fractional inspired o2 ratio 7 mmol / l, respiratory rate 30 breaths / min ; and diastolic blood pressure 60 mmhg or systolic blood pressure < 90 mmhg) was found to be a very quick and useful method for rapidly assessing the risk for dying. scores of 0, 12 and 34 were associated with mortality rates of 3%, 21% and 56%, respectively. this may well prove to be a useful ward / emergency room tool, but even the very best predictive tool is just a guide. leading on from these studies, the paper by bellomo and coworkers appreciates that part of the role of the icu is to prevent patients deteriorating before icu admission or indeed preventing admission. this approach has attracted much attention of late, and bellomo and coworkers performed a prospective controlled trial in order to assess whether the introduction of a medical emergency team (met ; slightly confusing for the exercise physiologists among us !) may reduce adverse outcomes following surgery. in this setting, the met consisted of the duty intensive care fellow and a designated icu nurse. specialist availability was provided on site for 12 hours but was also available after hours if needed. any member of the hospital clinical staff could activate the met team (including social workers), and the average response time was a phenomenal 1.7 min. this figure we find particularly astounding, and reflects australia 's resurgence as a sporting nation. of particular interest are the criteria for initiation of the met team. in an era in which increasingly complicated scoring systems are being employed to identify those ' at risk ', the authors must be applauded for using straightforward parameters that focus on drastic acute changes but also include the sensible caveat that a staff member is worried about a patient. the introduction of the met resulted in a relative risk reduction for adverse outcomes of 57.8%. the most striking reductions were in renal failure requiring renal replacement (relative risk reduction 88.5%), respiratory failure (79.1%) and severe sepsis (74.3%). somewhat surprisingly, there was also a dramatic reduction in the risk for acute stroke. unsurprisingly, this all translated into reductions in the number of postoperative deaths and in the length of stay. the authors themselves outlined the flaws in this study, some of which are almost impossible to circumvent. also, considerable effort was made to educate those on the wards with respect to the criteria needed to activate the met, which might have raised awareness on the wards. also, it is common that in one 's daily practice on the icu one is called to review patients on the wards in a more informal ' met - like ' arrangement. it is not clear whether such arrangements were in hand previously, but we assume so. these criticisms should not detract from the findings, although we would be interested to see whether the improvements are sustained. the power of these results is that they perhaps illustrate that the speed by which a patient is reviewed by individuals equipped to deal with any physiological deterioration may dictate the eventual outcome. now, if we could just get some more juniors and get rid of the european working time directive... the theme of early intervention in deteriorating patients was continued in a study conducted by esteban and coworkers. that prospective, randomized, multicentre trial of 221 patients compared non - invasive ventilation (niv) for respiratory failure, within 48 hours of elective extubation, versus standard medical therapy. those investigators demonstrated that not only did niv fail to prevent the need for reintubation, but that it also delayed reintubation and resulted in increased mortality. reintubation rates were 48% in both groups (relative risk 0.99, 95% confidence interval 0.761.30). median time to reintubation was 12 hours for the niv group, as compared with 2.5 hours for the standard therapy group (p = 0.02), and most importantly mortality almost doubled in the niv group (25% versus 14% ; relative risk 1.78, 95% confidence interval 1.033.20 ; p = 0.048). the bulk of the deaths in the niv group occurred in those who required reintubation (21 out of 28 deaths), suggesting that the delay in reintubation in this group may account for these findings. the moral of this study may be that if you are thinking of reintubation, then get on with it. it seems that things only get worse with time, and biting the bullet early helps to limit the risks. the choice of resuscitation fluid for those patients the athletic australians can not keep off the icu was made a little easier in may, thanks to the safe (saline versus albumin fluid evaluation) study group. in the largest multicentre, double blind, randomized controlled trial on this issue to date, 7000 patients were assigned to receive either 0.9% saline or 4% albumin solution for fluid resuscitation during their first 28 days on the icu. contrary to the original cochrane meta - analysis that sparked the whole debate on this issue, those investigators found no difference in 28-day mortality between patients resuscitated with saline and those with albumin. no significant differences were identifiable in the rate of new single or multiorgan failure (assessed using sequential organ failure assessment score), in the number of days spent on the icu, or hospital stay. there were also no differences in the time spent on mechanical ventilation or in the duration of renal replacement therapy between the two treatment groups. the study was pragmatically planned, dictating only the fluid resuscitation of the patients, leaving clinicians free to manage all other aspects of the patients ' care as they saw fit. this freedom was controlled by stratification of randomization such that individual units treated equal numbers of patients in each group. preplanned subgroup analysis suggested a benefit from albumin in septic patients, which was balanced by a detrimental effect in trauma patients with significant head injury. however, as the authors highlighted, further studies should look into such specific groups before any changes are introduced. with either fluid being shown to be equally safe and effective, the initial choice (for the moment at least) would seem to be down to the clinician (managers ?) involved. finally, although british telecom in the uk spent much money several years ago telling those of us on this small island that it is ' good to talk ', it appears that this may not be true in the immediate vicinity of a ventilated patient, at least on the telephone. shaw and coworkers demonstrated that some urban myths might be true ; 50% of ventilators they tested malfunctioned when in close (< 30 cm) proximity to a transmitting mobile phone. thankfully, only one model stopped completely. with the ever - increasing prevalence of wireless technology (computer networks, personal digital assistants, phones, pagers and radios, among other devices), better shielding by the manufacturers of icu equipment from electromagnetic interference would seem prudent. the manufacturers of the machine that stopped completely have already introduced hardware and software upgrades that remedy the problem. however, in the meantime, perhaps we should keep mobile phones and ventilators at a safe distance, or at least keep the conversation short. curb = confusion, urea, respiratory rate, blood pressure ; icu = intensive care unit ; met = medical emergency team ; niv = non - invasive ventilation.	early recognition of sick patients with a poor prognosis, and the rapid institution of appropriate therapy are tenets of good medical management across all specialties. here we highlight five recent papers that aid us in achieving such goals in and around the intensive care unit (icu). both score - generating clinical tools and clinical acumen are championed for identifying the sick, while appropriate, early intervention in acute deterioration is shown to be beneficial, before and after icu admission. saline or albumen for resuscitation ? the answer became clearer in may, as did what to do about all those mobile phones...
it is now clear that nucleic acids play several different roles in the living cell from genetic code storage to the catalysis of chemical reactions in ribosome. all of these particular behaviours are associated with various and very often transient structures of these polymers. the most prevalent secondary structure of nucleic acids is the double helix that can adopt either a- or b - type depending on the hydration level and/or the 2-deoxyribosyl or ribosyl nature of the hybridized strands. while the backbone organization of double - stranded dna and rna is normally quite regular, there are many other secondary and tertiary structures that dna and rna molecules can adopt in vivo. it is also well established that these disparate structures, which are predisposed to promote a significant local conformational heterogeneity in the sugar - phosphate backbone, play a crucial role in the fundamental biological processes where protein - nucleic acid interactions, folding, or catalytic activity are involved. as a consequence nucleic acids can fold into biologically relevant distinct structures such as bulges, hairpin loops, u - turns, adenosine platforms, branched junctions, or quadruplexes (figure 1). as proposed by few studies, the sugar / phosphate backbone of these unusual motifs exhibit a variety of conformations, which markedly differ from the regular conformational states of duplex dna and rna molecules [38 ]. however, the intrinsic role imparted to the phosphate diester backbone in respect with bases sequence in stamping these structures is still not properly defined. the determination of the precise biological role played by nonstandard helical conformations during the biochemically important processes (e.g., protein - dna complexation, dna processing, and dna packaging) is also an area of intense study [9, 10 ]. an important study based on an analysis of available high - resolution crystallographic data and molecular simulation techniques has shown that, in contrast to free b - dna structures, protein - bound b - dna oligomers regularly involve noncanonical backbone geometries. these unusual backbone states are believed to contribute to the specific recognition of dna by proteins in assisting, at some stages, the fine structural adjustments that are required between dna and proteins to form stable complexes. there are many examples in which dna / protein complex formation results in dna bending without disruption of the watson - crick base pairing [1215 ]. whereas this bending can be essential for complexes formation, it is generally sequence specific but with a strong impact on the sugar / phosphate backbone and can reach up to 90. unfortunately, experimental studies which aimed at determining the structural and functional implications of such helical deformations are somewhat complicated by the intrinsically transient nature of the corresponding backbone states. stable structural analogues of these distorted backbone geometries would be very useful in the elucidation of the role that helical deformations play in nucleic acid interactions with proteins. mainly driven by the need of antisens research, most of the conformationally restricted oligonucleotides have been designed to enhance duplex formation ability and stability. therefore, many efforts have been devoted to the synthesis of analogues with sugar - puckering conformational restriction of the north type [1618, 18, 19 ]. to our knowledge, less attention has been paid to the design of conformationally restricted nucleosides with the aim of mimicking nucleic acid secondary structures containing non - watson - crick pairs or unpaired nucleotides. we are interested in the development of conformationally constrained dinucleotide building units in which the backbone torsional angles can have predefined values that differ significantly from the typical values observed in dna and rna duplexes. in that context, the present paper will describe the last proposals and recent advances towards the introduction of conformational constraints into nucleotides by means of cyclic - phosphate structures. the introduction of constraint on the sugar - phosphate backbone by connecting a phosphate to a base, sugar moiety, or another phosphate of the same strand gave new opportunity to provide conformationally constrained nucleic acids mimics. the pioneering work of the sekine 's group in the late 90s illustrated this approach. this sharply bent conformation has been commonly found in the anticodon loop of trnas and later discovered at the active site of hammerhead ribozymes. therefore, they focused on the preparation of two cyclic diuridylates (compounds i and ii, figure 2), in which the two nucleosides moieties were connected either by an amide group or by a carbamate function for i, or by introducing a bridge between the 5-phosphate group and the 5-c position of the uracil moiety for ii. when incorporated within oligonucleotides, these modified nucleotides i were both able to induce a severe bent into the oligomer, whereas the cyclouridilic derivatives of type ii could either allow the formation of the duplex with the rp configured phosphotriester moiety or be a good motif to mimic the u - turn structure with the sp configured rigid - cyclouridilic acid derivative [22, 23 ]. later on, the poul nielsen 's group showed that the ring - closing metathesis (rcm) reaction was a suitable methodology towards the synthesis of conformationally restricted dinucleotide structures (compounds iii to vii) in order to preorganize a single - stranded nucleic acid and to either form stabilized duplexes or to induce stabilization in other secondary structures [2429 ]. the approach is based on the synthesis of dinucleotide units (or trinucleotide units) with a phosphotriester linkage constructed by rcm between an allyl - protected phosphate and another double - bond introduced at the appropriate location on the nucleoside either on the sugar or on the base moiety. whereas all the constrained dinucleotide structures evaluated in duplex context showed destabilizing behavior, the rp isomer of v provided the first example of stabilized three - way junction, in particular when the hairpin moiety was composed of ribonucleotides with an increase stability of + 2.2c rising to 2.7c with the addition of mg. the cyclic structures proposed there to modulate the sugar / phosphate backbone were composed of the smallest of a seven - membered cyclic phosphotriester to a very large macrocycle (up to eighteen members) and therefore exhibited rather flexible and undefined structures. in order to have a more rationalized approach to the design of covalently constrained nucleic acids (cna) with specific canonical or noncanonical backbone conformations, we have developed dimeric building units in which two or three backbone torsion angles are part of a well - defined six - membered ring structure (figure 3). the so - called d - cnas are dinucleotides, in which a set of backbone torsion angles is stereocontrolled to canonical or noncanonical values by a 1,3,2-dioxaphosphorinane ring structure. for a given dinucleotide step, there are fourteen possible [-d - deoxyribo]-configured d - cna stereoisomers which formally result from the introduction of a methylene or ethylene linker between a nonbridging phosphate oxygen and the 2/4-carbons (methylene linker) or the 3/5-carbons (ethylene linker) of the sugar moiety. herein, we disclose the synthesis of each member of the d - cna family, discuss their structural parameters which were established by means of x - ray diffraction analysis or nmr, and finally emphasize on the behaviour of,-d - cna within duplex or hairpin secondary structure. our retrosynthetic analysis for the synthesis of,-d - cna dinucleotides is based on the very simple strategy that consists of using both steric and anomeric effects to stereocontrol the cyclization reaction of a dinucleotide precursor, in which the phosphate oxyanions can attack an activated carbon atom. the preparations of the,-d - cna dithymidine diastereoisomers are disclosed in scheme 1 [30, 31 ]. the key compounds of these pathways are the diastereopure 5(s) and 5(r)-c - hydroxyethyl - substituted nucleosides 3 and 7, respectively. the former was obtained after reduction of the ester moiety of the product 2 of a diastereoselective mukayama 's reaction catalysed by bicl3/zni2 on the aldehyde 1 [3234 ]. the starting aldehyde 1 was prepared by a pfitzner - moffatt oxidation procedure of the primary hydroxyl function of the thymidine after a classical three - step protection / deprotection sequence [35, 36 ]. the 5(r) isomer 7 was generated from 1 through a sakura 's allylation with a -subsituted - allyltrimethylsilane [37, 38 ] followed by a three - step oxidative cleavage protocol of the double bond of the 5-c - hydroxypentenyl - thymidine 6 isolated by silica gel chromatography from its diastereoisomer. selective tosylation of the primary - hydroxyl function was achieved in good yield by reaction with tosyl chloride in the presence of pyridine to provide the corresponding 5-c - tosyloxyethylthymidines that were coupled with the commercially available thymidine phosphoramidite under a standard phosphoramidite procedure to give the acyclic dinucleotides 4 and 8 after the oxidation step, respectively. the cyclization reaction for the formation of the dioxaphosphorinane structure occurred by the treatment of 4 or 8 in basic medium to generate the phosphate anion that can displace the tosylate group. surprisingly, the (sc, rp) isomer 5 of,-d - cna was exclusively obtained from 4 whereas a lower stereoselectivity of 7/3 was observed for the formation of the (rc, sp) 9 and (rc, rp) 10,-d - cna from the 5(r)-c precursor 8. after deprotection of the hydroxyl functions both,,-d - cna were structurally characterized and revealed that the major isomers were those with the dioxaphosphorinane ring in the chair conformation (see section 6). following the same chemical synthesis pathway,,-d - cna analogues have been prepared by introducing a lna - modified nucleoside during the phosphoramidite coupling to lead to lna/,-d - cna (figure 4), while changing the oxidation procedure from water / iodine to sulfur provided after cyclisation thio - dioxa- and oxo - oxathiaphosphorinane structures (thio-,-d - cna). finally, starting from uridine or 2-ome - uridine, ribo-,-d - cna could be achieved with the same diastereoselectivity outcome during the cyclisation process. therefore, the high diastereoselectivity observed for the formation of 5 led us to develop a strategy to synthesize the missing isomer in order to complete the set of cna structural element. we turned our interest to phosphonate analogues of the d - cna,, in which the dioxaphosphorinane ring would be replaced by a cyclic phosphonate called phostone providing phostone - constrained nucleic acids building blocks (p - cna). we speculated that an intramolecular arbuzov reaction, performed on the phosphite dinucleotide intermediate 14 similar to that prepared for the synthesis of d - can, would be suitable to reach this target (scheme 2). starting from the thymidine aldehyde 1, allylation under hosomi and sakurai 's condition gave pure 5(s)-c - allylthymidine 11 that underwent a selective hydroboration / oxidation of the double - bond after protection of the secondary hydroxyl function to provide 12. the required 5-c - tosyloxypropylthymidine 13 was reached by tosylation of the primary hydroxyl function and removal of the trimethylsilyl protective group. the key phosphite intermediate 14 resulted from the standard coupling of 13 with the commercially cyanoethyl protected thymidine phosphoramidite using usual tetrazole activation and without oxidation step. in optimized arbuzov reaction conditions (micro - waves irradiation and addition of libr), the cyanoethyl group was eliminated after the attack of the phosphorus on the activated carbon, leading to the formation of a 2/1 diastereoisomeric mixture of phostones. the removal of the 5 and 3-hydroxyl function protective groups and silica gel chromatography led to the isolation of the p - cna 15 and 16. the (sc, sp),-p - cna isomer 16 was isolated as the minor isomer and was corresponding to the structural analogue of the missing,-d - cna. these two representatives of the d - cna family originate from the connection of the phosphate to the 4-c - carbon atom either of the downstream sugar moiety for the,,-d - cna or of the upstream sugar moiety in the case of the,,-d - cna (figure 3). therefore, their synthesis started from a common intermediate 4-c - hydroxymethyl - thymidine 17 obtained by a treatment of the thymidine aldehyde 1 under cannizzaro 's conditions (schemes 3 and 4). in the case of,,-d - cna thymidine dinucleotides, the dioxaphosphorinane ring structure was formed as previously described for,-d - cna by displacement of a tosyl group by a phosphate anion generated by the removal of a phosphate cyanoethyl protective group in basic medium (scheme 3). the acyclic precursor involved is the dithymidine 19 prepared by coupling 5-o - tosyl-4-c - hydroxymethylthymidine 18 with the commercially available thymidine phosphoramidite using standard phosphoramidite technology. a three - step procedure involving first a selective protection of the 5-hydroxyl function of 17, followed by tosylation of the residual primary 5-hydroxyl function, and finally removal under acidic conditions of the dimetoxytrityl group furnished the required 5-o - tosyl-4-c - hydroxymethyl thymidine 18. the removal of the cyanoethyl group from 19 by treatment with potassium carbonate in dimethylformamide generated the phosphate anion which by heating at 90c provided the formation of two cis- and trans- isomers of protected,,-d - cna in a 2/1 ratio in favor of the cis. after the removal of the 5- and 3-protective groups, the,,-d - cna cis 20 could be separated from the trans isomer 21 and characterized. a similar approach towards the synthesis of,,-d - cna, that is, introduction of a tosyl group on the 5-hydroxyl function, phosphoramidite coupling, and nucleophilic attack of the phosphate has been investigated, but it turned out to be troublesome and the desired 5-o - activated nucleoside could only be obtained in very poor yield. therefore, we choose to use the well - known phosphotriester methodology that allows the formation of a phosphoester from a phosphate with an alcohol in the presence of an activator such as 1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole (msnt). starting from the diol 17, both primary hydroxyl functions were protected as dimethoxytrityl ether and the 3-o - silyl protective group was removed by treatment with fluoride ion to produce the nucleoside 22 (scheme 4). a phosphoramidite coupling with a 5-o - phosphoramidite - thymidine gave dinucleotide 23 that was consecutively treated in acidic medium to remove the dimethoxytrityl protective groups and in basic medium with triethylamine to eliminate the cyanoethyl phosphate protective group. the key phosphodiester 24 was then available to undergo the cyclisation process according to the phosphotriester methodology. even if two primary hydroxyl functions were present, only the 5-hydroxyl reacted under the msnt catalyst to form the dioxaphosphorinane ring. this high regioselectivity was unfortunately combined with no diastereoselectivity in the neither ring formation nor the formation of a 1/1 mixture of (sc4, rp) and (sc4, sp) diastereoisomers 25 and 26, respectively. the poor diastereoselectivity could be explained by the fact that due to the fused sugar ring none of these compounds feature a chair conformation of the dioxaphosphorinane structure, which is indicative that there is not a more favorable intermediate during the cyclisation process. the synthesis of 2,,-d - cna implied the connection of the phosphate to the 2-c - carbon of the sugar moiety through a methylene link. to achieve this goal, instead of starting from a nucleoside precursor, we choose to reproduce a protocol previously described by marquez and coll. that used the commercially available 1,2 : 5,6-diisopropylidene - d - glucose and through an elegant rearrangement gave the pivotal protected 2-deoxy - c-(hydroxymethyl)-d - ribofuranose 30 (scheme 5). 's procedure could install the thymine base and a phosphoramidite coupling would provide the dinucleotide that could undergo the dioxaphosphorinane ring formation, here again by the phosphotriester method leading to the target 2,,-d - cna. the secondary 3-hydroxyl function of 1,2 : 5,6-diisopropylidene - d - glucose was oxidised to ketone to be substrate for a wittig homologation with methyltriphenylphosphonium on the 3-c position providing the sugar 27 with an exocyclic double bond. the hydroxymethyl function at 3-c was generated by a hydroboration / oxidation that occurred from the top - face of the sugar resulting in the formation of the required r - configured 3-carbon. benzoylation of the resulting hydroxyl function provided the fully protected 3-deoxy-3-hydroxymethyl - d - allose 28. acidic hydrolysis of the 5,6-isopropylidene followed by a tricky selective benzoylation of the primary hydroxyl function led to 29 with the unprotected 5-secondary alcohol. acetolysis of the 1,2-isopropylidene gave the 6-o - benzoyl-3-deoxy-3-benzoyloxymethyl - d - allose that was subsequently treated with sodium periodate to cleave the diol system. after rearrangement, the 2-deoxy-2-benzoyloxymethyl - d - ribose analogue has been isolated as a mixture of anomers. after protection of the anomeric position with an acetate function, thymine was introduced by a vorbrggen 's procedure and the thymidine analogue 31 was obtained in a 1/9 ratio of / anomers. removal of the residual - formyl group by aqueous ammonia gave the suitable nucleoside for a phosphoramidite coupling with a 5-o - phosphoramidite - thymidine ending in the formation of the acyclic dinucleotide 32. potassium carbonate treatment, to remove the base labile benzoyl and cyanoethyl protective groups proceeded with a concomitant loss of the t - butyldiphenylsilyl group and dinucleotide 33 was obtained as a 1/1 mixture of fully deprotected, and 3-o - silylated dinucleotide. these dinucleotides were separated and submitted to the cyclisation activated by 1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole (msnt) to furnish (rc2, sp) and (rc2, rp) 2,,-d - cna 34 and 35, respectively. while the ring formation occurred with a 1/1.8 ratio in the case of the partially protected dinucleotide, the diastereoselectivity was lowered to 1/1.4 for the fully deprotected dinucleotide. the restrains on only the torsional angles and requires the formation of a spiro connection between the sugar and the dioxaphosphorinane rings by introduction of an ethylene linker between the 3-c - carbon atom of the sugar moiety and the phosphate (figure 3). to date, on the four possible stereoisomers, we have reported the synthesis of the xylo - configured d - cnas because they represent a class of distorted structures directly available from commercially uridine (scheme 6). a similar approach to that proposed for,-d - cna has been followed for the preparation of the xylo-,-d - can, that is, aldol condensation to introduce the ethylene link on the 3-c and activation through a tosylation to form the dioxaphosphorinane ring after phosphoramidite coupling. uridine was selectively protected on the 5-o and 2-o by t - butyldimethylsilyl group following the ogilvie 's procedure before being oxidized the with dess - martin periodinane to give the keto - uridine 36. a stereoselective mukayama 's addition of the t - butyldimethylsilyl - methyl - ketene acetal occurred on the re face of the carbonyl as determined by noe experiments on the adduct 37. reduction of the ester function turned to be rather difficult using nabh4 and the solution came from dibah ; however ; in a modest yield. the primary hydroxyl function was then selectively tosylated to provide the 3-tosyloxyethyl - xylo - uridine 38 suitable to be engaged in the phosphoramidite coupling with the 5-o - phosphoramidite - thymidine. the acyclic 3-c -tosyloxyethyluridine / thymidine dinucleotide 39 was then submitted to basic treatment at room temperature, and the generated phosphate anion cleanly displaced the tosyl group to form a 1/1 diastereoisomeric mixture of protected (sc3, rp) and (sc3, sp) xylo-,-d - cna 40 and 41, respectively, which have been separated on reverse phase hplc after deprotection. whereas a relative instability of the phosphotriester could be expected due to the presence of the secondary hydroxyl function, the spiro structure with an s configuration of 3-c fixed their relative positions away to the necessary on line conformation avoiding any trans - esterification process [55, 56 ]. the determination of the values of the constrained torsional angles within d - cna structures relied on the establishment of the geometry of the dioxaphosphorinane ring whether in chair conformation or not. some of d - cnas were crystallized and solid phase structures were determined by x - ray diffraction analysis for (rc5, sp),-d - cna tt (compound 9, scheme 1), (sc5, rp),-d - cna tu (figure 4), and (sc4, sp),,-d - cna tt (compound 26, scheme 4). moreover, nmr analysis of the h / h and h / p coupling constants of the protons involved in the dioxaphosphorinane ring or in the sugar moieties either corroborated the results of the x - ray analysis or allowed for the establishment of the rings conformations. interestingly, jh / p coupling constants between relevant protons within the dioxaphosphorinane ring and the phosphorous gave important information because they exhibit specific values dependant on the relative axial or equatorial position of the proton within the six membered ring, that is, jhax / p < ca 3 hz and jheq / p < ca 20 hz, respectively. therefore, a careful examination of these data allowed for the determination of the dioxaphosphorinane ring conformation, whereas jh / h coupling constants gave also information on the sugar puckering. the conformational ranges of the constrained torsional angles within d - cna determined by these methods are summarized in table 1. torsional angles ' values depicted in a- or b - type duplex are given as reference and are considered as canonical values for the regular double - helix structure. among all the sets of constrained torsional angles, the values exhibited for and by the (rc5, sp) isomer of,-d - cna (or its analogue lna/,-d - cna) are identical to those observed for the a- or b- type duplex. in contrast, and as expected by the proposed approach, all the others constrained dinucleotides feature - torsional angle ' values greatly differ from the canonical ones. therefore, these members offer an extraordinary diversity in the relative spatial arrangement of the bases moieties allowing the description of an unusual local shape of nucleic acids. in order to illustrate this point, figure 5 shows a superimposition of (rc5, sp)-, (sc5, rp),-d - cna and cis-, trans-,,-d - cna featuring a (g, t), (g, t), (g, g), and (g, c / g) set of value for and, respectively. whereas (rc5, sp),-d - cna analogue stands for a good mimic of b - type dinucleotide with the thymine bases mostly stacked, it is nicely illustrated that the two bases can be oriented in rather different planes in the others d - cna dinucleotides. the sugar puckering of each nucleosides within d - cna was estimated by the empirical equation of altona - sundaralingan using the jh / h coupling constants : c2-endo (%) = [j1/2/(j1/2 + j3/4) ] 100. due to an increase of the electronegativity of the 3 oxygen by introduction of a neutral internucleotidic linkage, the sugar pucker of the upstream nucleoside the determination of the impact that a neutral phosphotriester linkage would display in the conformational north / south equilibrium is particularly important as it is well recognized that this conformational state is of major importance for the dna duplex formation ability. the examination of the relevant coupling constants showed that in all cases for d - cna built with 2-deoxyribose, the sugar puckering of the 5-upstream or 3-downstream nucleoside were in the c2-endo conformation. however, for,-,,,-, and 2,,-d - cna the 5-upstream nucleoside sugar puckering equilibrium was strongly displaced toward the c2-endo conformation (south) compared to natural 2-deoxyribose units. interestingly, in the cases of ribo-,-d - cna (figure 4), even the 2-ome - ribose unit was pushed into the c2-endo conformation upon the influence of the neutralized internucleotidic linkage. only the xylose - configured sugar within xylo-,-d - cna adopted a north conformation (c2-exo). dioxaphosphorinane - modified sugar / phosphate backbone of dinucleotide could therefore represent a promising methodology to provide alternative backbone conformations. it is likely that d - cna within dna or rna oligomers would be able to modulate the shape and the folding with significant - conformational distortion of secondary nucleic acid structures. we focused our interest on the study of the impact of the restraint on one specific torsional angle,, through the behavior within oligodeoxynucleotide (odn) of a couple of,-d - cna diastereoisomers featuring either canonical or noncanonical / combination. as shown previously (scheme 1 and table 1), (sc5, rp) and (rc5, sp),-d - cna derivatives 5 and 9 can be easily prepared and their structural assignment showed that the (rc5, sp),-d - cna 9 exhibited a canonical value set (gauche(), trans) for and, whereas its diastereoisomer (sc5, rp),-d - cna 5 differed only on the value which was changed to the gauche(+) conformation while maintaining in the trans configuration. therefore, we dispose of a unique couple of modified nucleotides that will give us new insight on the impact of backbone preorganization either in the b - type duplex geometry or with a strong torsional stress applied on corresponding to that observed in dna / protein complex or in unpaired secondary structures such as hairpin or bulges. a molecular dynamic simulation has been run on da10/dt10 duplex whether modified or not with one tt step constrained with (rc5, sp) or (sc5, rp),-d - cna denoted as odnref, odngm, and odngp, respectively (figure 6). this study gave us two main results : compared to unmodified duplex the structure seems to accommodate the canonical restraint on with a straightness of the double - helix whereas the gauche(+) conformation induced a bend without loss of the watson - crick base pairing. therefore, these observations let us speculate that controlling the torsion of an odn into its b - type canonical form should enhance the duplex formation ability, whereas displacing it to around + 70 might result in the formation of localized distortion able to stabilize unpaired conformations. interestingly, analysis of the atomic fluctuations derived from these simulations indicated that in odngm all of these fluctuations were diminished in both strands which could be indicative of a potential duplex stabilisation, whereas in odngp they were unchanged compared to those observed in odnref. therefore, we investigated the behavior of,-d - cna within odns by thermal denaturation studies by means of uv experiments. selected results are reported in table 2 for (rc5, sp),-d - cna (denoted to as cnagm), in table 3 for a comparative study between (rc5, sp) and (sc5, rp),-d - can, and in table 4 for hairpin structures stabilisation by (sc5, rp),-d - cna (denoted to as cnagp). all the odns containing d - cna were obtained by automated synthesis according to the phosphoramidite methodology. the phosphoramidite building blocks of (rc5, sp) and (sc5, rp),-d - cna were synthesized by conventional method and their incorporation within odns occurred similarly to standard phosphoramidite with no change in automated synthesis protocols but with a smooth deprotection in ammonia at room temperature. the introduction of a canonical constraint within odn resulted in a remarkable stabilizing effect on duplex formed with dna counterparts (tm = + 5.0 1c / mod, table 2, entries 2, 610). these increases in tm values are insensitive to salt concentration suggesting that the effects observed were primarily conformational rather than electrostatic. thus, cnagm represents a rare example of constrained nucleotide that significantly increases the hybridizing properties of odns without forcing the sugar pucker into the c3-endo conformation, demonstrating that the preorganization concept can also be successfully applied to other torsional angles than those involved in the sugar moiety puckering. the ability of cnagm to adapt to the b - conformation of the double - helix is outlined by its additive stabilizing effect (entries 24) when included in the same strand and also when the two strands are modified with one cnagm, with a maximum effect when constraints were close to the 3-ends preventing end frying (entries 1114). it is noteworthy that a rather moderate effect was observed with rna counterparts (tm = + 0.9 to + 3.0c / mod), which could originate from the reluctance of the upstream - furanose unit of the,-d - cna to undergo a significant conformational change from 2-endo to 3-endo in the hybrid duplex dna / rna due to the loss of the internucleotidic negative charge. on the other hand, when we prepared sequences either modified by cnagm or by cnagp we were able to have insight on the cost in terms of thermal stability of a dramatic change of restraint on from gauche() to gauche(+). as expected, incorporation of cnagp featuring noncanonical (gauche(+), trans) / combination resulted in an important loss in duplex stability (4.2 to 13.6c / mod, table 3) depending on the sequence length and composition. short decamer and rather unstable oligothymidilate exhibited the higher destabilized level (entries 13) while increasing the size to 18-mer (entries 410) and 24-mer (entries 11, 12) modulated the impact of the gauche(+) restraint around 5 1c / mod and also minimized the positive effect on duplex formation ability of cnagm from + 5 1c / mod to + 1 1c / mod. we showed that regardless of the type of restriction applied to odn a high level of sequence discrimination was maintained as natural duplexes do. interestingly, exceptions in the destabilization effect of cnagp (entries 6 and 13) and in the positive impact of cnagm (entry 13) appeared. however, the first sequence is hemipalindromic and the second is fully self - complementary. a further experiment showed that the observed transitions were indeed a combination of melting temperature from equilibrium made of high hairpin transition and lower duplex melting. in the case of the drew - dickerson sequence (entry 13), cnagm was able to displace the equilibrium in favor of the duplex, whereas cnagp displaced it to the hairpin structure because constraint was imposed within the loop. in order to emphasize the effect of stabilization of unpaired region of secondary nucleic acids structure by cnagp, we engaged the synthesis of modified t4 loop within hairpin that could differ in their stem composition and especially in the at or cg loop - closing base pair (table 4). in a hairpin, which is a single - stranded structure, the necessary torsional stress is not spread throughout all the loop constituents but ensured by a sharp - turn position called the turning phosphate that displays a gauche(+) transition of torsional angle. therefore, it was tempting to speculate that cnagp could play the role of a preorganized turning phosphate and as a consequence could induce hairpin stabilization. cnagp was installed in all the possible positions within the loop, and the thermal stabilities were evaluated by uv melting curves analysis. in the case of t4-looped hairpin structures with at closing base pair, the central position was best suited for cnagp (table 4, entry 3 versus 2, 4, and 5) with a maximum in tm of + 3.0c. however, if a constraint in the middle of the loop helped the hairpin folding, when installed at the 3-end of the loop, cnagp strongly was destabilized by 7.0c (entry 5). remarkably, with cg closing base pair, cnagp behaves as a stabilizing analogue in any position within the loop (entries 69). indeed, circular dichroism experiments showed that when cnagp was placed in the middle of the loop (entry 8), the stem structure was not altered, whereas when located in 3-end region a stem rearrangement occurred that could participate to the stabilization enhancement observed (entry 9). similar results were depicted for t5-looped hairpin structures with tm up to + 5.0c. eventually, we showed that the two diastereoisomers of,-d - cna featuring a fixed torsional angle alpha either in the b - type canonical value gauche() or in atypical gauche(+) conformation are powerful building blocks allowing high level of duplex or hairpin stabilization as expected according to the preorganization concept. this is evidence that controlling the sugar / phosphate backbone not only in terms of sugar puckering is a promising approach toward the control of nucleic acids secondary structures. the development of nucleotides analogues, for the purpose of mimicking nucleic acids secondary structures, started with the pioneering work of sekine with his approach toward u - turn loop, and then the design of conformationally constrained nucleotides grew up through the nielsen 's ring - closing metathesis pathway. finally, the introduction of dioxaphosphorinane element at key positions along the sugar / phosphate backbone proved to be a rational concept to gain control on torsional angle sets. we have synthesized most of the possible members of the d - cna family ; all this structural units provide control on to torsional angle associated with a broad range of backbone conformations. interestingly, the dioxaphosphorinane ring structures within d - cnas are reasonably stable towards the oligonucleotide synthesis conditions according to a special care during the final deprotection step and are especially inert towards enzymatic degradation such as snake venon phosphodiesterase as expected for phosphotriesters. as a consequence, they are potential elements for the elaboration of synthetic nucleic acids with programmable folding and stability either in the double - helix or in the unpaired secondary structures. as a proof of concept, we demonstrated that relying on the restrain applied within oligodeoxynucleotides by means of,-d - cna, high level of duplex formation ability or hairpin stabilization could be achieved. therefore, at least with these leading components of the family, torsional stress applied to the sugar / phosphate backbone could be used in probing the necessary flexibility, or in contrary rigidity, of the nucleic acids architecture during the interaction with ligands or biomacromolecules. however, d - cnas can be seen as a new alphabet for the conception of shape - defined nucleic acids, and if ten years ago c. leumann concluded a review by a large field that has not yet been tapped is the use of conformationally constrained nucleosides for the stabilization of secondary structural elements as, for example hairpin loops and bulges, there is still a long way before being able to properly address the use of each member of the family and to understand or predict the behavior of d - cna within nucleic acids. nevertheless, this new kind of nucleotide analogues could be the basis for the development of synthetic oligonucleotides for the modulation of protein / nucleic acids complex formation.	we describe a rational approach devoted to modulate the sugar - phosphate backbone geometry of nucleic acids. constraints were generated by connecting one oxygen of the phosphate group to a carbon of the sugar moiety. the so - called dioxaphosphorinane rings were introduced at key positions along the sugar - phosphate backbone allowing the control of the six - torsion angles to defining the polymer structure. the syntheses of all the members of the d - cna family are described, and we emphasize the effect on secondary structure stabilization of a couple of diastereoisomers of,-d - cna exhibiting wether b - type canonical values or not.
although a number of republican militant groups were active in this time, including the official irish republican army (ira), the irish national liberation army, the continuity ira, the real ira, and the irish people s liberation organization, we narrow our focus solely to pira. pira were the most prolific nonstate militant grouping during this period in terms of both terrorist events and fatalities caused. through a protracted campaign of violence, pira s overall objective was to force the removal of northern ireland as part of the united kingdom, thereby paving the way for the reestablishment of a thirty - two county republic of ireland. following a split in the republican movement in december 1969, pira was formed. originally, pira strategy sought to quickly force british troops out of northern ireland by inflicting a high death toll and substantial economic costs and thereby swaying british public opinion against maintaining an active presence in northern ireland. by 1977, pira s plans became more long term, and they started plotting a longer war of attrition that eventually led to an increased emphasis upon mainstream political mobilization through their political wing, sinn fin. in the years that followed, the republican movement came to embrace what it internally referred to as tactical use of the armed struggle. pira were active for twenty - nine years and had a long time to get very good at killing people. even if we only trace the development of skills and ties that could be exploited to target their enemies from the birth of pira in 1970 following state violence against protestors demanding civil rights (moloney 2003), by 1979 the organization had ten years of active and successful practice (sutton 1994). that year, pira operatives detonated a concealed trailer full of milk churns that had been packed with 227 kg of ammonium nitrate explosives as a convoy of five british army vehicles passed. revealing a great deal of malevolent creativity, the trailer had been surrounded by petrol cans that further enhanced the explosion s ferocity (oppenheimer 2009, 113). this detonation blew up the second truck in the convoy, killing six soldiers of the parachute regiment that had been responsible for the bloody sunday massacre that killed thirteen innocent civil rights marchers seven years prior. the surviving soldiers sought cover behind the gates of a nearby gatehouse. anticipating this retreat ahead of time, pira had already placed an even larger 450-kg ied using a homemade mixture of ammonium nitrate, nitroglycerine, and coal. upon additional rescue teams arriving to save the retreating survivors, pira operatives detonated the second bomb using a remote control trigger, killing a further twelve soldiers (oppenheimer 2009, 113 - 14). such examples however mask the fact that a large proportion of pira s acts of violence caused zero fatalities. for example, of the 5,461 pira ieds, we coded as part of the bigger data - driven project from which this article draws (from bomb to bomb - maker), only 8.7 percent killed at least one person (on many occasions at the beginning of the conflict, the sole victim was the ied planter who died due to a premature explosion such incidents are not counted in the following analyses). the relatively discriminative impression these results produce are a reflection of pira s strategy of aiming to limit civilian deaths (through the provision of advanced warnings), bombing economic targets in the middle of the night, and increasing the economic costs of the united kingdom s continued presence within northern ireland. we define an explosive device as an ied if any or all of the following are modified in any respect from its original expressed or intended function : explosive ingredient, initiation, triggering or detonation mechanism, delivery system. an explosive device is not considered an ied when no aspect of its deployment or fabrication is innovated upon (gill, horgan, and lovelace, 2011, 742). pira s use of soviet - manufactured rocket - propelled grenades called rpg-7s was not included in our analysis. as figures 1 through 3 illustrate, pira s use of ieds ebbed and flowed over the course of its twenty - nine - year conflict. the biggest spike occurred over the opening two years as violence on both sides reached its peak. during this time, pira s use of car bombs dramatically accelerated upon developing ammonium nitrate explosives requiring a delivery system capable of carrying hundreds of kilos of the mixture. the subsequent dip from 1972 to 1975 is largely attributable to british counterterrorism policy of blocking off pira s supply of commercial explosives (which were largely obtained through mining companies and robberies south of the border). also, pira s strategy of a quick military victory began to give way to a strategy based on a war of attrition. as such, the scale and intensity of ied attacks began to decrease in lethality but increased in frequency through the late 1970s as pira began to regularly use small, timer - based incendiary devices against commercial business premises. the idea behind these attacks was to minimize the possibility of civilian casualties by timing the ignition in the middle of the night but still to allow for maximum economic damage. pira hoped that the increasing costs of maintaining a visible british presence in northern ireland would ultimately tempt british voters to pressurize incumbent regimes to withdraw political and territorial interest from northern ireland. the growing politicization of the republican movement through the 1980s accompanied a perceived downgrading in pira s ability to consistently engage in fatal attacks (moloney 2010). interestingly, while private negotiations for peace with the british (between 1990 and 1994) were at their peak, pira s use of ieds gradually rose with 1993 experiencing the fourth - highest number of ied events in any given year of the conflict. in effect, pira had learned from previous negotiations that the only way to strengthen their position at the bargaining table while keeping active supporters and rank and file recruits happy was to gradually step up their violent (but not necessarily fatal) activities. bombing[s ] on and off like a tap came to represent one of their signature capabilities throughout both formal and informal negotiations (coogan 2002, 399). the remaining figures in this section disaggregate pira s campaign into five discrete phases of activity. our intention is to illustrate how organizational changes in structure and strategy impact the trajectory of violent events, types of events typically engaged upon, and their outcomes. pira structured themselves like an army composed of various brigades, battalions, and companies. each unit was responsible for specific geographical areas of operation, both combat and noncombat related. indiscriminate violence by both sides of the conflict marked this period, the most defining moment being bloody sunday when the british army shot and killed thirteen innocent civil rights marchers. this was an unprecedented propaganda coup for pira and led to mass recruitment and mobilization. civilian fatalities attributed to pira also peaked during this phase and included the events of bloody friday where, in under two hours, 22 ieds killed 9 (6 civilians, 2 british soldiers, and 1 ulster defence association member) and injured a further 130. the second phase, from 1977 to 1980, is significant for a number of reasons. first, there was a large - scale reorganization of pira s structure to a tighter cellular based network in which cells acted independently of one another. this change placed far less emphasis on the quantity of volunteers and far more emphasis on secrecy and discipline. almost instantly, the effects of the structural changes were noticeable. four hundred sixty - five fewer charges for paramilitary offences occurred within a year (smith, 1997, 145). second, a number of leadership changes occurred whereby younger northern born - and - bred members (such as gerry adams and martin mcguinness) became pira chiefs of staff in the late 1970s (moloney 2003, 513). the third phase covered the period from 1981 to 1989 and encompasses the growing politicization of the republican movement that occurred after the hunger strikes. in total, ten republicans died during hunger strikes in 1981 ; seven were pira members. additionally, pira s bobby sands was elected to westminster after winning a by - election while on a hunger strike. sympathy for pira began to rise again, and this was largely channeled toward pira s political wing, sinn fein, by organizational elites. phase 4 (19901994) includes the negotiations and pathway toward ceasefires undertaken secretly by organizational elites. phase 5 (19951998) incorporates the period in which the negotiations were made public and the march toward the final ceasefire and good friday agreement that symbolized for many the end of the northern ireland conflict. figure 2 neatly illustrates the substantial drop in the numbers of fatalities and injuries through pira s ied activity across time. phase 1 averages 70.57 fatalities a year, while the figures for phases 2 through 5 are 28, 32.5, 20.8, and 4.125, respectively. however, figure 3 illustrates that when calculated as a ratio, the average number of injuries attributed to pira ieds increases in the final phases of the conflict. one of the major findings in the quantitative analysis of the lethality of terrorist organizations is that organizational factors do indeed matter (asal and rethemeyer 2008c). asal and rethemeyer found that organizational size, connections, and ideology all had a significant impact on the lethality of terrorist organizations. however, their analysis reflects an aggregate cross - organizational perspective focusing upon a wide array of ideologies and does not take differences in tactics into account. what may be true of organizations in the aggregate may not be true if we disaggregate organizations (helfstein and wright 2011). one confounding factor may be that ideological differences hide important differences at the suborganizational level. drake (1998a, 53) notes that ideology relates to targeting practices of terrorist organizations because it sets out the moral framework within which they operate. similarly, a number of scholars note that the new terrorist organizations are more lethal and attribute this difference in lethality to religious ideology : religious ideologies are in some cases more permissive of violent and deadly acts (laqueur 1998). further, asal and rethemeyer (2008a, 438) argue that two factors shape how intrinsically linked any ideology is to deadliness. the first point relates to whether the organization s audience is earthly or supernatural. the second point relates to an organization s ability to clearly and cleanly define an other. ideology, however, can not explain divergences in lethality within a single organization. within a terrorist organization, the ideology s audience and ability to other the enemy remain relatively constant (although it must be stated that components of pira were more sectarian than others while some politicized earlier than others). the same is true for other variables often labeled root causes of terrorism, such as rates of democracy and state sponsorship (helfstein and wright 2011). our contention is that subunit capability (conceptualized as unit size, levels of professional training, and experience) and blue team (e.g., those actors charged with countering terrorism such as the military or police) activity combine to make particular forms of fatal violence more likely within particular components of a terrorist organization. the social movement literature has long established that resources are key to the success of organizations (mccarthy and zald 1977) and the lack of resources has been seen to limit what terrorist organizations can do (boyns and ballard 2004). human capital is often depicted as the key resource (boyns and ballard 2004 ; asal and rethemeyer 2008c). jackson points out that larger terrorist organizations should be better at adopting and making effective use of new technologies (jackson 2001). contrary to jackson (2001), oots (1986, 69 - 72) makes the case that when it comes to carrying out attacks, larger organizational structures are likely to be less effective because they require resources to maintain them. mccormick (2003) makes a similar argument about the tensions between an organization trying to protect itself while still trying to carry out its attacks. these arguments treat terrorist organizations as single entities and do not examine them as coexisting subunits. while we can measure terrorist attack counts by whole organizations, if the organization is big enough it is misleading to say that an attack is conducted by an entire organization. thus, when we look at a terrorist organization s component parts the logic may be different than for the organization as a whole. in the case of pira, attacks were carried out by different brigades (who themselves had specialized roles and responsibilities) and rarely as attacks orchestrated by the organization as a whole (horgan and taylor 1997). indeed, pira s claims of responsibility often attributed attacks to particular brigades or battalions. another possibility to consider is that the size of an organizational component will have a differential impact depending on the type of attack that is carried out. the level of expertise that a type of attack demands should have an impact on how human resources impact the success of that kind of attack (jackson 2009, 12 - 13). the level of technical expertise needed for an average shooting attack for example could be considered low on the other hand, complex attacks demand a higher level of organization, expertise, and security (if you assume that complex attacks have the potential to be more spectacular in their consequences ; drake 1998a, 1998b ; jackson 2009, 12 - 13). firsthand accounts of pira training suggest that a disproportionate amount of time was spent on ied training compared to shooting (see ocallaghan 1999), which leads us to suggest that ieds are generally more complex than shooting attacks and their successful execution depends more on knowledge at the individual bomb maker and his or her network affiliates level. further to this, johnson and braithwaite (2009) illustrate that ied attacks form tighter space - time clusters than do non - ied events, which indicates that ied attacks involve more planning, training, materials, expertise, and local support. knowledge at the individual level, rather than group size, may therefore be a more important factor for determining the effectiveness of ieds compared to shooting attacks. in fact, complex attacks have the potential to be more spectacular in their consequences, and certainly are more likely to portray the group responsible as more sophisticated with the ability to coordinate multiple, simultaneous efforts. however, group size also matters. on one hand, the larger the group, the more likely a group is to have in its ranks an individual with the requisite knowledge to build a sophisticated ied. on the other hand, larger groups are more likely to leak information to counterterrorism agencies and thus increase the chances of early detection. on balance, we believe the relationship is likely to be in favor of larger groups : larger groups contain more knowledge, and more knowledge leads to greater success. thus, group size and total knowledge should be related positively to one another from this perspective. asal and rethemeyer (2008a) have found that lack of experience reduces lethality in their cross - national study, as has jackson (2001 ; though jackson used the age of the organization as a proxy). as hoffman (1999, 25) argues, an almost darwinian principle of natural selection seems to affect terrorist organizations, whereby every new terrorist generation learns from its predecessors terrorists often analyze the mistakes made by former comrades who have been killed or apprehended. thus, we expect that having more experienced cadre in a brigade should also allow for an organization to be more effective in its use of ieds. given the relative lack of sophistication needed to perpetrate shooting attacks, the same should not hold true for such attacks at the brigade level. human capital can also be measured in outputs and not just inner traits such as experience and technical expertise. not all ieds and their constituent initiation systems are created equally, with some being more difficult than others. high - value targets such as military personnel, military infrastructure, police, or politicians are far more target hardened than most areas heavily frequented by civilian populations. while attacks against high - value targets may have a much higher pay off if they succeed, attacks that are easier (in both their deployment and who they target) should be more successful on average (jackson 2009 ; drake 1998a, 1998b). in other words, when a brigade tries to carry off an attack using a device that is harder to construct the likelihood of success should fall. by the same token, brigades intent on using an ied (as opposed to opting for a shooting attack with multiple offenders) to attack a hardened target are more likely to fail but also need a more sophisticated device to succeed. having limited resources (i.e., ied components and personnel), decision makers must choose between attacking soft or hard targets using complex or simple ieds and ied components. given the relative ease of attacking civilians, simple ieds are likely to be chosen. given the relative difficulty of attacking high - value targets, multifaceted ieds and complex attacks (defined as attacks involved more than one attack type) are more likely to be chosen despite the higher chance of failure. while the most lethal attacks are likely to be complex ones targeting civilians, there are fewer incentives to use such tactics given the fact that simpler means can also cause a great deal of death and destruction against weakly guarded targets (dolnik and bhattacharjee 2002). we derive the following hypotheses from this discussion : hypothesis 1 : larger brigades should kill more.hypothesis 2 : brigades with more knowledge (measured as age and training) should kill more people with ied attacks.hypothesis 3 : brigades with more knowledge (measured as age and training) should kill fewer people with shooting attacks.hypothesis 4 : complex attacks should kill more than simple attacks. hypothesis 1 : larger brigades should kill more. hypothesis 2 : brigades with more knowledge (measured as age and training) should kill more people with ied attacks. hypothesis 3 : brigades with more knowledge (measured as age and training) should kill fewer people with shooting attacks. hypothesis 4 : complex attacks should kill more than simple attacks. counterterrorism efforts are designed to impact how terrorists behave. yet, whether and how this precisely happens is often less than clear. a report from 2006 found that (1) very little rigorous empirical research exists, (2) what does exist provides little support for most common policies, and (3) the one policy that does seem to have an effect works in the wrong direction : retaliatory raids increased terrorism (lum, kennedy, and sherley 2006). more recent work focusing on particular cases or surveys of the literature has found either a complicated picture or a nonproductive : counterterrorism activities are ineffective or counterproductive (duyvesteyn 2008 ; feridun and shahbaz 2010 ; fielding and shortland 2010). an analysis of pira terrorism found that most counterterrorist efforts resulted in an increase in terrorist activity and thereby could be characterized by a backlash model of counterterrorism (lafree, dugan, and korte 2009). we should note that due to a specific interest in the impact of discriminate (e.g., the killing of pira members) versus indiscriminate (e.g., the killing of catholic noncombatants) violence our focus here is on proactive offensive state attacks and not on other types of repression that are not specifically violent in nature like situational crime prevention measures such as checkpoints and curfews or judicial deterrence measures such as punitive sentencing. benmelech, berrebi, and klor (2010) suggest an important distinction when it comes to counterterrorism. they found that targeted house demolitions that destroyed the homes of people engaged in suicide terrorism reduced suicide attacks, while house demolitions that were carried out against property not directly associated with the specific suicide attack increased subsequent terrorism. byman, examining israel s policies of targeted killing found that such assassinations reduced the effectiveness of hamas terrorism against israel (byman 2006), although others have found no effect (hafez and hatfield 2006). the benmelech, berrebi, and klor (2010) and lafree, dugan, and korte (2009) articles both suggest that counterterrorism actions can create a backlash. byman s articles introduce an interesting and potentially very important caveat. while we can assume that both kinds of attacks are likely to hurt the brigade, indiscriminate attacks compensate for this pain by making the group more popular and creating more support. mccauley and moskalenko (2008) argue that this is exactly one of the reasons why terrorist organizations stage attacks : attacks provoke repression and make terrorist organizations more popular and stronger something they label jujitsu politics (a term first coined by sharp in the context of nonviolent conflict resolution). on the other hand, violence directed specifically at the organization appears to lower the amount of subsequent terrorism. we derive the following hypotheses from this discussion : hypothesis 5 : indiscriminate counterterrorism killings should increase the number of people killed by ieds and shootings.hypothesis 6 : discriminate counterterrorism killings should reduce the number of people killed by ieds and shootings. hypothesis 5 : indiscriminate counterterrorism killings should increase the number of people killed by ieds and shootings. hypothesis 6 : discriminate counterterrorism killings should reduce the number of people killed by ieds and shootings. the data are an aggregation of 5,461 pira ied events, all fatal pira shootings, and the sociodemographic and operational behaviors of 1,240 pira members for the years 1970 to 1998. similar to many quantitative studies of terrorism and political violence, there exist a number of data constraints in this study. however, after an eighteen - month data collection effort across multiple data sources, we feel that this is the best data available. from a research perspective, focusing upon intraorganizational dynamics allows us to hold constant many of the environmental and systemic variables that may confound other studies. for this article, pira is disaggregated into six discrete subunits, each of which encompasses a county of northern ireland. although not an exact fit to pira s command and functional structure, it acts as the only realizable proxy measure. northern command includes both the six counties of northern ireland and the border counties of cavan, donegal, leitrim, louth, and monaghan. northern command therefore covered the main theater of conflict. in turn, northern command was composed of brigadiers, brigades, operations commanders, and active service units of typically four individual pira members. southern command constituted the other twenty - one counties of the republic of ireland and its duties largely encompassed logistical support for northern command activities. tasks included training, funding, storing, and moving arms as well as provision of safehouses (horgan and taylor 1997). often, active service units (asus) operated within their own locality (horgan and taylor 1997, 20). white and white s (1991) interviews with former senior pira figures explain why. reasons for this would be to avail of local facilities before, during and after operations, such as safehouses where they would be recognized without difficulty, and also because of familiarity with the operational area (a vital aspect of the operational cycletarget selection, planning, escape routes, etc.). perhaps, however, this may be seen to have a detrimental effect on the internal security of asus after all, it is far more difficult being required not to know the identity of one s asu colleague if, in fact, volunteers are operating in their own locality (horgan and taylor, 1997, 22). when questioned what makes a successful member of pira, sean macstiofain (a former member of pira chief of staff), noted that a person has got to be from the locality, right? he s got to be respected within his own community, right ? like he becomes a fish who can swim he has got to have an intimate knowledge of his the areas he s going to operate in. he has got to be considerate about the needs of his own community (white and white 1991, 107). for this study, we aggregated yearly counts of aspects of ied usage, fatal pira shooting events, fatal counterterrorism events, and a database of convicted pira members for each county. while pira s repertoires of violence also incorporated punishment beatings, kidnappings, and bank robberies, we specifically focus upon aggregate counts of ieds and shootings because they were the tactics most associated with lethal forms of violence each observation contains yearly counts of the ieds detonated as intended or were duds. we also count the number of each of the following : initiations systems used within a county in a given year : timer - initiated, wire - initiated, remote - initiated, projectile - initiated, booby trap initiated, impact - initiated, and victim - initiated devices. counts of the following ied types were also included in the analysis : letter bombs, pipe bombs, grenades, homemade bombs, static munitions, buried ieds, undervehicle ieds, car bombs, mortars, and rockets. we defined complex ied events as those where the ied was used in conjunction with another types of violent event such as machine gun fire or sniper fire. our metric of counterterrorism activity is a count of the pira members and innocent catholics killed by the british army and the royal ulster constabulary. it should be noted that these two counts do not reflect the whole counterterrorism picture. policies such as internment did not directly cause fatalities but caused a later backlash from the catholic community and ultimately increased pira s ability to recruit new individuals and mobilize mass support. on the other hand, prison - related policy changes such as the withdrawal of the special category status eventually led to the 1981 hunger strikes in which seven pira and three irish national liberation army members died. when counterterrorism policies indirectly lead to the deaths of pira members (such as the hunger strikes) these deaths were not counted in our analysis. other counterterrorism policies and actions such as the use of informants and the capture of bomb - making facilities would also impinge upon pira s ability to engage in lethal ied attacks, but such data were difficult to collect in a systematic way. our data set included a set of pira subunit traits, including a count of members in the brigade each year ; a measure of how big the subunit was in relation to the other five units each year ; the mean age of the subunits members ; the proportion of subunit members who possessed professional skills that could be applied to bomb making ; and the number of fatalities the subunit caused through ieds and shootings across target types (including civilians and high - value targets). high - value targets encompass a collection of northern ireland security forces such as the british army, the royal ulster constabulary (the northern ireland police force), the ulster defence regiment, and the royal irish regiment as well as government officials (both elected and unelected) and other political figures. the aggregate measures for the ied - related variables stem from a newly constructed data set of 5,461 events collected as part of the from bomb to bomb - maker project. the aggregate measures for deaths by the british army and other counterterrorism forces are collected through mckittrick.s definitive list of war dead from the northern ireland conflict. the subunit trait variables were aggregated by brigade from a database of 1,240 individuals who were either convicted of pira - related activities (including membership) or died on active service, a term used by pira to describe a member s involvement in pira - related activities. for the purposes of the data collection, being engaged in active service included both violent activities (e.g. bombing attacks) and nonviolent activities (e.g., training accidents). the individuals were identified from a number of open sources : (1) statements by pira including their annual roll of honor, which commemorates their war dead ; (2) the belfast graves publication that offers an account of republicans killed in combat ; (3) mckittrick. mentioned earlier ; and (4) historical accounts of pira from academic sources. these names were subsequently coded for a number of sociodemographic, operational, and network variables using the irish times archives. each piece of data (ied event, blue team activity, pira militant) was coded twice by separate coders and cross - checked for validity. four dependent variables were studied : total fatalities, civilian fatalities, fatalities among high - value targets (such as security forces, politicians, etc.), and fatalities from shootings. each dependent variable is a count of the fatalities attributed to each brigade during a year between 1970 and 1998. given that the dependent variables are counts generated by a rare event the data have two potential issues that must be accommodated during technique selection : (1) there is evidence of overdispersion of the dependent variable in all four cases, the mean of the counts is smaller than the standard deviation and (2) the presence of a large number of zeros in the dependent variable (long 1997 ; cameron and trivedi 1998 ; long and freese 2003). however, when we modeled the data using a zero - inflated negative binomial technique, the coefficient on, which captures the overdispersion, was not statistically significant in three of the four models. in the one case where was significant, for simplicity, we have thus used a poisson distribution for models reported below. with respect to problem (2), we compared the results from both the standard and the zero - inflated poisson (zip) models. the decision whether to kill or not is separated (analytically speaking) from the decision regarding how many people to kill. zip allows for the possibility that zeros in the model are present because brigades have chosen not to kill or because they were incapable of executing a fatal attack during a given year. zip is attractive precisely because it can accommodate these complexities in the data. in order to verify that the decisions are independent and should be modeled simultaneously but independently, a vuong (1989) test, which compares the fit of the zero - inflated model to the standard negative binomial regression model, was executed and is reported in the results table. in all four models, the vuong statistic clearly indicates that a zero - inflated model is superior. in all four models, we attempted to include phase and brigade controls to account for panel fixed effects. we also included controls for the mix of device and initiation types used in ieds over time, which could covary with our key training and age variables. however, because some types of attacks were highly correlated with brigade, phase, or type of attacks being modeled, the controls could not always be included. exposurethat is, length of time over which the count would be accumulated so there are no controls for exposure in these models. because there are clear reasons to believe there are commonalities across time in the behaviors of brigades, we adjusted the standard errors for brigade - level clustering (rogers 1993). statistical significance of coefficients was measured at the 0.1 percent, 1 percent, and 5 percent levels, except as otherwise noted. each of the four dependent variables was modeled independently, and the zero - inflation and count components of the estimations were modeled using a selection of variables that accorded to the understanding of factors that drove the respective dependent variables. across all four models, we also discovered that some variables had to be deleted to cope with high collinearity or problems with convergence of the model, and the ied - related controls are, of course, not included in the modeling of shooting fatalities. the central findings for each model revolve around a set of key variables see the first eight variables listed in both the count and zero - inflation components of the estimations. they capture the role of training, participation in pira, counterterrorism activities by british authorities, membership size, membership age, and number of members killed during a given brigade - year. however, in our analysis, we point to some interesting findings for each of the models that are not necessarily found across all four estimations. table 2 contains the estimated coefficients for the count component of the zip models for all four dependent variables. zero - inflated poisson (zip) estimates, total fatalities, civilian fatalities, fatalities among targeted groups, and fatalities for shootings. note : pira = provisional irish republican army ; ied = improvised explosive device. <.05, <.01, <.001. table 3 provides the zero - inflation estimation results plus standard model quality statistics such as n,, log likelihood, and the vuong statistics that pertain to the entire model (count and zero inflation). zero - inflated poisson (zip) estimates, total fatalities, civilian fatalities, fatalities among targeted groups, and fatalities for shootings (zero - inflation component). note : pira = provisional irish republican army ; ied = improvised explosive device. the block of eight key variables at the top of the count component of the analysis tells an interesting story. turning to hypotheses 2 and 3 first, knowledge matters to fatalities, but not in the manner most expect and as we anticipated originally. the variable % of brigade professionally trained is statistically significant and negative for both total and civilian ied casualties and unrelated to high - value ied and shooting casualties. in fact, this variable is negative across all four models, though not statistically significant in the models for ied high - value targets and fatalities for shootings. in all cases, training kept the body count down rather than causing it to go up. note that when the relative size of a brigade as compared to the rest of pira s northern command brigades went up, the total fatalities count also went down. thus, hypothesis 2 is contradicted fairly convincingly and there is no support for hypothesis 3 with respect to professional training. also contrary to expectations, brigade size does not tend to increase fatalities uniformly across all attack types. instead, the variable membership size (ct) has no effect on total ied fatalities and high - value target ied fatalities, a negative effect on civilian ied fatalities, but a positive effect on shooting fatalities. the results from the zero - inflation component of the estimation further nuance our findings. as the percentage of the brigade professionally trained (e.g., with training from professional occupations that can be turned to bomb making) increased, the probability that a brigade would kill no one went down and the probability that no members of the high - value target group (i.e., british security forces) also went down. stated another way, training made it more likely a brigade would kill a nonzero number of people with ieds and especially more likely that a nonzero number of high - value targets would be killed with ieds, but training did not drive up the number particularly. similarly, as brigade size increased, the probability that a brigade would kill a nonzero number of civilians would increase but again, membership size did not drive up the civilian body count. instead, the effect of size was to make it likely some civilians would be killed. a complementary pattern emerged in the zero - inflation results for the relative size of brigade in pira variable. as size of the brigade goes up in comparison to the other brigades in that year, the probability that the brigade killed nobody went down, but the probability that the brigade killed no civilians went up. the relatively larger brigades in the pira were more likely to kill a nonzero number of people in a given year, but being large did not translate into a drive to maximize killing, as the relative size of brigade in pira variable in the count model demonstrated. finally, in hypotheses 2 and 3, we suggested that age and/or professional training could be an important factor in driving ied fatalities. in fact, member average age was not a factor here (in either the count or the zero - inflation components), meaning that maturity and/or experience in the movement seemed to have little effect on fatality rates one way or the other. the upshot of this complex set of outcomes is that professional training and size create a capability for killing. as brigades become larger and more professionally trained, the likelihood that this instrument will be used for some killing increases. however, size and training does not lead inexorably to prolific killing with ieds or shooting attacks. instead, it appears that professional training may allow brigades to kill those people they wish to kill rather than to kill indiscriminately. pira was more generally committed to selected killings especially of british security forces rather than indiscriminate lethality. as asal and rethemeyer (2008a) found in their cross - national analyses, organizations can shape their lethality. looking cross - nationally between the years 1998 and 2005, asal and rethemeyer found that organizations with a religious ideology were more likely to kill prolifically, while organizations that had a combined ideology of religion and ethnonationalism were likely to kill even more. while an argument exists that the role of religion has not been given enough credence in the analysis of the northern ireland conflict (mitchell 2005), there is a more widely held position that religion merely differentiated catholics and protestants and acted as a proxy for what were essentially ethnonational identities (mcgarry and oleary 1995). our theoretical position largely concurs with the latter and our empirical analysis suggests that operational and ideological commitments helped to shape the impact of training toward focused, discriminate killing. hypothesis 4 addressed that nature of attacks : do complex attacks attacks that include both an ied and shooting component increase the body count ? our analysis suggests that when the number of complex attacks attempted went up, there was a particular pattern to fatalities (see the coefficients on complex attack (ct)). complex attacks did not affect the total number of fatalities. however, complex attacks tended to include a shooting component, so fatalities from shootings tended to increase. because such attacks involve additional terrorist actions (e.g., an ied attack and a shooting attack), the civilian fatality count tended to increase, but the fatality count among high - value target groups tended to decrease. ambitious attacks may be important for symbolic purposes and for recruiting, but the cost was a tendency for brigades to move away from the general preference for targeted killing of security personnel as outline above. turning now to counterterrorism effects (hypotheses 5 and 6), there are strong effects from the nature of british counterterrorism efforts. the data set contains two counterterrorism variables : a count of pira members killed by british forces (killing by ct, pira members (ct)) and a count of catholic civilian noncombatants killed by british forces (killings by ct, civilians (ct)). however, there is one twist from the zero - inflation component of the model : british killing of pira members tended to increase the probability that a brigade would kill a nonzero number of civilians in that period. thus, there is a two - step explanation : killing pira members reduced effectiveness and tended to reduce total killings (per hypothesis 6), but it made it more likely that a given brigade would lash out in retaliation that would end up killing civilians, which is more in line with the backlash models of benmelech, berrebi, and klor (2010) and lafree, dugan, and korte (2009). a topic for future research is to disentangle whether civilians were killed as a backlash or because the counterterrorism killings degraded the quality of pira members who were left, making the overall organization unable to carry out discriminate violence. killing of civilians by counterterrorism forces killings we term indiscriminate had a quite different effect : it tended to increase total fatalities, civilian fatalities, and fatalities from shootings, as anticipated in hypothesis 5. the implication from this finding is that killing of civilians by british counterterrorism authorities seemed to engender a more violent, less targeted response. killing of civilians tended to make it more likely that a brigade would kill no one during a given brigade year. so, we have contradictory results : british killing of civilians made it more likely that a brigade would kill no civilians during a given year, but if the brigade did kill civilians, it was likely to kill quite a few. drawing together these findings, this suggests that brigades may have acted like capacitors : british killing of civilians would generate a desire to retaliate that was held in check until a level of outrage was reached that triggered a response. once a response was triggered, it tended to be less planned and targeted than was the general norm for brigade activity. concurrent discriminate killing of pira members could have exacerbated the tendency toward civilian killings in response by degrading the quality of members left such that a discriminate, security - focused response became less possible. the zero - inflation results hint that brigades may have avoided tit - for - tat killing of civilians, possibly because there was a fear of triggering a cycle of violence. but the count component clearly indicates that civilian killings eventually sparked a more general, indiscriminate round of violence.. often this process begins with underlying systemic causes and is often sustained and driven by more proximate counterterrorism initiatives that feed the recruitment and mobilization of new cadre. within the cadre itself, individuals with technical expertise may facilitate particular forms of violence that the group could not engage in absence of this individual. the culmination of a terrorist attack is often preceded by tactical decisions such as who should be targeted, what methods should be used and in the case of an ied attack, what ied type and initiation system would be most effective. all of these processes occur within groups and subgroups of varying sizes and we illustrated that variations across these processes impact a terrorist organization s deadliness. while the scale and intensity of terrorist campaigns is often related in the literature to distal factors such as rates of poverty, unemployment, and democracy indicators, there remains a distinct lack of awareness that not all violence is equal in terms of the technical proficiency and psychological conditioning needed. similarly, not all ieds are equal, and some aspects may drive lethality indicators up or down dependent upon the prevailing strategic orientation of the terrorist group itself. in other words, existing studies tend to aggregate terrorist organizations as collective wholes and treat violent methods and fatality types homogeneously. our findings suggest that (1) rates of fatal attacks differ depending upon the type of oppressive counterterrorism policies employed, (2) fatality rates differ intraorganizationally, and finally (3) subunit variables such as membership age, professional technical expertise, and brigade size also affect lethality rates but in different ways dependent upon the type of violence, who is targeted, and the strategic choices the organization makes corporately. there are caveats to these findings, not the least of which is that our measures of ct activity are rather aggregated and can not fully capture intelligence interventions that reduce the number of attacks brought to fruition, thus also reducing the total fatalities. however, this issue does not affect the finding that killing of civilians by counterterrorism forces leads to increased violence if anything, our findings underestimate the increase in fatalities after ct forces kill civilians. together, the results may help inform both policy and operational decision making. at the policy level, the results show the negative impact likely to occur when nonaligned civilians are killed in the course of counterterrorism operations. such events embolden terrorist organizations to strike back, usually in an equally indiscriminate fashion. even more so, the results suggest that when organizations do decide to strike back in retaliation, it is likely to be at the local level, a finding that braithwaite and johnson (2012) and linke, witmer, and oloughlin (2012) also found in relation to insurgent activities in iraq. although the targeted killing of pira members reduces a brigade s ability to kill within a given year, there is no data available to suggest whether such events also lead to an increase in constituency support, and future recruits for the organization that may later breathe life into a depleted subunit. moreover, targeted killings may even have the perverse effect of making civilian casualties more likely if the net result is to reduce the skill level of the unit (though further research is needed to confirm this inference). the results also suggest that although pira can be described as a coherent, hierarchical organization, the variance among its subunits in terms of composition, capabilities, and targeting policies requires nuanced counterterrorism policies policies that are tailored to the local subunit s capacity for lethal activities. while there has been much theorizing about the structure of terrorist organizations, our results indicate that perhaps we should be more concerned with the composition and qualities inherent within a network of subunits. those subunits with a higher number of individuals with professional skills relevant to bomb making more often caused casualties, and those casualties are more likely to be among high - value targets rather than civilians. such findings may aid in decisions concerning what segments of a terrorist network should be prioritized for immediate postevent investigation and intelligence gathering. finally, we recognize that findings are not directly generalizable : we focused here upon one terrorist organization. generalizations to other groups may be difficult to make. that said, we believe the logic behind the relationship between counterterrorism killings and the lethality of units and subunits may apply more broadly. killing those whom the terrorist organization claims to represent is likely to encourage retaliation in the form of indiscriminate violence against civilians for a number of reasons elaborated upon above, whereas killing members of the group is likely to lead (at least in the short run) to reductions in the group s capacity to striking back. in terms of subunit composition, generalizations are more difficult. higher levels of skilled bomb makers within pira brigades led to both a higher likelihood of high - value targets being killed and civilians being spared both of which were long - standing strategic policies of pira. not every terrorist organization resembles pira in this regard, with many being far less reluctant to kill civilians. we might therefore expect that the more technical expertise within any given subunit in any terrorist organization, the closer that subunits fatality rates will mirror the organization s strategic logic as a whole.	this paper presents an analysis of the provisional irish republican army 's (pira) brigade level behavior during the northern ireland conflict (1970 - 1998) and identifies the organizational factors that impact a brigade 's lethality as measured via terrorist attacks. key independent variables include levels of technical expertise, cadre age, counter - terrorism policies experienced, brigade size, and ied components and delivery methods. we find that technical expertise within a brigade allows for careful ied usage, which significantly minimizes civilian casualties (a specific strategic goal of pira) while increasing the ability to kill more high value targets with ieds. lethal counter - terrorism events also significantly affect a brigade 's likelihood of killing both civilians and high - value targets but in different ways. killing pira members significantly decreases ied fatalities but also significantly decreases the possibility of zero civilian ied - related deaths in a given year. killing innocent catholics in a brigade 's county significantly increases total and civilian ied fatalities. together the results suggest the necessity to analyze dynamic situational variables that impact terrorist group behavior at the sub - unit level.
overdenture is defined as a removable partial or complete denture that covers and rests on one root of natural teeth and/or dental implants. aprosthesis that covers and is partially supported by natural teeth, natural tooth roots and or dental implant is called also overlay denture, overlay prosthesis, and superimposed prosthesis.1 overdentures supported by implants were the development of well - researched implant systems, providing a predictable success rate that made such restoration feasible. recently, new one - piece implant design has been fabricated and consequently the implant surgical technique has been changed into one - stage technique with its benefits for the patients and either immediate or progressive loading protocols were used for prosthetic appliances.2345 it is conceivable that implant materials, which are chosen because of their friendliness to tissue cells, offer particularly favorable grounds for bacterial adhesion and availability of cell - friendly adhesion - mediated infections developing on implanted biomaterials respond poorly to antimicrobial treatment and often require that the device be removed.6 dental implants, however, can be designed in such a way that the surfaces on which bacterial colonization occurs may be reached from the exterior. thus, in contrast to internal implants, there is a possibility for control of bacterial colonization on exposed surfaces and a potential for treatment of infectious conditions of peri - implant tissues.67 in a study, it was found that the microflora associated with stable osseointegrated implants serving successfully as abutments for overdentures was investigated, and 50% of the organisms cultured were facultatively anaerobic cocci, staphylococci, and the rest were facultatively anaerobic rods. separate samples taken within the same patient from different sites showed a similar composition of the microflora.89 the role of microorganisms in the development of peri - implant pathology has also been investigated in animals. differences in the presence of putative periodontal pathogens on titanium implants and teeth were determined in animals in experimental gingivitis and in peri - implantitis / periodontitis situation. ten completely edentulous male patients with age ranged from 50 to 60 years were selected from the outpatient clinic, prosthodontics department, faculty of dentistry october 6 university. the patients after preparation to receive an overdenture were randomly divided into two equal groups according to implant designs ; 1 group and the patients in this group received mandibular overdenture supported by two - piece implant system using delayed loading prosthetic technique. the 2 group and patients in this group received mandibular overdenture supported by one - piece implant system. surgical stent constructed using transparent acrylic resin on duplicated study castthreaded endosseous implant system (titanium plasma spray) coating root form with 13 mm length and 3.9 mm diameter were used for all patientsthe implant was inserted manually and observing the correct insertion angle [figure 1]4 months after implant placement, all patients were recalled. for group 1, the abutments were inserted in place and attached to the fixture using abutment screw, and for group 2, the patients were ready for prosthetic procedures. surgical stent constructed using transparent acrylic resin on duplicated study cast threaded endosseous implant system (titanium plasma spray) coating root form with 13 mm length and 3.9 mm diameter were used for all patients the implant was inserted manually and observing the correct insertion angle [figure 1 ] 4 months after implant placement, all patients were recalled. for group 1, the abutments were inserted in place and attached to the fixture using abutment screw, and for group 2, the patients were ready for prosthetic procedures. upper and lower primary impressions were made for all patients followed by a secondary impression that was made for the lower arch in the preconstructed special tray after proper border moldingcentric occluding relation following the interocclusal wax wafer technique was made and a try in stage was made successfullythe denture was processed, laboratory remounted, finished, polished, and delivered to the patient in the usual manner after clinical remounting [figure 2 ]. upper and lower primary impressions were made for all patients followed by a secondary impression that was made for the lower arch in the preconstructed special tray after proper border molding centric occluding relation following the interocclusal wax wafer technique was made and a try in stage was made successfully the denture was processed, laboratory remounted, finished, polished, and delivered to the patient in the usual manner after clinical remounting [figure 2 ]. the insertion appointment (considered as zero readings), 6, 12, and 18 months of postinsertion successively considered as follow - up periods. manual insertion of implant final denture in patient mouth a swap sample was taken from area around the implants by means of sterile swap, transferred and immersed in reduced transport media ; all samples were carefully homogenized without aeration into 1 mm of saline and centrifuged for 30 s. one plate from each media was aerobically incubated at 37c.films were prepared from various colonies, stained with gram 's stain, and examinedcertain organisms required special tests as staphylococcus aureus, which was further identified by coagulase test done through the tube methodcatalase production to differentiate between staphylococci and streptococci.12 one plate from each media was aerobically incubated at 37c. films were prepared from various colonies, stained with gram 's stain, and examined certain organisms required special tests as staphylococcus aureus, which was further identified by coagulase test done through the tube method catalase production to differentiate between staphylococci and streptococci.12 visible colonies of each organism were counted in every plate, and the number of colonies - plate was multiplied by the corresponding dilution factor and by 10 to determine the total colony forming units per ml of suspension. surgical stent constructed using transparent acrylic resin on duplicated study castthreaded endosseous implant system (titanium plasma spray) coating root form with 13 mm length and 3.9 mm diameter were used for all patientsthe implant was inserted manually and observing the correct insertion angle [figure 1]4 months after implant placement, all patients were recalled. for group 1, the abutments were inserted in place and attached to the fixture using abutment screw, and for group 2, the patients were ready for prosthetic procedures. surgical stent constructed using transparent acrylic resin on duplicated study cast threaded endosseous implant system (titanium plasma spray) coating root form with 13 mm length and 3.9 mm diameter were used for all patients the implant was inserted manually and observing the correct insertion angle [figure 1 ] 4 months after implant placement, all patients were recalled. for group 1, the abutments were inserted in place and attached to the fixture using abutment screw, and for group 2, the patients were ready for prosthetic procedures. upper and lower primary impressions were made for all patients followed by a secondary impression that was made for the lower arch in the preconstructed special tray after proper border moldingcentric occluding relation following the interocclusal wax wafer technique was made and a try in stage was made successfullythe denture was processed, laboratory remounted, finished, polished, and delivered to the patient in the usual manner after clinical remounting [figure 2 ]. upper and lower primary impressions were made for all patients followed by a secondary impression that was made for the lower arch in the preconstructed special tray after proper border molding centric occluding relation following the interocclusal wax wafer technique was made and a try in stage was made successfully the denture was processed, laboratory remounted, finished, polished, and delivered to the patient in the usual manner after clinical remounting [figure 2 ]. the insertion appointment (considered as zero readings), 6, 12, and 18 months of postinsertion successively considered as follow - up periods. manual insertion of implant final denture in patient mouth a swap sample was taken from area around the implants by means of sterile swap, transferred and immersed in reduced transport media ; all samples were carefully homogenized without aeration into 1 mm of saline and centrifuged for 30 s. one plate from each media was aerobically incubated at 37c.films were prepared from various colonies, stained with gram 's stain, and examinedcertain organisms required special tests as staphylococcus aureus, which was further identified by coagulase test done through the tube methodcatalase production to differentiate between staphylococci and streptococci.12 one plate from each media was aerobically incubated at 37c. films were prepared from various colonies, stained with gram 's stain, and examined certain organisms required special tests as staphylococcus aureus, which was further identified by coagulase test done through the tube method catalase production to differentiate between staphylococci and streptococci.12 visible colonies of each organism were counted in every plate, and the number of colonies - plate was multiplied by the corresponding dilution factor and by 10 to determine the total colony forming units per ml of suspension. comparison between group 1 (two - piece implants) and group 2 (one - piece implants) regarding staphylococcal bacteria : at 6, 12, and 18 months, the mean values of staphylococcus count around the implants revealed significant difference revealed between the two studied groups [graph 1 ]. at 6, 12, and 18 months, the mean values of staphylococcus count around the implants revealed significant difference revealed between the two studied groups [graph 1 ]. at 6, 12, and 18 months, the mean values of staphylococcus count around the implants revealed significant difference revealed between the two studied groups [graph 1 ]. using the sterile swap in taking specimen from the area around the implant abutment would help in direct spreading the specimens on the culture plates, and the biochemical reactions were carried out to differentiate the microscopically resembled microorganisms. films were stained with gram stain to see the morphology of the organism and its reaction to gram 's stain. catalase production to differentiate between staphylococci and streptococci.12 regarding group1, the microgap, which is the most effective factor for bacterial colonization at this critical area around implant, provides a good media for inflammation around implant neck and subsequently leads to bone resorption, and regarding group2(one - piece implant), the design eliminates the microgap and that decrease the possibility for bacterial colonization.13 despite strict oral hygiene measures and patient instructions about proper cleansing of the prosthesis and implant neck area along the 18 months follow - up period, there is tiny unreachable areas, especially in the microgap area at fixture - abutment connection, regarding group 1 that area can not be reached by the patient especially the subgingival portion of implant neck ; that subsequently increase the possibility for bacterial colonization and that may explain significant difference in bacterial count around the implants between the two groups as regarding the four main types of bacteria that had been examined in the study (staphylococci, streptococci, l. bacilli, anaerobes).1415 within the limitation of the results of this study, it could be concluded that the complete mandibular overdentures supported by two osseointegrated one - piece implant design showed better effect on bacteriological changes around the implants abutments when compared with two - piece implant design. october sixth university, egypt - future university, egypt - al - farabi colleges, ksa. october sixth university, egypt - future university, egypt - al - farabi colleges, ksa.	background : this study evaluated and compared the bacteriological effect of two - piece implants and one - piece implants in complete overdenture cases on supporting structures.materials and methods : ten male completely edentulous patients were selected and randomly divided into two equal groups according to the implant design and surgical technique for this study ; group 1 : patients were rehabilitated with complete mandibular overdenture supported by two - piece implants one on each side of the lower arch following two - stage surgical technique and group 2 : patients were rehabilitated with complete mandibular overdenture supported by one - piece implants one on each side. evaluation was made at the time of insertion, 6, 12, and 18 months after overdenture insertion, by measuring bacteriological changes around implants abutments.results:complete overdenture supported by one - piece implants showed better effect on the bacteriological changes as compared to that supported by two - piece implants.conclusion:complete overdenture supported by one - piece implants one on each side of the lower arch showed better effect on the bacteriological changes than using the same prosthesis supported by two - piece implants.
	matr3 is an rna / dna binding protein that interacts with tdp-43, a major disease protein linked to amyotrophic lateral sclerosis (als) and fronto - temporal dementia. using exome sequencing, we identified mutations in matr3 in als kindreds. we also observed matr3 pathology in the spinal cords of als cases with and without matr3 mutations. our data provide additional evidence supporting the role of aberrant rna processing in motor neuron degeneration.
patients who underwent curative surgery for hnscc at inha university hospital between january 1996 and december 2011 were selected for this study. the primary sites of the tumors were the oral cavity, oropharynx, hypopharynx, and larynx. the patients ' clinical and pathological characteristics regarding age, sex, smoking history, alcohol consumption, histologic types, pathologic tnm staging, relapse - free survival, and overall survival were obtained by a review of medical records. thus, a total of 118 patients were eligible, according to the following criteria : histology of squamous cell carcinoma and the availability of hematoxylin and eosin - stained glass slides and paraffin blocks for construction of a tissue microarray (tma). however, the smoking history of eight patients and the status of lymph node metastasis in one patient were not available. this study protocol was approved by the ethics committee (institutional review board) of inha university hospital. we obtained formalin - fixed paraffin - embedded tissues of 118 patients for this study. the criteria for defining the representative area were as follows : 1) invasive front of the tumor and 2) high percentage of tumor cells compared to surrounding stromal cells. to create tmas, we punched two tissue columns (2.0 mm in diameter) from each original paraffin block and inserted them into the recipient paraffin blocks (each containing 30 to 69 holes). the sections were processed in an automated machine (benchmarkxt, ventana medical systems, tucson, az, usa) for deparaffinization and then re - hydrated through graded alcohol. epitope retrieval was performed by heating for 30 minutes and then incubating the slides for 32 minutes (37) with monoclonal antibody, followed by an incubation with a visualization reagent., san francisco, ca, usa), anti - vimentin (1:300, dako, carpinteria, ca, usa), and anti - ezh2 (1:400, novocastra, bannockburn, il, usa) were stained by the same method. additionally, anti - p16 (bd transduction laboratories, bd biosciences, mtm laboratories ag, heidelberg, germany) antibody was used to stain only the group with oropharynx cancer. for the evaluation of the expression of e - cadherin and vimentin, e - cadherin with membranous staining was classified into three categories6 : 1) strong (s) pattern : almost all tumor cells showed diffuse patterns and strong positive staining in the membrane ; 2) weak and homogeneous (w&h) pattern : tumor cells were uniformly, but more weakly stained ; 3) heterogeneous (heg) pattern : tumor cells showed focal staining with variable intensity. w&h and heg patterns were considered to represent a loss of e - cadherin expression. vimentin expression was interpreted as positive when cytoplasmic staining was observed, even in a small portion of the tumor cells (at least 5%) at the invasive front. however, the tumor cells in the basal layer were excluded in the interpretation, because they were frequently positive for vimentin in almost all cases. the nuclear staining of ezh2 was evaluated semi - quantitatively on the basis of staining intensity and distribution using the immunoreactive score13,14,17 : immunoreactive score = intensity scoreproportion score. the intensity score was defined as follows-0, negative ; 1, weak ; 2, moderate ; or 3, strong, and the proportion score was defined as 0, negative ; 1, 80% positive cells. the proportion of the immunoreactive tumor cells was estimated in one high - power field (400) of the hot spot. low expression of ezh2 was defined as a total score of 0 to 4, and high expression was defined as a total score > 4. the p16-positive cases showed diffuse and strong nuclear expression in all cases, and the other cases were negative for p16 without any ambiguous cases. a pearson 's chi - squared test and independent - sample t - test were used to determine the statistical significance of differences between the positive and negative immunoreactive groups for e - cadherin, vimentin, and ezh2 of hnscc in terms of sex, age, smoking history, primary tumor site, histological differentiation, tumor stage, resection margin status, node metastasis, recurrence, and survival rate. the overall survival was determined by measuring the time interval from the beginning of the treatment to the date of death. we censored the patients who were alive or were lost during the follow - up in the data analysis., chicago, il, usa) and p - values less than.05 were considered statistically significant. patients who underwent curative surgery for hnscc at inha university hospital between january 1996 and december 2011 were selected for this study. the primary sites of the tumors were the oral cavity, oropharynx, hypopharynx, and larynx. the patients ' clinical and pathological characteristics regarding age, sex, smoking history, alcohol consumption, histologic types, pathologic tnm staging, relapse - free survival, and overall survival were obtained by a review of medical records. thus, a total of 118 patients were eligible, according to the following criteria : histology of squamous cell carcinoma and the availability of hematoxylin and eosin - stained glass slides and paraffin blocks for construction of a tissue microarray (tma). however, the smoking history of eight patients and the status of lymph node metastasis in one patient were not available. this study protocol was approved by the ethics committee (institutional review board) of inha university hospital. we obtained formalin - fixed paraffin - embedded tissues of 118 patients for this study. the criteria for defining the representative area were as follows : 1) invasive front of the tumor and 2) high percentage of tumor cells compared to surrounding stromal cells. to create tmas, we punched two tissue columns (2.0 mm in diameter) from each original paraffin block and inserted them into the recipient paraffin blocks (each containing 30 to 69 holes). the sections were processed in an automated machine (benchmarkxt, ventana medical systems, tucson, az, usa) for deparaffinization and then re - hydrated through graded alcohol. epitope retrieval was performed by heating for 30 minutes and then incubating the slides for 32 minutes (37) with monoclonal antibody, followed by an incubation with a visualization reagent., san francisco, ca, usa), anti - vimentin (1:300, dako, carpinteria, ca, usa), and anti - ezh2 (1:400, novocastra, bannockburn, il, usa) were stained by the same method. additionally, anti - p16 (bd transduction laboratories, bd biosciences, mtm laboratories ag, heidelberg, germany) antibody was used to stain only the group with oropharynx cancer. for the evaluation of the expression of e - cadherin and vimentin, the proportion of positive tumor cells was visually estimated in two total cores. e - cadherin with membranous staining was classified into three categories6 : 1) strong (s) pattern : almost all tumor cells showed diffuse patterns and strong positive staining in the membrane ; 2) weak and homogeneous (w&h) pattern : tumor cells were uniformly, but more weakly stained ; 3) heterogeneous (heg) pattern : tumor cells showed focal staining with variable intensity. w&h and vimentin expression was interpreted as positive when cytoplasmic staining was observed, even in a small portion of the tumor cells (at least 5%) at the invasive front. however, the tumor cells in the basal layer were excluded in the interpretation, because they were frequently positive for vimentin in almost all cases. the nuclear staining of ezh2 was evaluated semi - quantitatively on the basis of staining intensity and distribution using the immunoreactive score13,14,17 : immunoreactive score = intensity scoreproportion score. the intensity score was defined as follows-0, negative ; 1, weak ; 2, moderate ; or 3, strong, and the proportion score was defined as 0, negative ; 1, 80% positive cells. the proportion of the immunoreactive tumor cells was estimated in one high - power field (400) of the hot spot. low expression of ezh2 was defined as a total score of 0 to 4, and high expression was defined as a total score > 4. the p16-positive cases showed diffuse and strong nuclear expression in all cases, and the other cases were negative for p16 without any ambiguous cases. a pearson 's chi - squared test and independent - sample t - test were used to determine the statistical significance of differences between the positive and negative immunoreactive groups for e - cadherin, vimentin, and ezh2 of hnscc in terms of sex, age, smoking history, primary tumor site, histological differentiation, tumor stage, resection margin status, node metastasis, recurrence, and survival rate. the overall survival was determined by measuring the time interval from the beginning of the treatment to the date of death. we censored the patients who were alive or were lost during the follow - up in the data analysis. all statistical analyses were conducted using statistical software pasw statistics ver. 18.0 (spss inc., chicago, il, usa) and p - values less than.05 were considered statistically significant. we analyzed 118 patients (101 men [85.6% ] and 17 women [14.4% ]), with a median age of 58 years (range, 27 to 94 years). the tumors were located in the oral cavity (33.9%, 40 cases), oropharynx (18.6%, 22 cases), hypopharynx (19.5%, 23 cases), and larynx (28.0%, 33 cases). eighty - six cases (72.9%) exhibited strong and homogenous membranous e - cadherin expression. the loss of e - cadherin was found in 32 cases (27.1%) (fig. the expression of vimentin was frequently observed in tumor cells of the invasive front, especially abutting adjacent stroma. twenty - nine cases (24.6%) exhibited focal or diffuse cytoplasmic immmunoreactivity for vimentin (fig. 1e - h). according to the immunoreactive score, high expression of ezh2 was observed in 29 cases (24.6%) (fig. 1i - l). expression of p16 was observed in 14 of 22 cases (63.6%) of oropharyngeal cancer. the results of immunohistochemical staining and its association with clinicopathological parameters are summarized in table 1. the e - cadherin - negative group showed more moderately / poorly differentiated cell types than the higher e - cadherin - expressing group (62.8% vs 87.5%, p=.016). high ezh2 expression was significantly correlated with nodal metastasis (p=.012). in the subgroup composed of only oral cavity tumors, the expression of vimentin was associated with a higher tumor stage (t stage [vimentin negative / positive ] ; t1 (10/5), t2 (14/5), t3 (2/0), t4 (0/4), p=.027). the association between vimentin, e - cadherin, ezh2 expression and clinicohistologic parameters with survival rate was evaluated by cox proportional hazard model. comparing the clinicohistologic parameters, pathologic tumor stage (odds ratio, 2.541 ; p<.001), pathologic nodal stage (odds ratio, 2.043 ; p=.009), tmn stage (odds ratio, 2.233 ; p=.006), extracapsular extension (odds ratio, 1.982 ; p=.045), and margin status (odds ratio, 2.956 ; p<.001) were shown to be significantly associated with survival rate. no significant differences were found for sex, alcohol consumption, smoking history, and histologic differentiation of tumors. furthermore, the expression of e - cadherin, vimentin, and ezh2 was not significantly associated with overall survival. however, in an analysis according to primary tumor site subgroups, the loss of e - cadherin was associated with lower overall survival in oropharyngeal and hypopharyngeal tumors (p=.001 and p=.038, respectively) (fig. the recurrence rate was significantly higher than that in the e - cadherin - negative group (loss, 2/4 [50% ] ; e - cadherin - expressing group, 1/18 [5.6% ] ; p=.019). twenty - nine cases of all 118 hnscc showed overexpression of ezh2, especially in the oropharynx tumor group (41.4%, p=.002). hpv infection is a well - known biomarker in oropharyngeal squamous cell carcinoma. in our study, expression of p16, a well - known surrogate marker in oropharyngeal cancer, was found in 14 of 22 cases of oropharynx cancer. in these patients, the rate of ezh2 expression according to p16 status was not statistically different (p=.225). in contrast, three of eight cases (37.5%) showed recurrence in the p16-negative group. oropharyngeal squamous cell carcinoma patients who had a smoking history showed more frequent, but not statistically significant differences in, ezh2 expression compared patients who had never smoked (72.7% vs 36.3%, p=.094). the results of immunohistochemical staining and its association with clinicopathological parameters are summarized in table 1. the e - cadherin - negative group showed more moderately / poorly differentiated cell types than the higher e - cadherin - expressing group (62.8% vs 87.5%, p=.016). high ezh2 expression was significantly correlated with nodal metastasis (p=.012). in the subgroup composed of only oral cavity tumors, the expression of vimentin was associated with a higher tumor stage (t stage [vimentin negative / positive ] ; t1 (10/5), t2 (14/5), t3 (2/0), t4 (0/4), p=.027). the association between vimentin, e - cadherin, ezh2 expression and clinicohistologic parameters with survival rate was evaluated by cox proportional hazard model. comparing the clinicohistologic parameters, pathologic tumor stage (odds ratio, 2.541 ; p<.001), pathologic nodal stage (odds ratio, 2.043 ; p=.009), tmn stage (odds ratio, 2.233 ; p=.006), extracapsular extension (odds ratio, 1.982 ; p=.045), and margin status (odds ratio, 2.956 ; p<.001) were shown to be significantly associated with survival rate. no significant differences were found for sex, alcohol consumption, smoking history, and histologic differentiation of tumors. furthermore, the expression of e - cadherin, vimentin, and ezh2 was not significantly associated with overall survival. however, in an analysis according to primary tumor site subgroups, the loss of e - cadherin was associated with lower overall survival in oropharyngeal and hypopharyngeal tumors (p=.001 and p=.038, respectively) (fig. in the oropharynx tumor group, the recurrence rate was significantly higher than that in the e - cadherin - negative group (loss, 2/4 [50% ] ; e - cadherin - expressing group, 1/18 [5.6% ] ; p=.019). twenty - nine cases of all 118 hnscc showed overexpression of ezh2, especially in the oropharynx tumor group (41.4%, p=.002). hpv infection is a well - known biomarker in oropharyngeal squamous cell carcinoma. in our study, expression of p16, a well - known surrogate marker in oropharyngeal cancer, was found in 14 of 22 cases of oropharynx cancer. in these patients, the rate of ezh2 expression according to p16 status was not statistically different (p=.225). in contrast, three of eight cases (37.5%) showed recurrence in the p16-negative group. oropharyngeal squamous cell carcinoma patients who had a smoking history showed more frequent, but not statistically significant differences in, ezh2 expression compared patients who had never smoked (72.7% vs 36.3%, p=.094). in this study, we found that the emt - associated protein expression profile was a strong prognostic marker for the entire hnscc spectrum. loss of e - cadherin expression is significantly associated with recurrence rate in oropharyngeal tumors, as well as overall survival in oropharyngeal and hypopharyngeal tumors. our study also suggests that this protein expression profile analysis may be helpful to identify patients at high risk of developing distant metastasis in early stage node - negative hnscc patients. e - cadherin is a key molecule involved in the maintenance of intracellular adhesion, and down - regulation of e - cadherin is associated with tumor progression in diverse human cancer types.18 there have been several reports regarding the inverse correlation between ezh2 and e - cadherin expression in cancer cells. however, the exact underlying mechanism by which ezh2 causes a poor prognosis is not known and further analyses are necessary to elucidate how ezh2 regulates e - cadherin expression. in our study, each of the factors was found to be associated with a pattern of metastasis and prognosis. however, we could not confirm a significant correlation between ezh2/vimentin expression and the recurrence rate or overall survival. there was a trend of a lower distant metastasis rate in patients with lower ezh2 expression compared to patients with high e - cadherin expression. the significance of e - cadherin expression as a predictive factor in metastatic spread is not clear. there is in vivo and in vitro data demonstrating that ezh2 plays a crucial role in several steps of the metastatic process and that it is activated in endothelial cells in response to pro - angiogenic signals.19 in our study, we found that there was a statistically significant difference in metastatic pattern according to mesenchymal markers expression. given these hypotheses, our results suggest that mesenchymal markers could be very important biomarkers of distant metastasis in hnscc. thus far, there is no conclusive proof that the markers related to progression are directly correlated with prognosis in hnscc. a recent tma study of e - cadherin expression in oropharyngeal squamous cell carcinoma20 failed to show a significant correlation between the expression of e - cadherin and histologic type, nodal and distant metastasis, suggesting that e - cadherin expression may not be a predictor of nodal or distant metastasis in these tumors. the inconsistent results in several studies are limit the use of these markers to predict patient outcome. we think that the differences in methods for analyzing immunohistochemistry may be an important factor affecting the results. our analysis included all types of hnscc and none of the subtypes had enough cases. a large cohort study is necessary to confirm the significance of these markers. in conclusion, emt - associated protein expression is related to aggressive pathological features, even in early stage hnscc. therefore, emt - associated proteins could be useful markers for determination of additional treatment (e.g., adjuvant chemotherapy and/or radiotherapy) in curatively - resected hnscc patients in the future.	backgroundepithelial mesenchymal transition (emt) has an important role in invasion and metastasis of tumor cells. the purpose of this study was to evaluate the roles of emt - associated proteins on progression and metastasis as a prognostic / predictive factor in curatively - resected (r0) head and neck squamous cell carcinoma (hnscc).methodsa total of 118 patients who received curative surgery for hnscc at inha university hospital between january 1996 and december 2011 were included. we used protein immunohistochemistry to evaluate the expression of e - cadherin, vimentin, and ezh2 on tissue microarrays. also, we reviewed all medical records and analyzed the relationship between the expression of emt - associated proteins and prognosis.resultsthe e - cadherin - negative group showed more moderate / poor differentiation of cancer cell type than the higher e - cadherin - expressing group (p=.016) and high ezh2 expression was significantly correlated with nodal metastasis (p=.012). our results demonstrate a significant association between high expression of ezh2 and vimentin and presence of distant progression (p=.026). however, expression of e - cadherin, vimentin, and ezh2 was not significantly associated with overall survival.conclusionsthese findings suggest that an emt - associated protein expression profile is correlated with aggressiveness of disease and prognosis, and could be a useful marker for determination of additional treatment in curatively - resected hnscc patients.
the maxillomandibular relationship (mmr) record is a critical step to establish the new occlusion in implant supported complete mouth rehabilitation. using patients existing denture for recording the mmr requires implant definitive cast to be modified extensively to completely seat the denture (with unaltered flanges) on it. this may influence the correct seating of the denture on the implant definitive cast causing faulty recording of the mmr. elastomeric record bases, reinforced with the resin framework, are fabricated and relined with the light body elastomeric material when all the healing abutments are in place. the mmr is recorded with these elastomeric record bases using vacuum formed facial surface index of the occluded existing dentures as a guideline. the elastomeric record bases with facial surface index of the existing dentures can allow clinicians to record mmr records without removing the healing abutments from the mouth with acceptable accuracy. the record of facial surfaces of existing complete denture in the form of vacuum formed sheet helps to set the occlusal vertical dimension. use of facial surface index together with the elastomeric record bases can be the useful alternative technique to record the mmr in patients with implant supported full mouth rehabilitation. conventional complete denture principles are frequently used to restore edentulous patients receiving implants for complete mouth rehabilitation. following the reliable osseointegration is achieved ; the sequential clinical steps including elastomeric impressions, maxillomandibular relationship (mmr) records, mounting of the mmr records onto the articulators, fabrication and try - in of the implant fixed restoration framework, verification of mmr records by taking another records, veneering of the metal framework with the ceramics or indirect composites are well - described in various clinical reports in the literature. all these routine procedures have been carried out with few modifications and alternate techniques. using patients existing denture for recording however, the implant definitive cast needed to be modified extensively to completely seat the denture on it (without altering the flanges). the mmr record is a critical step to establish the new occlusion in such situations. however, these procedures are time consuming due to repeated removal and replacement of healing / prosthetic abutments, impression copings or similar components during the clinical appointments. using patients existing denture for recording the mmr requires implant definitive cast to be modified extensively to completely seat the denture (with unaltered flanges) on it. this may influence the correct seating of the denture on the implant definitive cast causing faulty recording of the mmr. to overcome this problem, the elastomeric record bases, reinforced with the resin framework, are fabricated on both the arches and mmr is recorded with the vacuum formed index adapted on the facial surfaces of the occluded dentures. at the time of stage ii surgery (uncovering), place titanium healing abutments (adin implants, israel) of appropriate heights in the patient 's mouth to prevent the soft tissue from closing over the implantwhen the surgical site is completely healed, modify the intaglio surface of an existing denture at healing abutment level by selective grinding and addition of the soft (sofreliner tough ; tokuyama dental, tokyo, japan) or hard denture reline material (tokuyama rebase ii ; tokuyama dental, tokyo, japan). note that, alternately the denture can be remade at this stageconfirm the correct mmr records by keeping the dentures in mouth and fix them with the help of sticky wax (national keystone products, cherry hill, nj, usa). once the fixed denture removed outside the mouth, the secure the dentures together with the help of the instant sticking glue (fevi quick, pedilite, australia) in minimum three different contacting areas of the denturesplace the joined dentures on the vacuum former machine and adapt 2 mm thick vacuum formed polyethylene sheet (tray - vac complete, buffalo dental manufacturing, ny, usa) to cover the entire facial polished surfaces of both the dentures (secured together with the instant sticking glue) to make a facial surface index (fsi) [figure 1 ] remove the fsi from the dentures and cut it along the flanges of the dentures to make it look like a mouth guard [figure 2 ] remove the healing abutments (adin implants, israel) and replace them with the impression copings. make an impression with open tray techniqueconnect the implant lab analogs (adin implants, israel) to the impression copings. pour a soft tissue simulating material (gi - mask, coltene / whaledent inc., mahwah, nj, usa) and type iv gypsum (ultra - rock, kalabhaikarson, mumbai, maharashtra, and india) to make a definitive castseat all the healing abutments on the implant definitive casts on the respective implants [figure 3 ]. prepare a horse - shoe shaped resin framework on the definitive cast just enough to cover the areas of all the healing abutments [figure 4 ]. apply tray adhesive and the silicone putty (zetaplus soft, zhermack, badia polesine, italy) to the framework and make an impression of abutments by keeping minimum flange extensions on facial and lingual side [figure 5 ]. note that, alternately light - body impression material can be applied on the healing abutments while making the putty impression (single step putty - wash impression technique)repeat step 8 to make similar putty record base of the opposite arch. note that a polyvinyl - siloxane or polyether material can be used instead of condensation silicone to prepare the record basesseat the maxillary and mandibular silicone putty record bases in mouth. add or remove putty impression material on the elastomeric record bases to adjust the vertical relation. every time during addition of a new material to either of the record base, apply separating media like vaseline petroleum jelly to the opposite record base to prevent sticking them with each other. thus repeated addition and removal the putty material and the use the fsi (seated along the labial and buccal vestibules) help recording the desirable vertical relation (closely matching with the denture vertical dimensions) [figure 6 ] record and verify the centric relation in conventional mannerapply the tray adhesive and silicone putty on the inner aspect of the fsi. apply impression putty to the inner aspect of the fsi and carry it inside the mouth to seal the already established mmr records as shown in figure 7after setting of the silicone putty, remove the entire block of elastomeric mmr record [figure 8 ] cut the flange extensions of the elastomeric mmr record block with a cutter and examine for the proper seating of the implant definitive casts on respective record bases [figure 9 ] mount the maxillary casts on the semi - adjustable articulator and proceed with fabrication of the metal / ceramic / metal - ceramic restorations. at the time of stage ii surgery (uncovering), place titanium healing abutments (adin implants, israel) of appropriate heights in the patient 's mouth to prevent the soft tissue from closing over the implant when the surgical site is completely healed, modify the intaglio surface of an existing denture at healing abutment level by selective grinding and addition of the soft (sofreliner tough ; tokuyama dental, tokyo, japan) or hard denture reline material (tokuyama rebase ii ; tokuyama dental, tokyo, japan). note that, alternately the denture can be remade at this stage confirm the correct mmr records by keeping the dentures in mouth and fix them with the help of sticky wax (national keystone products, cherry hill, nj, usa). once the fixed denture removed outside the mouth, the secure the dentures together with the help of the instant sticking glue (fevi quick, pedilite, australia) in minimum three different contacting areas of the dentures place the joined dentures on the vacuum former machine and adapt 2 mm thick vacuum formed polyethylene sheet (tray - vac complete, buffalo dental manufacturing, ny, usa) to cover the entire facial polished surfaces of both the dentures (secured together with the instant sticking glue) to make a facial surface index (fsi) [figure 1 ] remove the fsi from the dentures and cut it along the flanges of the dentures to make it look like a mouth guard [figure 2 ] remove the healing abutments (adin implants, israel) and replace them with the impression copings. make an impression with open tray technique connect the implant lab analogs (adin implants, israel) to the impression copings. pour a soft tissue simulating material (gi - mask, coltene / whaledent inc., mahwah, nj, usa) and type iv gypsum (ultra - rock, kalabhaikarson, mumbai, maharashtra, and india) to make a definitive cast seat all the healing abutments on the implant definitive casts on the respective implants [figure 3 ]. prepare a horse - shoe shaped resin framework on the definitive cast just enough to cover the areas of all the healing abutments [figure 4 ]. apply tray adhesive and the silicone putty (zetaplus soft, zhermack, badia polesine, italy) to the framework and make an impression of abutments by keeping minimum flange extensions on facial and lingual side [figure 5 ]. note that, alternately light - body impression material can be applied on the healing abutments while making the putty impression (single step putty - wash impression technique) repeat step 8 to make similar putty record base of the opposite arch. note that a polyvinyl - siloxane or polyether material can be used instead of condensation silicone to prepare the record bases seat the maxillary and mandibular silicone putty record bases in mouth. add or remove putty impression material on the elastomeric record bases to adjust the vertical relation. every time during addition of a new material to either of the record base, apply separating media like vaseline petroleum jelly to the opposite record base to prevent sticking them with each other. thus repeated addition and removal the putty material and the use the fsi (seated along the labial and buccal vestibules) help recording the desirable vertical relation (closely matching with the denture vertical dimensions) [figure 6 ] record and verify the centric relation in conventional manner apply the tray adhesive and silicone putty on the inner aspect of the fsi. apply impression putty to the inner aspect of the fsi and carry it inside the mouth to seal the already established mmr records as shown in figure 7 after setting of the silicone putty, remove the entire block of elastomeric mmr record [figure 8 ] cut the flange extensions of the elastomeric mmr record block with a cutter and examine for the proper seating of the implant definitive casts on respective record bases [figure 9 ] mount the maxillary casts on the semi - adjustable articulator and proceed with fabrication of the metal / ceramic / metal - ceramic restorations. vacuum formed facial surface index of denture master cast with gingival formers in place resin framework for reinforcement of elastomeric record bases resin reinforced elastomeric record bases with minimum extensions recording vertical relation maxillomandibular relationship record sealed completed maxillomandibular relationship record excess flanges of elastomeric maxillomandibular relationship records cut and implant definitive casts seated securing the record base to the implants is a useful way to obtain an accurate mmr registration. there are various methods to reseat the record bases intraorally and on the cast alternatively depending on the prosthetic need, implant systems, ease of procedure, supporting healing / prosthetic abutments. several clinicians have used resin record bases on healing abutments, screwed record bases, relined denture, or duplicating denture in various forms to record the mmr. most of the techniques use methods to secure the record bases to the implants to record the accurate mmr records. these procedures can be time consuming due to removal of the healing abutments and placement of other components for mmr recording and vice versa. however, rungcharassaeng and kan uses light polymerized resin record bases and papaspyridakos and lal used existing dentures at abutment level in which fixation of the record bases to the implant components is not required. described the technique of application of plastic sprues that fit on the top of implants to hold the record bases and allows for easy recording of mmr with an implant - supported record base. usually, the procedures are more time consuming due to screwing / unscrewing of the abutments and complexity of fabrication of the record bases that are fixable to the implants. nimmo and nimmo used two widely spaced implants as the optimal number of implants to stabilize record bases. parnia. described a technique to transfer an ideal vertical dimension and centric relation of the patient 's previous denture to the definitive cast with the help of individual silicone cones. cranin. and misch that described the use of plastic template to capture the final denture contour. in addition, the vacuum formed index was adapted on the facial surfaces of the occluded dentures to prepare the fsi. the fsi copies (1) exact vestibular shape, (2) shape and contour of the facial polished surfaces and (3) the level and ca nt of the occlusal plane of the existing dentures in occlusion. hence, the fsi can be a direct and/or indirect guide for recording the mmr for complex fixed implant restorations. the fsi is useful in initial evaluation and verification of the vertical relation as it copies the relation from the patient 's existing denture. later the same fsi can be used to transport the elastomeric material to seal the already recorded mmr. the direct use of the previous dentures thus can be avoided to record the mmr in such situation. the fsi engages into the vestibules and can not be repeatable at the same position as there are possibilities to close the mandible in slightly different positions each time. the fsi being transparent, the clinician can easily look through it to confirm that the record bases are in correct position before they can be sealed. the elastomeric material can be transported with the help of fsi to seal the record bases in the already recorded and verified (by fsi) position. while sealing the mmr, anterior area of the fsi can be left empty to look through the fsi for better visibility. it is recommended that the record bases should rest on maximum or all the healing / prosthetic abutments to achieve stability to the bases. in atrophic alveolar bone limits the placement of implants as well as contribute to unstable record bases. the resin base frameworks are used to reinforce the elastomeric bases as elastomeric bases alone can not have sufficient strength. this technique recommends to prepare the resin framework to avoid unnecessary flexion / distortion / bending of the elastomeric bases. though this technique provides accurate mmr records for a single case, further study is required to prove its routine clinical utility. the long term success of implant fixed restorations lies in accurately reproduced centric and eccentric occlusal contacts and the accurate occlusion is produced with accurate mmr records. use of fsi together with the elastomeric record bases can be the useful alternative technique to record the mmr in patients with implant supported full mouth rehabilitation. the record of facial surfaces of existing complete denture in the form of vacuum formed sheet can be considered as an initial guideline to set the occlusal vertical dimension.	introduction : the maxillomandibular relationship (mmr) record is a critical step to establish the new occlusion in implant supported complete mouth rehabilitation. using patients existing denture for recording the mmr requires implant definitive cast to be modified extensively to completely seat the denture (with unaltered flanges) on it. this may influence the correct seating of the denture on the implant definitive cast causing faulty recording of the mmr.materials and method : elastomeric record bases, reinforced with the resin framework, are fabricated and relined with the light body elastomeric material when all the healing abutments are in place. the mmr is recorded with these elastomeric record bases using vacuum formed facial surface index of the occluded existing dentures as a guideline.results:the elastomeric record bases with facial surface index of the existing dentures can allow clinicians to record mmr records without removing the healing abutments from the mouth with acceptable accuracy. this can save chair - side time of the procedure. the record of facial surfaces of existing complete denture in the form of vacuum formed sheet helps to set the occlusal vertical dimension.conclusion:use of facial surface index together with the elastomeric record bases can be the useful alternative technique to record the mmr in patients with implant supported full mouth rehabilitation. further study is required to prove its routine clinical utility.
	visualization of the reaction coordinate undertaken by glycosyltransferases has remained elusive, but is critical for understanding this important class of enzyme. using substrates and substrate mimics, we describe structural snapshots of all species along the kinetic pathway for human o - glcnac transferase, an intracellular enzyme that catalyzes installation of a dynamic post - translational modification. the structures reveal key features of the mechanism and show that substrate participation is important during catalysis.
migraine patients experience severe, recurrent headache that is accompanied with some symptoms such as nausea, vomiting, photophobia, phonophobia, neck pain, and muscle tension. migraine headaches are one - sided and pulsating that last usually 23 h. other related symptoms are alteration in sensory sensitivity, autonomic dysfunction, dysregulation of mood, and focal neurological symptoms. one of four migraine patients has visual disturbances in every attack that is known as aura. migraine is prevalent in around 14.7% of the european population and affects 3% of men and 10% of women in asian countries. in iran, several studies have shown a prevalence of 9.5%, which is considered to be high. the etiology of migraine is still unknown, but several theories about the cause of this disease have been proposed. these include genetic defects and environmental factors. among environmental factors, alcohol and coffee consumption, smoking, low physical activity, nutritional deficiencies, and psychological difficulties in addition, some evidences reported that obese migraine patients have higher severity and frequency of migraine attacks compared with normal weight patients. earlier studies have mainly considered the effects of general obesity on the characteristics of migraine attacks and the effects of central obesity has been paid less attention. one study showed that central obesity increases the frequency of migraine attacks but does not have any effect on the severity and duration of migraine. if the mentioned relation is confirmed, medical team members can use it as a proper approach in managing migraine attacks and nursing care processes. given the few studies in this regard and the high prevalence of migraine and central obesity in the iranian population, this study was performed to assess the relationship between central obesity and characteristics of migraine attacks among migraine patients in isfahan, iran. this cross - sectional study was performed on migraine patients who referred to khorshid and emam mosa sadr clinics in isfahan city, iran. patients with migraine for a long time and a 1-year history of severe, recurrent attacks (at least one attack per month) were selected. migraine diagnosis was carried out by a qualified clinical neurologist using the international headache society (ihs) criteria. we collected data on age, medical history [chronic diseases such as diabetes mellitus (dm), cardiovascular diseases (cvd), hypertension (htn), and kidney disease ], anti - migraine drug consumption (corticosteroids and analgesics drugs), anthropometric measurements, and family history of migraine from each patient. we determined characteristics of migraine attacks such as headache severity, frequency, duration, and headache diary result (hdr) for each participant. waist circumference (wc), waist hip ratio (whr), and waist height ratio (whtr) as the indicators of central obesity were determined for each participant. wc was measured at the midpoint between the top of the iliac crest and the last floating rib in the midaxillary line using an inelastic tape. moreover, hip circumference (hc) was measured at the widest part of the buttocks or hip. central obesity was defined based on who classification : wc more than 102 and 88 cm in men and women, respectively, was defined as abdominal obesity. quantitative and qualitative variables are presented as mean (sd) and number (percent), respectively. we evaluated the differences between men and women with respect to age, anthropometric measurements, and characteristics of migraine attacks using independent sample t - test. to assess the proportion of chronic diseases, intake of anti - migraine drugs for a long term, and family history of migraine between both genders, we used chi - square test. we examined the association between anthropometric measurements with the severity, frequency, duration of migraine attacks, as well as hdr by multiple linear regression tests in crude and adjusted models. in the first model, we adjusted age (continuous) as a confounding variable which can affect the mentioned associations. in the second model, we additionally adjusted intake of anti - migraine drugs for a long term and history of chronic diseases including cvd, htn, dm type 2, and kidney disorders. all analyses were conducted using spss statistical software (version 18.0 ; spss, inc., chicago, il, usa). the study was approved by the ethics committee of isfahan university of medical sciences, isfahan, iran. we determined characteristics of migraine attacks such as headache severity, frequency, duration, and headache diary result (hdr) for each participant. visual analog scale (vas) waist circumference (wc), waist hip ratio (whr), and waist height ratio (whtr) as the indicators of central obesity were determined for each participant. wc was measured at the midpoint between the top of the iliac crest and the last floating rib in the midaxillary line using an inelastic tape. moreover, hip circumference (hc) was measured at the widest part of the buttocks or hip. central obesity was defined based on who classification : wc more than 102 and 88 cm in men and women, respectively, was defined as abdominal obesity. quantitative and qualitative variables are presented as mean (sd) and number (percent), respectively. we evaluated the differences between men and women with respect to age, anthropometric measurements, and characteristics of migraine attacks using independent sample t - test. to assess the proportion of chronic diseases, intake of anti - migraine drugs for a long term, and family history of migraine between both genders, we used chi - square test. we examined the association between anthropometric measurements with the severity, frequency, duration of migraine attacks, as well as hdr by multiple linear regression tests in crude and adjusted models. in the first model, we adjusted age (continuous) as a confounding variable which can affect the mentioned associations. in the second model, we additionally adjusted intake of anti - migraine drugs for a long term and history of chronic diseases including cvd, htn, dm type 2, and kidney disorders. all analyses were conducted using spss statistical software (version 18.0 ; spss, inc., chicago, il, usa). the study was approved by the ethics committee of isfahan university of medical sciences, isfahan, iran. in this study, out of the 136 migraine patients, we had incomplete data on 7 patients which were not considered for statistical analysis. of the remaining 129 participants, 28 were men and 101 were women with a mean age of 34.00 11.11 and 34.07 10.68 years, respectively. differences between men and women in age, central obesity indicators, and characteristics of migraine attacks are shown in table 1. prevalence of chronic disorders such as cvd, htn, dm (type 2), and kidney diseases was higher in women than men. there was no difference between men and women in age, hc, whr, whtr, features of migraine attacks, family history of migraine, and intake of anti - migraine drugs for a long term. anthropometric measurements and characteristics of migraine attacks in men and women results of multiple linear regression test for the association between central obesity indicators and severity of migraine attacks are presented in table 2. there was a significant positive association between wc, whr, and whtr with the severity of migraine attacks. this relationship remained significant even after adjustment for potential confounding variables such as age, history of chronic disorders, taking anti - migraine drugs for a long term, and family history of migraine. we observed that one unit increase in wc, whr, and whtr was accompanied with 0.028, 5.36, and 5.70 units increase in the severity of migraine attacks. multiple linear regression analysis for the association between central obesity indicators and severity of migraine attacks the association between central obesity indicators and the frequency of migraine attacks is presented in table 3. high wc, whr, and whtr values were significantly associated with high frequency of migraine attacks. no change was observed in this association after controlling the potential confounders, such that one unit increase in wc, whr, and whtr was associated with 0.211, 31.81, and 36 units increase in the frequency of attacks. moreover, such relationship was found among men for wc and whtr, but it was not significant for whr ; however, adjustment for potential confounders made it significant. among women, a significant positive association was found between wc, whr, and whtr with the frequency of attacks ; however, adjustment for potential confounding variables attenuated this relationship. multiple linear regression analysis for the association between central obesity indicators and frequency of migraine attacks multiple linear regression analyses revealed no significant association between central obesity indicators and duration of migraine attacks either in crude or adjusted models. in addition, wc, whr, and whtr were not significantly associated with duration of attacks either in men or women [table 4 ]. multiple linear regression analysis for the association between central obesity indicators and frequency of migraine attacks the relationship between central obesity indicators and hdr is shown in table 5. sex - stratified analysis revealed a significant positive relationship between wc and hdr in both sexes. among men, no significant association was found between whr and whtr with hdr, but after adjustment for potential confounders, this relationship was marginally significant for whr. among women, there was a significant positive association between whr and whtr with hdr ; however, controlling for potential confounding variables attenuated this relationship for whr. in this study, we found a significant positive association between central obesity indicators with the severity and frequency of migraine attacks in the total population and in both sexes, separately. moreover, high wc, whr, and whtr values were associated with high hdr. sex - stratified analysis revealed a significant positive association between wc and hdr in both sexes. no significant relationship was observed between central obesity indicators and duration of migraine attacks, and this association remained non - significant in sex - stratified analysis also. this is one of the few studies that examined the association between central obesity and characteristics of migraine attacks. in addition, some studies have demonstrated that obesity may worsen the features of migraine attacks. recent studies have mainly assessed the association between general obesity and characteristics of migraine attacks, and data about central obesity are scarce. also, the studies conducted have just focused on wc and there is no evidence on the association between whr and whtr with the features of migraine attacks. in addition, to our knowledge, no studies have examined the association between obesity and hdr and the present study is the first one to examine this relationship. we found a significant positive association between wc, whr, and whtr with the severity of migraine attacks, frequency of migraine attacks, and hdr, but not with duration of migraine attacks. in addition, among men, the association between whr and whtr with hdr was non - significant or partially significant and it can be due to the less number of men with central obesity. in line with our findings, rossoni de oliveira. reported that migraine patients with high wc have higher frequency of migraine attacks, compared to patients with normal wc. moreover, they reported no significant association between wc with the severity and duration of migraine attacks. in another similar study, both kinds of obesity were associated with migraine symptoms in patients between 22 and 55 years of age. in a clinical trial, verrotti. assessed the effect of weight loss on the characteristics of migraine attacks. weight loss decreased abdominal fat as well as the severity and frequency of migraine attacks. reported that women with body mass index (bmi) 35 had increased incidence of active migraine, compared to women with bmi 23. moreover, obese migraine women had higher attacks frequency, photophobia, and phonophobia than non - obese migraine women. in contrast, some evidences showed no significant association between obesity and characteristics of migraine attacks. in addition, mattsson showed no significant relationship between obesity and the incidence of migraine. it should be mentioned that several factors including genetic polymorphisms, diet, physical activity, and health status can affect characteristics of migraine attacks ; therefore, different results obtained in previous studies may be due to changes in these effective factors. the main mechanism explaining the association between obesity and characteristics of migraine attacks is unknown. first, several inflammatory factors that are secreted from adipose tissue, especially in visceral adipose tissue, are involved in the pathogenesis of migraine and may affect the symptoms of migraine. calcitonin - related peptide and some interleukins as inflammatory factors can increase the frequency of migraine attacks and central nervous system sensitivity. second, adipose tissue secretes some hormones that affect the functioning of hypothalamus as an important center for regulation of body weight, food intake, and initiation of migraine attacks. therefore, alteration in hypothalamus function due to obesity status may change the incidence of migraine attacks. it seems that controlling body weight can be a useful strategy in reducing migraine attacks and its outcomes, and nursing care can use that in this regard. the present study was performed in a cross - sectional design and we can not confer a causal link between central obesity and characteristics of migraine attacks. moreover, sample size of our study was small and more studies with a larger number of participants are required to confirm the associations. although we controlled some potential confounders, further adjustment made for other confounding variables such as dietary pattern, physical activity, and psychological factors will be effective in reaching an independent association between central obesity and characteristics of migraine attacks. however, strength of this study is that for the first time, the relationship between whr and whtr with migraine attack features such as its severity and frequency has been assessed. in addition, we evaluated the relationship between central obesity and characteristics of migraine attacks in iran for the first time. from the findings of the present study, it can be concluded that obesity, especially abdominal obesity, aggravates the migraine symptoms and the severity and frequency of migraine attacks. therefore, we suggest prevention of obesity, especially abdominal obesity, in migraine patients and weight loss need to be taken by obese migraine patients. food security research center and department of community nutrition, school of nutrition and food sciences, isfahan university of medical sciences, isfahan, iran. this study was extracted from an msc dissertation which was approved by the school of nutrition and food sciences, isfahan university of medical sciences, code 392363. food security research center and department of community nutrition, school of nutrition and food sciences, isfahan university of medical sciences, isfahan, iran. this study was extracted from an msc dissertation which was approved by the school of nutrition and food sciences, isfahan university of medical sciences, code 392363.	background : migraine is a primary headache disorder that affects the neurovascular system. recent studies have shown that migraine patients with general obesity have higher characteristics of migraine attacks compared with normal weight patients, but data on central obesity are scarce. this study was done to assess the relationship between central obesity and the characteristics of migraine attacks in migraine patients.materials and methods : this cross - sectional study was conducted on 129 migraine patients (28 men and 101 women), aged 1567 years, in isfahan, iran. anthropometric measurements such as waist circumference (wc), hip circumference (hc), waist hip ratio (whr) and waist height ratio (whtr), as well as characteristics of migraine attacks such as severity, frequency, duration, and headache diary result (hdr) was determined for each participant. linear regression was used to examine the association between anthropometric measurements and characteristics of migraine attacks. p value less than 0.05 was considered significant.results:wc, whr, and whtr were positively associated with the severity (p - value : wc : 0.002, whr : 0.002, whtr : 0.001) and frequency (p - value : wc : 0.006, whr : 0.01, whtr : 0.002) of migraine attacks. moreover, we found a significant association between wc (p = 0.001), whr (p = 0.004), and whtr (p < 0.001) with hdr. no significant relationship was observed between central obesity indicators and duration of migraine attacks.conclusions:central obesity indicators were positively associated with the severity and frequency of migraine attacks as well as hdr, but not with duration of attacks. based on our findings, it can be concluded that weight loss may decrease the characteristics of migraine attacks.
birdshot chorioretinopathy (bscr) is a relatively rare form of uveitis, occurring in approximately 8% of posterior uveitis in the united states [1, 2, 3, 4, 5, 6 ]. it is correlated with the histocompatibility leukocyte antigen (hla)-a29 class i type, even though this is not required for the diagnosis. it has been reported that 8595% of bscr patients express hla - a29, whereas the prevalence of hla - a29 is only 7% in the general population [1, 8, 9, 10 ]. the positive predictive value of hla - a29 has been evaluated to be less than 50%. the results of the international consensus conference highlighted that hla - a29 is only a supportive finding for diagnosis, and, thus, it is not a required criteria. even if hla - a29 typing may be useful clinically in the identification of bscr, physicians should be aware that in some cases hla - a29-positive uveitis could be something other than typical bscr. the aim of the present study is to retrospectively review the ocular manifestations associated with the presence of hla - a29. all consecutive patients with uveitis who were found to be positive for hla - a29 and seen at the ocular immunology and uveitis service of the scientific institute san raffaele, milan, italy, between may 2006 and january 2011 were retrospectively reviewed. data were obtained by reviewing the patients charts, from a database and, in selected cases, using fluorescein angiograms and optical coherence tomography scans. at baseline and follow - up visits, all patients underwent a complete ophthalmologic evaluation, including best - corrected visual acuity, tonometry, slit - lamp examination and fundus biomicroscopy, with assessment of the grade of inflammation in the anterior chamber and vitreous according to the sun classification. laboratory tests for infection (including syphilis, toxoplasmosis, tuberculosis and herpetic infections) and autoimmune diseases (including sarcoidosis, behet 's disease and vogt - koyanagi - harada syndrome) were performed at baseline on all patients. birdshot lesions inferior or nasal to the optic disk in 1 eye, a low - grade anterior segment intraocular inflammation (defined as 1 + cells) and finally a low - grade vitreous inflammation (defined as 2 + vitreous haze). birdshot lesions were defined as cream - colored, irregular or elongated, choroidal lesions with indistinct borders, the long axis of which is radial to the optic disk. the ocular inflammatory activity was assessed according to the standardization of uveitis nomenclature working group. supportive findings for the diagnosis of bscr were as follows : hla - a29 positivity, retinal vasculitis and cystoid macular edema (cmo). exclusion criteria for the diagnosis of bscr were the presence of keratic precipitates, posterior synechiae and the presence of infectious, neoplastic or other inflammatory diseases that can cause multifocal choroidal lesions, as reported by levinson.. demographic data of the pooled patients, including age, sex, and duration of follow - up were recorded. main outcome measures included the percentage of bscr and non - bscr cases in the pooled hla-29-positive uveitis cases, based on the referred guidelines for bscr. in case of non - bscr uveitis, final definite ocular diagnosis was reported, based on all the findings of the intraocular inflammation. a total of 18 patients with a diagnosis of intraocular inflammation who were found to be positive for the presence of the hla - a29 were included in the analysis (table 1). five patients were male and 13 were female, the mean age was 48 16 years (range 1980 years) and mean follow - up was 25.7 21.3 months (range 260 months). only 7 (38.9%) patients met the criteria for a definite diagnosis of bscr and hla - a29 positivity (table 2, fig. six were female and only 1 was ma the mean age at onset of ocular inflammation in this group was 54 years. five out of 7 patients had retinal vasculitis, and 4 out of 7 had cmo as supportive findings. furthermore, 2 out of these 7 patients had papillitis, 2 had inflammation in peripheral retina, 2 had choroidal neovascularization and 1 had branch retinal vein occlusion. in the other 11 (61.1%) patients (7 female and 4 male), the mean age at onset of intraocular inflammation was 44 19 years, with a range from 19 to 80 years (table 3). birdshot lesions were found in 3 out of 12 patients, a mild anterior ocular inflammation (1 + cells) was detected in all patients and a low - grade vitreous inflammation (2 + vitreous haze) was present in 10 out of 11 patients. one patient was initially diagnosed with bscr, but during follow - up the lack of response to immunosuppressive treatment required a further work - up assessment. the patient underwent a brain mri and lumbar puncture showing a definite diagnosis of intraocular and cns lymphoma (masquerade syndrome, fig. one patient had a positive ppd skin test and a definite diagnosis of posterior tubercular uveitis with occlusive vasculitis ; the following diagnoses were also made : latent ocular tuberculosis in 1 patient, fuchs uveitis in 1 patient, herpetic panuveitis (pcr - analysis positive for hsv - dna on aqueous humor) in 1 patient and hla - b27 anterior uveitis in another patient. bscr is strongly associated with hla - a29, and at least 96% of bscr patients are hla - a29 carriers. the prevalence of hla - a29 is about 7% in the general population [1, 8, 9, 10 ], while in the normal italian population a frequency of 4.76% has been reported. although hla - a29 seems to play a major role, the prevalence of the disease in the hla - a29-positive population remains low, indicating that there are additional susceptibility factors for disease development. in everyday clinical practice, nevertheless, hla typing alone should not be considered as a diagnostic tool for definite diagnosis of bscr. in fact, even if bscr is strongly related to the hla - a29 phenotype, and its presence confers a relative risk of disease, the definite diagnosis requires other specific ocular characteristics. in the present study, in fact, less than 40% of patients with hla - a29 met the criteria for a definite diagnosis of bscr. the other 61.1% of patients expressed different ocular findings and consequently had a different diagnosis. the subgroup with a bscr diagnosis was for the most part female, with a mean age of 54 7.3 years. our data are consistent with those from earlier studies with a wider population, describing a female predominance and a mean reported age of 53.0 years. the interest of the current study is to estimate the frequency of bscr in the whole hla - a29-positive uveitis population. the results of our small study indicate that almost one third of patients with ocular inflammation actually have bscr. to our knowledge, this is the first study that analyzes the percentage of non - bscr hla - a29-positive uveitis patients in clinical practice and evaluates the ocular manifestations of the other clinical entities. in 6 out of 11 patients, a specific etiological classification was established : intraocular and cns lymphoma, lupus, latent ocular tuberculosis and hla - b27 anterior uveitis. diagnosis of bscr is most challenging at the onset of the disease, particularly if the typical birdshot lesions are subtle. in this study, 1 patient with an initial diagnosis of bscr was found to be affected instead by cns lymphoma. in clinical practice, different confounding features of ocular inflammation may be present and this should prompt the ophthalmologist to include other important differential diagnoses. the differential diagnosis of uveitis is extensive and undergoes change with time. in the present work, no definite etiological diagnosis could be determined in 5 out of 18 patients. our study has several limitations : it is short term, retrospective and evaluates a small study population. however, hla - a29-positive uveitis is a relatively uncommon disease, and therefore it will take a long time to recruit a larger sample. nevertheless, these results are valuable and reflect the demographic and causative pattern of uveitis in the pooled hla - a29-positive patients. in conclusion, our findings demonstrated that 61.1% of hla - a29-positive uveitis could be something other than bscr. frequently, in clinical practice, hla - a29 positivity leads ophthalmologists to erroneously formulate the diagnosis of bscr, while they should consider other different entities of ocular inflammation. hla - a29 typing alone is not a diagnostic requirement for the definite diagnosis of bscr and should only be considered as a supportive finding. none of the authors has a conflict of interest and no financial support was received for this work.	birdshot chorioretinopathy (bscr) is a relatively rare form of uveitis, which is strongly correlated with the histocompatibility leukocyte antigen (hla)-a29 class i type. nevertheless, hla typing is not diagnostic. the purpose of the present study was to retrospectively evaluate the ocular manifestations associated with the presence of hla - a29 other than typical bscr. charts of consecutive patients with a diagnosis of intraocular inflammation and who were found to be positive for the presence of hla - a29 were retrospectively reviewed. only 7 patients met the criteria for a definite diagnosis of bscr. among the other 11 patients, the disease was bilateral in 7 patients and unilateral in 4 patients. a definite diagnosis of the following conditions were found : intraocular and cns lymphoma in 1 patient, posterior tubercular uveitis with occlusive vasculitis in 1 patient, latent ocular tuberculosis in 1 patient, fuchs uveitis in 1 patient, herpetic panuveitis in 1 patient and hla - b27 anterior uveitis in another patient. although bscr is strongly related to the hla - a29 phenotype, and its presence confers a relative risk of disease, the definite diagnosis requires specific ocular characteristics. hla - a29 typing alone is not a diagnostic requirement for the definite diagnosis of bscr and should only be considered as a supportive finding.
bladder carcinoma (bc) is the most common malignancy of the urinary tract. in the united states, more than 70,000 new cases are diagnosed every year ; one out of five patients dies of this disease. noninvasive diagnosis of bc relies on bladder ultrasounds (us) and urinary cytology, but, whenever these investigations are negative or just suspicious of malignancy, cystoscopy remains the gold standard diagnostic method. cystoscopy also remains the gold standard followup investigation in patients with nonmuscle - invasive bc ; due to the high rate of disease recurrence, these patients are likely to undergo several cystoscopies during their life. unfortunately, cystoscopy is invasive, relatively expensive, and flexible instruments have reduced but have not eliminated patients ' discomfort ; therefore, noninvasive diagnostic tests comparing well with cystoscopy findings would be extremely desirable. in the last decades, the search for noninvasive tests for the diagnosis and followup of bc has concentrated onto urinary molecular tumor markers. though some of them, such as nmp22, urovision, and immunocyt, appear particularly promising, they all remain far from the goals of having such a high positive predictive value (ppv) to avoid unnecessary workup or, most important, to have such a high negative predictive value (npv) to avoid the risk of failing to detect tumors. in this scenario, little attention has been paid to a new device for noninvasive analysis of electromagnetic anisotropy in biological tissues (the tissue - resonance interaction method, trim - prob ; trimprobe, turin, italy). briefly, the device generates an alternating electromagnetic field that interacts with charged particles (molecules, ions, electrons, and nuclei) in a target tissue leading to a secondary radiation that varies for normal and neoplastic biological tissues. several pilot clinical studies have shown that trim - prob scanning may be a valuable tool in diagnosing prostate [48 ], breast, gastric, thyroid, rectum, and even bladder cancer. the present study therefore aimed to determine the diagnostic accuracy of trim - prob in detecting bc as compared to cystoscopy, the gold standard diagnostic method. following internal institutional review board approval, consecutive patients referred for cystoscopy were informed about the study purpose and asked to provide a written consent to be enrolled. inclusion criteria were (i) microscopic or gross hematuria and/or irritative voiding symptoms and (ii) bladder us and urinary cytology findings negative or just suspicious of malignancy. exclusion criteria were (i) pace - makers and/or any other active implantable device, (ii) a history of previous or coexistent pelvic neoplasm, and (iii) psa > 4 ng / ml in males. all patients were first submitted to trim - prob bladder scanning by a single investigator (gl) and then to cystoscopy by another investigator blind to the trim - prob findings. trim - prob bladder scanning was carried out in a room free from relevant electromagnetic interference, including other electric device and mobile phones, and the patients were asked to remove personal metal objects. the physical principles and the technique of trim - prob bladder scanning have already been described in detail. briefly, the trim - prob system consists of a battery - operated probe, a receiver, and a computer display. the probe, which is about 30 cm long and can easily be held in one hand, contains a tuneable oscillator and an antenna emitting a very weak electromagnetic wave at several frequencies (465, 930, and 1395 mhz). with the patient in standing position and a partly full bladder, the electromagnetic wave stimulates minute electrical oscillations that resonate inducing changes in the amplitude of one or more frequencies, depending on the pathological state of the tissue. such changes are detected by the receiver, a multifrequency radiation pattern analyzer located at approximately 150 cm from the probe, at the same height of the bladder. resonance values are visualized on the computer display on a logarithmic scale and expressed in arbitrary units (au) ranging from 0 to 255. when the signal reduction at the frequency of 930 mhz confirms the exact position of the probe, the response to the 465 mhz frequency is recorded ; the test is positive for bc when the signal intensity at 465 mhz is below 45 au. the study population consisted of 125 patients, 100 males and 25 females, with a mean (standard deviation) age of 62.1 11.2 years. eighty - two patients were referred because of gross hematuria, whereas 43 were referred because of irritative urinary symptoms that were associated or not with microscopic hematuria in 38 and 5 cases, respectively. bladder us were negative in 102 patients and suspicious of bladder tumor in 23 ; urinary cytology was negative in 114 and suspicious of malignancy in 11. trim - prob bladder scanning was negative in 72 patients and positive in 53, whereas cystoscopy was negative in 78 and positive in 47. all lesions visible at cystoscopy were small (< 1 cm in diameter) and/or flat, thus explaining negative or just suspicion us findings. comparing the findings of the two diagnostic methods, 46 (86.8%) of the 53 patients positive to trim - prob were also positive to cystoscopy (true positive), whereas 7 (13.2%) were negative (false positive) ; conversely, 71 (98.6%) of the 72 patients negative to trim - prob were also negative to cystoscopy (true negative), whereas only 1 (1.4%) was positive (false negative). therefore, trim - prob bladder scanning had an overall sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of 97.9%, 89.9%, 86.8%, 98.6%, and 93.6%, respectively. of the 47 lesions visible at cystoscopy, 6 were just coagulated ; of the remaining 41, 22 (53.7%) were found to be a low - grade ta transitional cell carcinoma (tcc), 9 (22%) a low grade t1 tcc, 3 (7.3%) a tis, 5 (12.2%) a high - grade t1 tcc, 1 (2.4%) a high - grade t2 tcc, and 1 (2.4%) a t0. the dielectric characteristics of biological tissues constitute a long standing research line in the field of physics. already in 1926, fricke and morse evaluated the electromagnetic properties of breast cancer, benign breast tumors and normal breast tissue in human. at a frequency of 20 khz, the frequency - dependent function called dielectric constant was tenfold greater for tumors than for normal tissue, whereas the other frequency - dependent function called conductivity was nearly the same for all tissues. the increase in for actively growing malignant tumors was attributed to an increased proportion of cell membrane surfaces per unit volume. chaudhary and coworkers measured and of breast carcinoma and homologous normal tissue between 3 mhz and 3 ghz and found that both functions were always greater for tumors. however, increased towards frequencies below 30 mhz, whereas increased towards frequencies above 300 mhz. the effect on was again attributed to cell membranes, while the effect on was attributed to reorientation of dipolar particles due to the increased water content of tumors [17, 18 ]. it was subsequently confirmed that electromagnetic energy absorption of malignant tumors of various origins was significantly higher than that of normal tissues, particularly at frequencies around 400 mhz. when the existence of significant differences between the electromagnetic properties of malignant and normal tissues was further confirmed by sophisticated in vitro measurements [20, 21 ], it became clear that a simple and noninvasive device was needed to apply these principles to cancer diagnosis. such device for electromagnetic detection of biologic tissues anomalies, consisting of a nonlinear oscillator concealed into a cylindrical probe and a radiofrequency spectrum analyzer, was invented and patented by one of us (clarbruno vedruccio) who named it bioscanner, while the commercial name trim - prob was imposed by the producer, galileo avionica a finmeccanica company [22, 23 ]. were the first to evaluate the feasibility and diagnostic accuracy of trim - prob scanning in detecting prostate cancer ; in their study population of 757 men, the test had a 95.5% sensitivity, a 42.7% specificity, a 63.6% ppv, and an 89.8% npv. these results have subsequently been replicated by other studies [68 ] and even by a multicenter trial, thus supporting also the reproducibility of the technique in detecting prostate cancer. similar results have been achieved in the study testing the diagnostic accuracy of trim - prob in detecting breast cancer, whereas much better results, with sensitivity and specificity up to 100%, have been achieved in the studies dealing with thyroid, gastric, and rectal cancer [1012 ]. since there is a single study for each of these organs, these findings await to be replicated by other investigators in a larger number of patients. also for bc there is a single study in the literature comparing trim - prob with cystoscopy findings and showing a highly significant (p < 0.001) level of agreement (cohen 's k = 0.77) between the two diagnostic procedures. the present study confirmed the high level of agreement between trim - prob and cystoscopy findings, as the comparison of the two diagnostic methods showed that trim - prob scanning had a 97.9% sensitivity, an 89.9% specificity, an 86.8% ppv, and a 98.6% npv, with an overall diagnostic accuracy of 93.6%. the potential clinical relevance of these findings is obvious. due to its high (86.8%) ppv, trim - prob bladder scanning could be used to screen patients at high risk of bc, such as smokers or people subjected to professional or environmental exposure to carcinogenic agents. on the other hand, its even higher (98.6%) npv suggests that this test has the potential to replace cystoscopy in evaluating patients at low risk of harboring bc or in those under surveillance after transurethral resection of bc, with significant reductions in healthcare costs, patients ' discomfort, and urologists, burden. such a high npv, ranging from 84% for prostate cancer to 100% for thyroid and gastric cancer [413 ], seems to be the common denominator in the studies dealing with electromagnetic detection of cancer and the real strength of this new technology, as it may lead to a reduction of unnecessary invasive tests or biopsies. the present study, however, shares also some limitations of the previous similar studies. the first is the absence of true negative cases, as it included only patients suspected of having bc. we already designed a new study whereby patients undergoing cystoscopy as the first part of transurethral resection of prostate will serve as controls ; meanwhile, it should be noticed that a previous study demonstrated a significant difference in the signal intensity at 465 mhz between patients referred for infertility and those referred for suspected prostate cancer. another limitation common to previous studies is the blind scanning of the target organ, as the system is not fused with an imaging technique. the possibility to combine trim - prob with us scanning of the bladder by a fusion software, thus to visualize the organ to be electromagnetically scanned, would certainly help to simplify the introduction of this new technology in clinical practice. specific limitations of the present study were as follows : (i) not having correlated clinical with pathological findings, but this was felt to be beyond the scopes of a study comparing two diagnostic methods and not having a centralized pathological evaluation ; (ii) not having explored the problem of intra- and interobserver reproducibility of results, but this was, again, beyond the scopes of a pilot study ; (iii) not having correlated the trim - prob diagnostic accuracy with tumor size and location, but this turned to be of little relevance in view of the extremely low rate (1.4%) of false negatives. in spite of all these limitations, we believe that electromagnetic detection of cancer is a fascinating research field that deserves, like all new and surprising developments, scientific enthusiasm as well as critical assessment and appropriate validation. fortunately, these steps are made easy by the fact that this new technology is, like ultrasonography, noninvasive, simple, safe (the electromagnetic radiation is 1/10 of that of mobile phones) and has low costs (approximately 50.000 euros the machine in italy, no disposables). trim - prob bladder scanning was found to be an accurate method for noninvasive electromagnetic detection of bc, having its main strength in its very high npv. this finding was shared by all neoplasms evaluated with this new technology and, if confirmed in further well - designed studies, would suggest that the introduction of trim - prob bladder scanning in clinical practice could avoid a large number of unnecessary diagnostic and followup cystoscopies.	objectives. normal and neoplastic human tissues have different electromagnetic properties. this study aimed to determine the diagnostic accuracy of noninvasive electromagnetic detection of bladder cancer (bc) by the tissue - resonance interaction method (trim - prob). patients and methods. consecutive patients were referred for cystoscopy because of (i) microscopic or gross hematuria and/or irritative voiding symptoms and (ii) bladder ultrasounds and urinary cytology findings negative or just suspicious of malignancy. patients were first submitted to trim - prob bladder scanning by a single investigator and then to cystoscopy by another investigator blind to trim - prob data. results. in 125 evaluated patients cystoscopy was positive for bc in 47 and negative in the remaining 78 ; conversely, trim - prob bladder scanning was positive for bc in 53 and negative in 72. in particular, trim - prob scanning yielded 7 false positives and only one false negative ; therefore, its overall sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy were 97.9%, 89.9%, 86.8%, 98.6%, and 93.6%, respectively. conclusions. trim - prob bladder scanning was a simple and quite accurate method for non - invasive electromagnetic detection of bc. if the elevated positive and negative predictive values will be replicated in further well - designed studies, it could be used to screen asymptomatic patients at high risk of bc.
healthy men and women aged 2140 years with a bmi between 19 and 27 kg / m who had at least one parent with type 2 diabetes were recruited through local advertisements. fasting body weight and height were measured in the morning using a calibrated scale (scale - tronix inc., wheaton, il) and stationary harpenden stadiometer (holtain, crymych, wales). we excluded subjects who had any acute or chronic medical condition ; self - reported sleep problems (pittsburgh sleep quality index global score > 6), night work, or habitual daytime naps ; recent (15 confirmed by clinical interview) ; current tobacco use or excessive alcohol intake (> 14 drinks / week for men ; > 7 for women) ; use of prescription, over - the - counter, or illegal drugs and supplements that can affect sleep or metabolism ; and abnormal findings on physical exam or laboratory testing, including complete blood counts, comprehensive metabolic panel, thyroid function tests, 12-lead electrocardiogram, and a 75-g oral glucose challenge. the study protocol was registered and approved by the institutional review board of the university of chicago. participants were asked to complete 14 days of sleep monitoring while following their usual lifestyle at home. a small accelerometer equipped with an event marker (actiwatch-64, mini - mitter - respironics, bend, or) was attached with a wrist band to the nondominant arm and actigraphy data were collected continuously in 1-min epochs to measure usual sleep duration under free - living conditions (1). since reduction in physical activity related to sleep loss may contribute to the association of short sleep with metabolic morbidity, we also measured the amount and intensity of body movement of each participant using a small accelerometer (actical, mini - mitter - respironics) attached with an elastic waistband over the iliac crest. detailed physical activity data from a subset of the participants in this study have been reported elsewhere (17). to be included in the analysis, participants were required to wear the actiwatch for at least seven full nights and the actical for at least six full days. a total of 61 subjects were enrolled ; 8 discontinued their participation before completing the study (because of new job, family illness, or moving), 4 had 10). overnight recordings were obtained in 41 subjects (87%) who kept their study appointments (table 1). sleep was scheduled between 23002400 h and 07300830 h with a fixed time in bed of 8.5 h. records were scored in 30-s epochs of wake ; movement ; stage 1, 2, 3, and 4 sleep ; and rapid eye movement (rem) sleep according to standard criteria. respiratory events, periodic leg movements, and arousals were scored using current clinical guidelines. sleep efficiency was calculated as the percent of scheduled time in bed that was scored as sleep. sleep onset latency was defined as the time between lights - off and the first epoch of stage 1 sleep. p values reflect comparisons between participants with habitual short sleep and matched subjects with sleep duration > 6 h / day using pearson test for categorical and student independent - samples test for continuous variables. a distal forearm intravenous catheter was placed upon the arrival of each participant in the morning after an overnight fast. two baseline blood samples were collected (15 and 0 min) for measurement of glucose and insulin, and 75 g dextrose dissolved in 296 cc of orange - flavored water (nerl diagnostics llc, east providence, ri) was ingested in 6 h / day (table 1). the primary outcome variable (isi) and four secondary measures selected based on the outcomes of the previous regression analysis (homa - ir, cir, area under the 3-h ogtt insulin curve, and fasting insulin) were compared between the two groups using ancova, controlling for time spent in physical activity of moderate - and - vigorous intensity (table 3). although the difference was not statistically significant, short sleepers were slightly older (26 3 vs. 28 5 years ; p = 0.137), and we repeated the ancova after entering age as an additional covariate to minimize its influence (table 3). the level of statistical significance was set at p 6 h / day data are mean sd (n = 15 in each group). p values based on ancova with sleep group (short vs. control) as a between - subject factor controlling for daily time spent in physical activity of moderate - and - vigorous intensity, which was included as a covariate. p values based on ancova as in with additional control for participant age, which was included as a covariate. # square root participants were asked to complete 14 days of sleep monitoring while following their usual lifestyle at home. a small accelerometer equipped with an event marker (actiwatch-64, mini - mitter - respironics, bend, or) was attached with a wrist band to the nondominant arm and actigraphy data were collected continuously in 1-min epochs to measure usual sleep duration under free - living conditions (1). since reduction in physical activity related to sleep loss may contribute to the association of short sleep with metabolic morbidity, we also measured the amount and intensity of body movement of each participant using a small accelerometer (actical, mini - mitter - respironics) attached with an elastic waistband over the iliac crest. detailed physical activity data from a subset of the participants in this study have been reported elsewhere (17). to be included in the analysis, participants were required to wear the actiwatch for at least seven full nights and the actical for at least six full days. a total of 61 subjects were enrolled ; 8 discontinued their participation before completing the study (because of new job, family illness, or moving), 4 had 10). overnight recordings were obtained in 41 subjects (87%) who kept their study appointments (table 1). sleep was scheduled between 23002400 h and 07300830 h with a fixed time in bed of 8.5 h. records were scored in 30-s epochs of wake ; movement ; stage 1, 2, 3, and 4 sleep ; and rapid eye movement (rem) sleep according to standard criteria. respiratory events, periodic leg movements, and arousals were scored using current clinical guidelines. sleep efficiency was calculated as the percent of scheduled time in bed that was scored as sleep. sleep onset latency was defined as the time between lights - off and the first epoch of stage 1 sleep. p values reflect comparisons between participants with habitual short sleep and matched subjects with sleep duration > 6 h / day using pearson test for categorical and student independent - samples test for continuous variables. a distal forearm intravenous catheter was placed upon the arrival of each participant in the morning after an overnight fast. an oral glucose tolerance test (ogtt) two baseline blood samples were collected (15 and 0 min) for measurement of glucose and insulin, and 75 g dextrose dissolved in 296 cc of orange - flavored water (nerl diagnostics llc, east providence, ri) was ingested in 6 h / day (table 1). the primary outcome variable (isi) and four secondary measures selected based on the outcomes of the previous regression analysis (homa - ir, cir, area under the 3-h ogtt insulin curve, and fasting insulin) were compared between the two groups using ancova, controlling for time spent in physical activity of moderate - and - vigorous intensity (table 3). although the difference was not statistically significant, short sleepers were slightly older (26 3 vs. 28 5 years ; p = 0.137), and we repeated the ancova after entering age as an additional covariate to minimize its influence (table 3). the level of statistical significance was set at p 6 h / day data are mean sd (n = 15 in each group). p values based on ancova with sleep group (short vs. control) as a between - subject factor controlling for daily time spent in physical activity of moderate - and - vigorous intensity, which was included as a covariate. p values based on ancova as in with additional control for participant age, which was included as a covariate. multiple linear regression analysis showed a significant association between reduced sleep duration and increased insulin resistance (lower isi) in the entire study sample independent of age, bmi, sex, race / ethnicity, familial diabetes risk, and daily time spent in physical activity of moderate - and - vigorous intensity (table 2). regression analyses of secondary end points confirmed this finding using homa - ir as a measure of insulin resistance and showed that participants with shorter sleep duration had higher indices of insulin secretion (cir and area under the 3-h ogtt curve for insulin) (table 2). there was no significant association between objectively measured sleep duration and markers of glucose tolerance (table 2). the addition of control for alcohol and caffeine consumption in these models produced similar results (data not shown). during an average night at home, the 15 participants with measured sleep duration in the lowest tertile of the study sample obtained 1 h 29 min less sleep (p < 0.001) compared with the group of 15 reference sleepers with similar age, bmi, sex, race / ethnicity, and family risk for diabetes (table 1). self - reported sleep exceeded objectively measured sleep duration in both groups (table 1), but short sleepers overestimated their usual sleep duration considerably more than the participants in the reference group (average overreporting : 106 58 vs. 63 42 min / day ; p = 0.026). both groups had good subjective sleep quality (table 1) ; however, short sleepers reported significantly more daytime sleepiness (p = 0.022). when time in bed was fixed to 8.5 h in the laboratory, the measured quantity and quality of sleep of the short sleepers was comparable to that of the reference sleep group (table 1). consistent with the presence of increased sleep pressure, short sleepers tended to have faster onset of rem sleep (p = 0.095) (table 1). compared with the subjects in the reference group, short sleepers had significantly lower isi and higher homa - ir (table 3). the 32% lower index of insulin sensitivity in the short - sleep group was accompanied by higher fasting insulin and cir values and a larger area under the 3-h ogtt insulin curve (table 3). participants in the short- and reference sleep groups had comparable di (13,784 8,565 and 14,046 9,312), fasting (87 7 and 84 6 mg / dl) and 2-h blood glucose (105 21 and 101 15 mg / dl), and area under the 3-h ogtt glucose curve (19,257 2,059 and 17,800 2,206 mg dl min). this study examined the relationship between objectively documented reduced sleep duration and ogtt - based measures of insulin sensitivity, insulin secretion, and glucose tolerance in healthy free - living adults with parental history of type 2 diabetes. consistent with previous population - based data (1), 40% of the participants in our convenience sample had habitual sleep duration < 6 h / day. compared with their counterparts in the reference group, participants in the short - sleep group obtained an average of 1.5 h less sleep per night. however, when assessed formally by polysomnography, short sleepers did not have any sleep problems or biologically lower need to sleep (table 1). instead, they reported significantly more daytime sleepiness consistent with the presence of a behavioral pattern of habitual sleep curtailment and chronic sleep debt. study participants who habitually curtailed their sleep in this fashion were considerably more insulin resistant (table 2). the increased insulin resistance of the short sleepers was associated with a compensatory rise in insulin secretion (table 3), which allowed them to maintain normal glucose tolerance similar to that of the participants in the reference sleep group. adults with parental history of type 2 diabetes have decreased insulin - mediated glucose disposal and nonoxidative glucose metabolism compared with individuals without family history of diabetes (11,13,14). our data indicate that this insulin resistance may be considerably higher among those members of this group who habitually curtail their sleep. several laboratory experiments have documented that short - term sleep deprivation can result in reduced systemic insulin sensitivity, and some have speculated that this may be related to changes in adrenocortical or autonomic nervous system activity (3,58). whether similar mechanisms may contribute to the increased insulin resistance in free - living adults who habitually curtail their sleep has not been studied. other determinants of systemic insulin resistance include obesity, advancing age, and familial and ethnic risk factors. however, short sleep duration was found to predict the presence of increased insulin resistance when the contribution of these factors was controlled for in our multiple regression analysis (table 2). it is notable that short sleepers had lower levels of physical activity of moderate - and - vigorous intensity (table 1). nevertheless, inclusion of the average daily time spent in moderate - and - vigorous activity as an independent variable in our multiple regression analysis did not attenuate the association between objectively measured short sleep duration and increased insulin resistance (table 2). the 32% difference in insulin sensitivity between otherwise comparable groups of short and reference sleepers was also statistically significant when time spent in moderate - and - vigorous activity was entered in that analysis as a covariate (table 3). results were similar when we used daily counts of total body movement instead of the average time spent in moderate - and - vigorous physical activity (data not shown). despite this, our findings remain compatible with the possibility that longer - term effects of physical activity on predictors of insulin resistance, such as aerobic fitness, metabolic flexibility, and accumulation of fat in visceral and ectopic organ sites, may explain some of the association between habitual sleep duration and isi. reduced physical fitness and total aerobic capacity, lower resting metabolic rate, increased abdominal adiposity, and defects in lipid metabolism have been described in healthy normoglycemic offspring of type 2 diabetic parents (13,15,16,23). since chronic sleep insufficiency alone, or in association with lower physical activity, may have an adverse effect on these variables (8,24,25), additional studies are needed to define the metabolic risk profile of habitual sleep curtailment in this high - risk population. irrespective of the underlying mechanisms, the finding that healthy lean adults with parental history of type 2 diabetes who habitually sleep < 6 h / day are considerably more insulin resistant compared with those who obtain more sleep may have important health implications. since insulin resistance is a strong predictor of diabetes incidence in the offspring of type 2 diabetic parents (11), a behavioral pattern of habitual sleep curtailment in this group may be associated with a higher risk of developing the disease relative to the risk conferred by family history alone (2). the maintenance of normal glucose homeostasis involves reciprocal changes in insulin secretion and sensitivity (20). the young and lean short sleepers in our study were able to secrete more insulin to compensate for their higher insulin resistance (tables 2 and 3). some studies of experimental sleep deprivation in healthy volunteers have observed similar changes in insulin secretion and sensitivity (3,8), whereas others found reduced glucose tolerance and either unchanged or decreased insulin secretion (46). individuals with parental history of type 2 diabetes can exhibit deficits in insulin secretion long before the development of overt diabetes, and progressive loss of -cell function is another key factor for the development of type 2 diabetes in this susceptible population (12,15,16). our findings now raise the question whether the demand for increased insulin secretion related to higher insulin resistance in at - risk adults who habitually curtail their sleep may result in earlier -cell failure as they grow older, gain excess weight, and continue to maintain a more sedentary lifestyle. we collected proof - of - concept data from a carefully screened sample of healthy individuals at risk for type 2 diabetes, while avoiding the confounding effects of obesity and poor general or emotional health. the use of laboratory polysomnography and objective monitoring of habitual sleep and free - living physical activity also allowed us to exclude the presence of sleep pathology and avoid assessments based on unreliable self - reports of these behaviors (see results for an example of systemic bias in self - reported sleep). finally, it was important to study a population with high risk for type 2 diabetes, which may inform future behavioral research on sleep and metabolic risk reduction. despite its strengths, this was an exploratory study that included a relatively small number of subjects and used indirect ogtt - based techniques to assess insulin secretion and sensitivity. although our free - living sleep data were similar to those of other population - based reports (1), study participants were not randomly selected and results may not be entirely representative of the relationship between sleep and insulin sensitivity in this high - risk group. in conclusion, a large number of healthy young adults with familial risk for type 2 diabetes habitually curtail their sleep. compared with a similar group of participants with parental history of type 2 diabetes and habitual sleep 6 h / day, those who slept less were considerably more insulin resistant and had increased insulin secretion a pattern that has been associated with a higher risk of developing diabetes in this population (11). future studies are needed to elucidate the link between habitual sleep curtailment as a behavioral risk factor for insulin resistance and increased metabolic morbidity in individuals with familial predisposition for type 2 diabetes.	objectiveexperimental sleep deprivation is accompanied by changes in glucose regulation. however, the effects of chronic sleep insufficiency on insulin secretion and action in populations at high risk for type 2 diabetes are not known. this study examined the relationship between objectively documented habitual sleep curtailment and measures of insulin sensitivity, insulin secretion, and oral glucose tolerance in free - living adults with parental history of type 2 diabetes.research design and methodsa total of 47 healthy participants with parental history of type 2 diabetes (26 female/21 male, mean [sd ] age 26 [4 ] years and bmi 23.8 [2.5 ] kg / m2) completed 13 (sd = 2) days of sleep and physical activity monitoring by wrist actigraphy and waist accelerometry while following their usual lifestyle at home. laboratory polysomnography was used to screen for sleep disorders. indices of diabetes risk based on oral glucose tolerance tests were compared between participants with habitual short sleep and those with usual sleep duration > 6 h / day.resultsconsistent with a behavioral pattern of habitual sleep curtailment, short sleepers obtained an average of 1.5 h less sleep per night and showed signs of increased sleep pressure. participants who habitually curtailed their sleep had considerably higher indices of insulin resistance and increased insulin secretion but maintained normal glucose tolerance similar to that of subjects who slept more.conclusionsyoung lean adults with parental history of type 2 diabetes who habitually curtail their sleep have increased insulin resistance and compensatory hyperinsulinemia a pattern that has been associated with higher risk of developing diabetes in such susceptible individuals.
as more and more genomes are automatically sequenced, comprehensive protein annotation is a needed step after gene identification. even in good annotated genomes (human, mouse) about 30% of all proteins are not functionally identified (13), and thus often a similarity search will not be sufficient. here, we present a suite of protein tasks, protsweep, domainsweep and 2dsweep, which perform analysis from sequence similarity to small domains and structural elements. this includes similarity searches against protein sequence databases and specialized motif collections, prediction of secondary structural elements, attributing each sequence to known super - families, protein localization prediction, physicochemical protein characteristics and domain functional assignation. our strategy for assigning relevant functional roles is based on the joint use of both global (homology similarity) and local (domain and motif) sequence similarities (4). several query sequences can be uploaded by the usual copy & paste procedure into the input box using fasta format. if more than one sequence is to be queried, a multiple fasta file can be used. then the user will be redirected to an application page, and the runbutton can start the task. additionally, there is a link to an online help, indicated with a ?, with the following topics : short description, programs employed, algorithm, output, additional options and acknowledgments. results can be received by selecting the tab go to results page. the results are provided as html for visual inspection or can be downloaded as xml for storage in private databases. in case of error when clicking on the application name in the results manager page, a log - file is displayed where more human readable error messages can be found. standard protein databases used by the tasks like uniprot / swissprot, uniprot / trembl and refseq are automatically updated whenever new versions become available. concerning ensembl, the situation is more complex. due to possible inconsistencies between the different ensembl api versions, which are used in the tasks, it is not possible to automatically update the ensembl data and this needs to be done by hand. the different databases and the way they were used in these pipelines are described in table 1. table 1databases used in the different pipelines, and the programs and parameters used to search themserverdatabaseslinksprogramparametersprotsweepuniprot / swissprothttp://www.ebi.ac.uk / swissprotblastp - nofilter -exp = 10.0uniprot / tremblhttp://www.ncbi.nlm.nih.gov / refseq / blastp - nofilter -exp = 10.0refseqftp://ftp.ebi.ac.uk / pub / databases / trembl / sptrembl / blastp - nofilter -exp = 10.0ensemblhttp://www.ensembl.org / blastp - nofilter -exp = 10.02dsweepdssphttp://www.sander.ebi.ac.uk / dssp / blastp - exp = 0.001 -extension = 10 - nogappedalignnrpep (non - redundant ncbi protein database)ftp://ftp.ncbi.nih.gov / blast / db / fastapsiblast - b20000 -a5 -j2 -e0.001uniprot / swissprot + uniprot / trembl + updateshttp://www.expasy.ch / sprot / sprot - top.html http://www.ebi.ac.uk/swissprotmsfgenerator-exp = 0.001 -overlap = 75 -customrange = 5 custompercentage = 80,60,50,45,40,35domainsweepprositeftp://ftp.expasy.ch / databases / prositemotifs & pfscandefaultpfamhmmhttp://www.sanger.ac.uk / software / pfam / hmmscan - lib = pfam.hmm -dprintsftp.bioinf.man.ac.ukhmmscan - lib = prints.hmm -dsmarthttp://smart.embl - heidelberg.de / hmmscan - lib = smart.hmm -dtigrfamshttp://www.tigr.org / tigrfams / index.shtml / hmmscan - lib = tigrfams.hmm -dscophttp://scop.mrc - lmb.cam.ac.uk / scopscopscandefaultblocksftp://ftp.ncbi.nih.gov / repository / blocks / unixblockssearcher - cutoff = 0.01 dinterprohttp://www.ebi.ac.uk / interpro / srs queriesprodomhttp://www.toulouse.inra.fr / prodom.htmlprodomblastdefault databases used in the different pipelines, and the programs and parameters used to search them standard protein databases used by the tasks like uniprot / swissprot, uniprot / trembl and refseq are automatically updated whenever new versions become available. concerning ensembl, the situation is more complex. due to possible inconsistencies between the different ensembl api versions, which are used in the tasks, it is not possible to automatically update the ensembl data and this needs to be done by hand. the different databases and the way they were used in these pipelines are described in table 1. table 1databases used in the different pipelines, and the programs and parameters used to search themserverdatabaseslinksprogramparametersprotsweepuniprot / swissprothttp://www.ebi.ac.uk / swissprotblastp - nofilter -exp = 10.0uniprot / tremblhttp://www.ncbi.nlm.nih.gov / refseq / blastp - nofilter -exp = 10.0refseqftp://ftp.ebi.ac.uk / pub / databases / trembl / sptrembl / blastp - nofilter -exp = 10.0ensemblhttp://www.ensembl.org / blastp - nofilter -exp = 10.02dsweepdssphttp://www.sander.ebi.ac.uk / dssp / blastp - exp = 0.001 -extension = 10 - nogappedalignnrpep (non - redundant ncbi protein database)ftp://ftp.ncbi.nih.gov / blast / db / fastapsiblast - b20000 -a5 -j2 -e0.001uniprot / swissprot + uniprot / trembl + updateshttp://www.expasy.ch / sprot / sprot - top.html http://www.ebi.ac.uk/swissprotmsfgenerator-exp = 0.001 -overlap = 75 -customrange = 5 custompercentage = 80,60,50,45,40,35domainsweepprositeftp://ftp.expasy.ch / databases / prositemotifs & pfscandefaultpfamhmmhttp://www.sanger.ac.uk / software / pfam / hmmscan - lib = pfam.hmm -dprintsftp.bioinf.man.ac.ukhmmscan - lib = prints.hmm -dsmarthttp://smart.embl - heidelberg.de / hmmscan - lib = smart.hmm -dtigrfamshttp://www.tigr.org / tigrfams / index.shtml / hmmscan - lib = tigrfams.hmm -dscophttp://scop.mrc - lmb.cam.ac.uk / scopscopscandefaultblocksftp://ftp.ncbi.nih.gov / repository / blocks / unixblockssearcher - cutoff = 0.01 dinterprohttp://www.ebi.ac.uk / interpro / srs queriesprodomhttp://www.toulouse.inra.fr / prodom.htmlprodomblastdefault databases used in the different pipelines, and the programs and parameters used to search them protsweep is an approach to the functional characterization of unknown proteins based on a cascade of similarity searches. it is well known that protein databases do not completely overlap and differ in their annotation quality (5). this task takes into account the significant differences among databases (supplementary table 1) to improve the quality of the protein characterization. it selects the order in which the databases have to be searched and combines the annotation found depending on the results. protsweep classifies proteins into the following categories : identical, homolog, similar, weakly similar and putative proteins. the query protein starts the blast (6) cascade against swissprot (7) first (figure 2). we do take into account three parameters to classify the blast hits : (i) percentage of identity, (ii) spercent. the two last parameters are related to the length of the total alignment, being qpercent the percentage of the query sequence length covered in the alignment with the database hit and spercent the percentage of the hit (subject) sequence length covered by the alignment (figure 2). depending on the classification of the blast hits according to these parameters and the hit protein annotation if the hit has 100% qpercent and spercent and more than 98% identity, it is considered an identical protein and the swissprot i d will be searched in ensembl (8). if it is successful, all information from both databases will be combined (supplementary table 2) and stored in the xml output. if the i d can not be found in ensembl then a blast search is performed with the query protein against ensembl. the best ensembl hit is selected and compared against the swissprot hit using the smith if the identity between sequences is greater than 98%, then the information from both sources and the blast alignment will be added to the final output, if the identity is less, only swissprot annotation and the alignments will be added to the xml. if the qpercent and spercent is between 80% and 98% and the identity is between 85% and 98%, the hit is classified as homologous and follows the same strategy with ensembl as already described (figure 2). in case, spercent are greater than 85%, then the blast cascade continues with sptrembl and refseqprot. in the case that no identical or homologous hits can be found in any of the databases, the best similar hit among the three databases is selected and classified as similar, weakly similar or putative (figure 2). if the protein is characterized, information concerning the coding gene, about the splicing variants and orthologous genes is also provided. depending on the degree of homology, protein function, transcript of origin, genomic localization, and go annotation or partial similarities will also be shown. hypothetical proteins will only be presented in the result when no other information about identical or homologous proteins can be found in any of the databases (supplementary figure 1). the web output of protsweep (supplementary figure 1) is divided in five sections : (i) general information, (ii) identified protein and transcripts, (iii) features and functions, (iv) genomic localisation and (v) homology to other organisms / genes. the information provided in each of these sections is provided in figure 2 and supplementary table 2. the user has immediate access to all complete application outputs and database entries via hyperlinks. at the bottom of the html output there is a link to the explanatory legend as well as to the xml output containing all the generated information. domainsweep identifies the domain architecture within a protein sequence and therefore aids in finding correct functional assignments for uncharacterized protein sequences (figure 3). it employs different database search methods to scan a number of protein / domain family databases. among these models, in increasing complexity, are : prodom (10), automatically generated protein family consensus sequences, prosite (11) regular - expression patterns, blocks (12), ungapped position - specific scoring matrices of sequence segments, prints (13) sequence motifs, prosite profiles (7), gapped position - specific scoring matrices and hidden markov models like pfam (14), smart (15), tigrfams (16) and scop (17). each database covers a slightly different, but overlapping set of protein families / domains. each model has its own diagnostic strengths and weaknesses and for each of these protein / domain family databases used we have established different thresholds. for example, in the case of the database pfam - a, we compare the input sequence against the hidden markov model profile of each pfam protein family. in principle, it is possible to decide the significance of a match upon its e - value. however, there are a few complications such as that there is no analytical results available for accurately determining e - values for gapped alignments, especially profile hmm alignments. we use as threshold the trusted cut - off value (tc) existing for each pfam family. this value is the lowest score for sequences included in the family (e.g. in the full alignment). therefore, we consider a hit very significant if scores better than the trusted cut - off and at the same time has a significant e - value. in the case of scop, we use the scop filtering mechanism to look for consistency in the hmmscan output, and filtering out inconsistent hits. in the case of smart we use only the e - value. for each of the protein / domain databases used afterwards domainsweep takes all true positive hits of all individual database searches for further data interpretation. domain hits are listed as significant : if two or more hits belong to the same interpro family. the task compares all true positive hits of the different protein family databases grouping together those hits, which are members of the same interpro family / domain.if the motif shows the same order as described in prints or blocks. both databases characterize a protein family with a group of highly conserved motifs / segments in a well - defined order. the task compares the order of the identified true positive hits with the order described in the corresponding prints or blocks entry. only hits in correct order the task compares all true positive hits of the different protein family databases grouping together those hits, which are members of the same interpro family / domain. if the motif shows the same order as described in prints or blocks. both databases characterize a protein family with a group of highly conserved motifs / segments in a well - defined order. the task compares the order of the identified true positive hits with the order described in the corresponding prints or blocks entry it is clear that any automatically produced sequence analysis implies a reasonable compromise between sensitivity and selectivity, and that no ideal recognition threshold exists that would allow for perfect separation of true and false similarities. our thresholds tend to be rather conservative and stringent and thus the possibility of extending false positives is very limited. the output in the web consists of two groups of graphs, those corresponding to the significant and putative hits, and one table output containing all reported protein domains (supplementary figure 2). putative hit a cartoon of the sequence with the domain corresponding to the match, the hit i d, description, begin, end and gene ontology (go) annotation. the user has immediate access to all complete application outputs and database entries (via hyperlinks) by clicking on the corresponding part of the picture. at the bottom of each graph the table output contains all hits, ids, descriptions and links to the original output. the xml output containing all the generated information is available via hyperlink at the bottom of the task output. 2dsweep identifies the structural domains in the protein and therefore aids in finding structural elements. it reports on predictions for alpha - helix, beta - strand, coiled - coil and helix - turn - helix motifs, transmembrane regions, signal sequences, hydrophobicity, antigenicity, protease cleavage sites and more. when predicting the secondary structure of a protein, it is useful to exploit the features of several available prediction algorithms rather than to rely on a single program. unfortunately, combining prediction methods on a large scale is complicated by the fact that prediction programs have very different input requirements and output formats. some of them perform much better when they have a multiple sequence alignment covering different degrees of similarity as input instead of a single sequence. we have developed msfgenerator, a program, which creates a multiple sequence alignment for a single protein sequence according to user, defined rules (supplementary data msf). it performs a blast search against a non - redundant protein database following different strategies that will generate different kind of alignments (supplementary data msf, figure 4). the output of msfgenerator is an alignment in msf format (multiple sequence file). the generated msf will be used as input for four different structure prediction programs : psipred (18), jnet (19), prof (20), and dsc (21). psipred is a two - stage neural network that bases its prediction on position specific scoring matrices, jnet is a neural network method that works by utilizing an alignment as input, alongside psiblast (22) and hmm profiles. dsc is based on decomposing secondary structure prediction into basic concepts and then uses simple and linear statistical methods to combine them. since dsc is known to perform worse than the other prediction methods employed in 2dsweep, the usage of dsc is optional. figure 4.2dsweep flowchart. as a second concept, 2dsweep searches for dssp (definition of secondary structure of the protein, (23) annotation for the input protein. found it extracts secondary structure elements (if any) from the structure definition of the dssp database. if there is more than one element covering the same sequence region, 2dsweep uses a simple majority vote to determine the structure at each position. the result of this procedure is shown together with the prediction of the different secondary structure prediction tools. first, the distribution of small, charged and hydrophilic amino acids are shown and probable antigenic regions are indicated. furthermore, the task searches for transmembrane helices and intervening loop regions using four different methods : tmhmm (24), das (25), tmap (26) and tmpred. in eukaryotic protein sequences finally, information is given about molecular weight, isoelectric point, the distribution of protease cleavage - sites, and the possible sub - cellular localization of the protein. the web output of 2dsweep (supplementary figure 3) is divided in five sections : (i) general information, (ii) secondary structure, (iii) features and (iv) cleavage sites. the information provided in each of these sections is shown in figure 4 and supplementary table 3. the complete results can be viewed by clicking on the corresponding part of the picture. at the bottom of each graph there is a link to the corresponding explanatory legend. as in the other tasks the xml output containing all the generated information is available via a hyperlink at the bottom of the task output these servers have been implemented using the w3h task framework (27), which allows the execution of compound jobs using work and data flow descriptions in a heterogeneous bioinformatics environment using meta - data information. the system regulates the dataflow by specifying dependency rules between the used applications in the meta - data, which allows the design of high complexity bioinformatics tasks, and stores the results of the different applications together with the new results computed during the process. the final output of the task is an xml file which contains all relevant information generated. the xml information is transformed by means of w2h 's (28) post - processing mechanism into an html page for the task report using the extensible style - sheet language transformations (xslt ; http://www.w3.org/tr/xslt for facilitating a final visual inspection of the results. furthermore, the xml output can be also required and used for further analysis (i.e. direct integration in user 's databases, additional pipeline analysis). all public databases used by these servers are installed under the sequence retrieval system (srs) at the dkfz (29). the dkfz srs server contains more than 500 databases that are automatically updated whenever new releases become available ; this means that the webservers will be using the very last version of each database. therefore, as new and improved algorithms and methodologies are developed, they are incorporated into the protein analysis process without having to redesign the entire task. it is also possible to incorporate specific sets of databases as they become available, and to implement arbitrary configuration parameters. the development of the three pipelines presented here, has been user - driven from the beginning. their functionalities are continually being updated and extended in response to requests and suggestions emerging from our core users like lifedb (30,31), where these servers are actively used in their protein analysis and annotation. we are currently developing checks especially through the application of filtering strategies and algorithms that will take into account the relationships between domain structure and homology searches. at the moment we are starting to develop a filtering system for the homology searches results taking into account the different quality of annotation in different protein databases with the idea to assign confidence levels and cross - checking results between tasks. we are additionally working on the implementation of directed text mining using the keywords of the proteins description.	the wealth of transcript information that has been made publicly available in recent years has led to large pools of individual web sites offering access to bioinformatics software. however, finding out which services exist, what they can or can not do, how to use them and how to feed results from one service to the next one in the right format can be very time and resource consuming, especially for non-experts.automating this task, we present a suite of protein annotation pipelines (tasks) developed at the german cancer research centre (dkfz) oriented to protein annotation by homology (protsweep), by domain analysis (domainsweep), and by secondary structure elements (2dsweep). the aim of these tasks is to perform an exhaustive structural and functional analysis employing a wide variety of methods in combination with the most updated public databases. the three servers are available for academic users at the husar open server http://genius.embnet.dkfz-heidelberg.de/menu/biounit/open-husar/
alopecia areata (aa), a nonscarring type of hair loss, is the most prevalent autoimmune disease, with a lifetime prevalence of 1.7% [1, 2 ]. men and women are equally affected, and onset of the disease can occur at any age ; however, most cases start before the age of 30. fifty percent of children and adolescents with aa suffer from depression. autoimmune diseases such as vitiligo a high concordance rate among monozygotic twins was reported [7, 8 ], and a positive family history is linked to aa [9, 10, 11 ]. additionally, human leukocyte antigen (hla)-dqb1, hla - drb1, hla - a, hla - b, hla - c and also the genes notch4, mica, ptpn22 and aire were found to be associated with aa [2, 12 ]. the first genome - wide association study found 8 loci (table 1) with genome - wide significance containing multiple genes involved in the adaptive t cell - driven immune response. the current view is that both genetic and immune factors contribute to the development of aa (fig. much less well - defined environmental and psychologic elements are sure to have some influence as well. a cochrane review analyzing 17 randomized controlled trials concluded that there is currently no effective evidence - based treatment for aa. even though topical minoxidil, cyclosporine, corticosteroids (as well as systemic corticosteroids) and photodynamic therapy are used, there is no firm evidence of superiority compared to placebo. however, in daily clinical use, all these drugs are used with apparent success. performed microarray and rt - pcr of 27 lesional and 17 nonlesional samples of patients with aa. it was shown that th1, th2, and il-23 cytokine were increased, while th17/th22 skewing was lacking. additionally, also ustekinumab, a monoclonal il-12/23 inhibitor, is of interest as a potential treatment of aa. there have been case reports that ustekinumab causes aa [15, 16, 17 ], but in contrast, successful treatments with significant increase of hair growth were reported [18, 19 ]. the possibility of reversal of aa by janus kinase (jak) inhibitors was successfully shown in the murine model. additionally, craiglow and king published a case of a 25-year - old patient with psoriasis vulgaris and alopecia universalis, a type of aa in which complete loss of hair of the entire body is observed. after treatment with tofacitinib, a jak1/3 inhibitor approved for the treatment of rheumatoid arthritis, complete regrowth of hair was observed. also, one case from germany responded well to tofacitinib (u. mrowietz, personal communication). in another case report, 3 patients suffering from aa were successfully treated with ruxolitinib, a jak1/2 inhibitor approved for myelofibrosis. his past medical history revealed a bilateral chronic retinal vasculitis and uveitis, for which he had been treated in the past with various drugs such as methotrexate, azathioprine, oral prednisolone and infliximab. two years before, while receiving infliximab and azathioprine, sudden loss of hair had occurred on the temples, and drug - induced aa was suspected. even though the drug treatment was stopped, four months later, the retinal vasculitis showed progression of disease as well, so infliximab and azathioprine were started again. subsequently, treatment with topical and oral steroids, followed by topical diphenylcyclopropenone as well as oral methotrexate (up to 30 mg per week) was initiated. however, no regrowth of hair was observed after 6 months. upon his first consultation in our clinic, a biopsy confirmed sparse lymphocytic infiltrates along nonsclerotic fibrous tracts extending along the site of previous follicles. 2a), but after 3 months of treatment, growth of short terminal pigmented hair was detected (fig. 2b). these, however, disappeared again within a single month, resulting in renewed complete alopecia (fig. 2c) the efficacy of tofacitinib has been suggested by murine experiments and by one case presentation [20, 21 ]. tofacitinib citrate (xeljanz) abrogates il-15 signaling and thus mediates il-15 activation of lymphocytes. even though the initial clinical results were promising, the efficacy of tofacitinib waned again in our patient. this was even more striking when considering that the patient had additional immunosuppression by methotrexate for his retinal vasculitis. notably, methotrexate monotherapy has been shown to be a safe treatment option for aa as well. another potential reason for treatment failure could have been the presence of antibodies specific for hair follicles [23, 24 ] ; however, we were unable to measure and rule them out. the clinical observation in this patient could be interpreted as follows : suppression of aa by tofacitinib is an active process that, if too weak, may not tip the balance towards stable hair regrowth but instead allow a reversion to a completely alopecic state. although here we report only on a single case with all its limitations, it will be interesting to analyze the outcome of randomized clinical trials, especially in patients not showing efficacy to tofacitinib. also, our observation may prompt the question whether combinations of immunosuppressive drugs potentiate or inhibit each other in aa. f.a. is funded by a hsm2 (hochspezialisierte medizin) grant awarded by the kanton of zurich, switzerland.	alopecia areata is a common autoimmune disorder that targets hair follicles. swarms of lymphocytes surround the basis of the follicles, inducing loss of pigmented terminal hair and subsequently inhibit further hair growth. depending on the extent of involvement, alopecia areata can be associated with a dramatic reduction of quality of life. currently, no targeted treatment option is available, and topical immune therapies or immunosuppressive drugs are typically used with mixed success. recently, several cases of alopecia areata responding to janus kinase inhibitors were published. here, we report on a businessman with alopecia areata universalis who was treated with tofacitinib. we observed initial signs of hair regrowth in the same timeframe as previously reported, but efficacy quickly waned again, leading to renewed effluvium. thus, even though tofacitinib and ruxolitinib are a promising new treatment option, we have yet to learn more about their potential role in each particular patient 's individual treatment strategy.
although well - differentiated thyroid carcinoma (wdtc) is the most commonly diagnosed thyroid malignancy, it accounts for only 2% of all cancers in the body and is responsible for less than 0.5% of cancer - related deaths. combination therapy with thyroidectomy and adjuvant i is the treatment of choice at most institutions. the majority of patients have an excellent prognosis, with disease - specific survival rates at 10 years greater than 90%. however, 8%23% of patients will fail initial therapy and go on to develop a recurrence of their disease [14 ]. mortality rates among patients with disease recurrence have been reported to be as high as 38%69% [46 ]. in a previous study, palme. showed that wdtc patients who had either no recurrence of their disease or only one recurrence after initial therapy had no difference in disease - specific or overall survival (100% versus 94%, 89% versus 83%, resp.). in addition, patients with multiple treatment failures had significantly reduced survivals (60% and 58%, resp.. there has been a large body of research compiled over the past few decades examining various prognostic factors for both recurrence and mortality in patients with wdtc. factors such as age > 45, male sex, large tumor size, histological type, advanced stage of disease, extrathyroidal extension, lymphatic invasion, and presence of distant metastasis have all been cited as indicators of poor outcome [14, 6, 820 ]. several groups have attempted to classify patients into low-, intermediate-, and high- risk groups based on the presence of these factors [10, 17 ]. prognostication is therefore used to identify patients at high risk who require close follow - up and prompt therapy for any evidence of disease recurrence. to our knowledge, there are no reports in literature delineating prognostic factors to predict disease outcome in patients who have suffered with multiple treatment failures of wdtc. thus, the purpose of this investigation is to examine patient, tumor, treatment, and recurrence factors that may predict for mortality among patients with multiple recurrences of wdtc. thirty - one patients with multiple recurrences of wdtc were retrospectively identified from the thyroid cancer database at mount sinai hospital, toronto (19632000). recurrence was defined as any evidence of disease requiring further treatment after initial curative therapy. patient (age, sex), tumor (histology, size, stage, solitary / multifocal, extrathyroidal spread, vascular invasion, lymphatic invasion), and treatment (extent of initial surgery, adjuvant i, and external beam radiation) characteristics were collected. information about the site of each recurrence (local, regional, distant, unspecified), mode of detection (clinical, imaging, thryoglobulin estimation), and treatment (surgery, i therapy) were also recorded. extent of disease at presentation was staged according to the american joint committee on cancer (ajcc) staging system for wdtc. in addition, each patient was scored according to the metastasis, age, completeness of resection, invasion, and size (macis) prognostic index. extent of initial surgery was recorded as either a subtotal or total thyroidectomy with or without an accompanying neck dissection. a recurrence was classified as unspecified if thyroglobulin levels were elevated in the presence of a negative clinical exam and failure of localization with available imaging modalities (i.e., ultrasonography, i scanning, ct, and mri). final outcome was recorded as alive, no evidence of disease (aned), alive with disease (awd), dead, no disease (dnd), and dead of disease (dod). follow - up was counted from completion of initial therapy to the last known clinical encounter or date of death. statistical analysis of survival data was performed using the kaplan - meier method, and curves were compared using the log - rank test. univariate analysis was performed in order to identify prognostic factors significant for the development of a poor outcome (i.e., death) in patients with multiple recurrences of wdtc. multivariate analysis using the cox proportional hazards model was not possible due to the limited number of events in this study. all statistics were carried out using spss software (spss inc, chicago, ill). thirty - one patients with multiple recurrences of wdtc were identified from treatment records at the department of otolaryngology head & neck surgery, mount sinai hospital (toronto), with a median follow - up of 12.6 years (range 9 months35.5 years). there were 20 (64.5%) female patients and 11 (35.5%) male patients (median age 43, range 1683 years ; table 1). the final histopathologic diagnosis was papillary carcinoma in 19 (61.3%), tall cell variant in 5 (16.1%), follicular carcinoma in 4 (12.9%), and mixed in 3 (9.7%) cases. the median size of the dominant nodule was 3.3 cm (range 0.55.5 cm). seven of the charts did not contain a report of tumor size and thus could not be scored with the macis prognostic index. these patients were excluded from further analysis. of the 31 patients identified, 6 (19.4%) had evidence of distant metastasis at diagnosis. other tumor characteristics present at first surgery included multifocal disease in 21 (67.7%) patients, extrathyroidal spread in 18 (58.1%) patients, lymphatic invasion in 18 (58.1%) patients, and vascular invasion in 3 (9.7%) patients. according to the ajcc staging system, stage i disease was present in 4 patients (12.9%), stage ii in 8 patients (25.8%), stage iii in 11 patients (35.5%), and stage iv in 8 patients (25.8%). the macis prognostic index was applied to 24 cases in the series with complete pathology records (median score 6.03, range 3.2511.82). extent of initial surgery was dependent on both disease severity and the prevailing treatment philosophy at the time of diagnosis. a total thyroidectomy was performed in 19 (61.3%) patients, whereas a subtotal thyroidectomy was performed in 12 (38.7%) cases. a neck dissection accompanied thyroidectomy in 15 patients with evidence of nodal metastasis at initial surgery. almost all patients in this series were treated with adjuvant i (87.1%). ten patients had residual disease severe enough after initial operation to warrant external beam radiation therapy (ert). the average time to first recurrence was 25.4 months (range 0.2185.4 months). recurrences were classified as local (19.4%), regional (48.4%), distant (19.4%), and unspecified (12.9%). neither the mode of detection (i.e., clinical, imaging, or elevated thyroglobulin) nor the method of treatment (surgery, i, or ert) for recurrent disease was found to be a significant predictor of survival in this study population. thirty - two percent of patients with multiple recurrences of wdtc died of their disease (dod). other outcomes included alive, no evidence of disease (aned) in 29% and alive with disease (awd) in 38.7%. no patients in this series died of causes unrelated to their thyroid carcinoma. actuarially predicted disease - specific survival among patients with multiple treatment failures at 20 years was 60%, a significant reduction from that of patients with either no recurrences or only one recurrence of their disease. univariate analysis revealed that age > 45, stage iii / iv disease, distant metastasis, vascular invasion, macis score > 6, and a time to recurrence of 45, stage iii / iv disease, distant metastasis, vascular invasion, macis score > 6, and a time to recurrence of 4 cm, has been shown to adversely affect mortality in multiple trials [911, 14 ]. although greater tumor size (i.e., > 4 cm) was found to be a predictor of multiple recurrences in patients with wdtc, it did not appear to predict for mortality in this population. the ajcc staging system, which incorporates the tnm (tumor, lymph nodes, metastases) classification, is the current standard in staging thyroid malignancies. stage iii / iv disease (iii = ets or nodal metastases, iv = distant metastases) appears to portend an increased risk of mortality among patients with multiple recurrences of wdtc. this is in agreement with other authors, who have found that advanced stage disease not only increases the risk of recurrence but aslo significantly reduces disease - specific survival [79, 12, 13, 15 ]. in the present study, there appears to be a trend toward significance for the adverse effect of extrathyroidal extension (t3) on mortality (p =.07). in addition, we found that neither lymphatic invasion nor initial neck dissection showed a statistical significance for mortality among patients with wdtc. however, the presence of vascular invasion did appear to portend a poor prognosis on survival in this cohort (p =.002). lastly, distant metastases were found to be a highly significant predictor of mortality among patients with multiple recurrences of wdtc (p =.0002). patients who have their first treatment failure 6) have a significantly higher risk of mortality. in addition, time to first recurrence within 12 months of initial therapy conveys a worse prognosis. interestingly, mortality rates in this study were not influenced by the method of detection nor the type of therapy chosen for first recurrences. one potential explanation is that these patients have a more biologically aggressive variant of wdtc which does not readily respond to treatment of the primary tumor or the initial recurrence. although the failure to cure these cases after several attempts may cause frustration in both the treating physician and the patient, close follow - up and aggressive treatment of further recurrences is still warranted, as approximately 30% of these will go on to be free of disease after subsequent therapies. further research is needed into the biological and molecular markers of tumor severity in order to provide an understanding of why some patients with wdtc have an excellent prognosis with complete cure, while others are plagued by multiple treatment failures and eventual death.	introduction. patients with multiple recurrences of well - differentiated thyroid carcinoma (wdtc) have markedly reduced overall survival when compared with those who have 1 recurrence of their disease. the purpose of this investigation is to identify prognostic factors for mortality in this subgroup. methods. patients with multiple recurrences of wdtc were retrospectively identified from the thyroid cancer database at mount sinai hospital, toronto (19632000). data on patient, tumor, and recurrence characteristics were collected, and each patient was given a macis score. results. a total of 31 patients were identified (11 male, 20 female ; 1683 years). using univariate analysis, age > 45, stage iii / iv disease, distant metastasis, vascular invasion, macis score > 6, and time to recurrence of < 12 months were found to be significant predictors for mortality in this subgroup. conclusions. patients with multiple recurrences of wdtc follow a distinct clinical course, marked with multiple treatment failures and a substantial risk of mortality.
cryptococcosis is a life - threatening opportunistic infection caused by the yeast like fungus, cryptococcocus neoformans affecting both the immunocompetent and immunocompromised individuals. among the immunocompromised individuals, as seen in hiv patients, central nervous system and lungs are the commonly affected sites. few of the unusual sites involved are optic nerve, bone, liver and lymph node. we describe a case of disseminated cryptococcosis presenting as generalized lymphadenopathy, clinically mimicking tuberculous lymphadenitis, diagnosed on fine needle aspiration cytology (fnac). a 32-year - old male individual, an old treated case of tuberculous lymphadenitis presented to the medicine outpatient department with chief complaints of progressive breathlessness, weight loss and low - grade fever on and off for 3 months. on examination, multiple bilateral cervical, axillary and inguinal lymph nodes were present, the largest of them involving the axillary group measuring 3 2 cm. the blood film showed dual deficiency anemia, leukocytosis with toxic changes in neutrophils and thrombocytopenia. chest radiograph showed miliary mottling and right pleural effusion suggestive of tuberculosis ; ultrasonography of the abdomen showed hepatosplenomegaly and enlarged para - aortic lymph nodes. routine examination with may - grnwald - giemsa (mgg) [figure 1a ] and hematoxylin and eosin (h and e) stained smears demonstrated numerous spherical yeast cells of variable size with occasional budding forms surrounded by a clear halo suggestive of a capsule, lymphocytes and histiocytes in a necrotic background. (a) numerous spherical yeast cells of variable size with occasional budding forms surrounded by a clear halo suggestive of a capsule, lymphocytes and histiocytes in a necrotic background (mgg, 200).(b) budding yeast forms surrounded by a thick capsule (yellow arrow) (india ink preparation, 400). (c) budding yeast forms (red arrow) of variable size surrounded by a clear halo (gomori 's methenamine silver, 400) thick capsule was demonstrated on india ink preparation [figure 1b ] which was further highlighted by mucicarmine stain, imparting it a red color. other special stains used to demonstrate the yeast forms were periodic acid schiff stain (pas) and gomori 's silver methenamine stain [figure 1c ]. cryptococcus was also demonstrated in the patient 's skin lesions from skin scrapings and bone marrow aspiration including biopsy. the definitive diagnosis of c. neoformans was proven by isolating the fungus from the lymph node aspirate culture. - grnwald - giemsa (mgg) [figure 1a ] and hematoxylin and eosin (h and e) stained smears demonstrated numerous spherical yeast cells of variable size with occasional budding forms surrounded by a clear halo suggestive of a capsule, lymphocytes and histiocytes in a necrotic background. (a) numerous spherical yeast cells of variable size with occasional budding forms surrounded by a clear halo suggestive of a capsule, lymphocytes and histiocytes in a necrotic background (mgg, 200).(b) budding yeast forms surrounded by a thick capsule (yellow arrow) (india ink preparation, 400). (c) budding yeast forms (red arrow) of variable size surrounded by a clear halo (gomori 's methenamine silver, 400) thick capsule was demonstrated on india ink preparation [figure 1b ] which was further highlighted by mucicarmine stain, imparting it a red color. other special stains used to demonstrate the yeast forms were periodic acid schiff stain (pas) and gomori 's silver methenamine stain [figure 1c ]. cryptococcus was also demonstrated in the patient 's skin lesions from skin scrapings and bone marrow aspiration including biopsy. the definitive diagnosis of c. neoformans was proven by isolating the fungus from the lymph node aspirate culture. cryptococcosis caused by c. neoformans was first described in the 1890s though its increased prevalence was reported in the early 1980s owing to hiv pandemic, increased medical awareness and advanced diagnostic facility. the first indigenous case of cryptococcosis was reported in kolkata, india. c. neoformans traditionally has two varieties. c. neoformans var. cryptococcus is a fungus existing as yeast form, both in vitro and in vivo. the yeast cells show variable sized diameter ranging from 5 to 25 some of which show budding daughter yeast cells attached by a narrow base with a characteristic halo - like thick capsule made of polysaccharides. the capsule is antiphagocytic and the capsular polysaccharide released in to the tissue initiates immune dysfunction. the cryptococcal polysaccharide is the antigen that is measured as the diagnostic marker of c. neoformans. the mode of spread is by inhalation of the aerosolized infectious particles. c. neoformans is frequently found in the soil contaminated with pigeon droppings. the differential diagnosis to be considered in an immunocompromised patient with generalized lymphadenopathy can be broadly divided in to non - neoplastic and neoplastic causes. the non - neoplastic entities include mainly infective etiologies and reactive hyperplasia (follicular). infective etiologies include bacteria (tuberculosis), fungi (histoplasmosis, cryptococcosis), virus and parasites (toxoplasmosis). the neoplastic causes include lymphomas (hodgkin 's and non - hodgkin 's lymphoma), kaposi sarcoma and metastatic cancers. fnac of the largest lymph node is performed subjecting the aspirates to routine stains (mgg, h and e). special stains for tuberculosis (ziehl - neelsen stain) and fungi (pas and gomori 's silver methenamine stain) must be performed as and when indicated based on cytological findings. if the fnac is inconclusive, excision biopsy of the lymph node is done. if cytological features of neoplastic etiology are demonstrated, excision biopsy of the lymph node must be performed for categorical diagnosis with use of supportive ancillary tests like immunohistochemistry, flow cytometry analysis and cytogenetics. the diagnosis of cryptococcus is made by microscopically demonstrating yeast cells in tissue specimens like cerebrospinal fluid (csf), sputum, lymph node aspirate and skin scrapings. the yeast cells are surrounded by a thick halo - like capsule demonstrated with india ink preparation and other special stains like meyer 's mucicarmine which imparts red color to the capsule. other special stains used to demonstrate the yeast cells are pas, gomori 's silver methenamine stain and alcian blue. the definitive diagnosis can be made by culturing the blood, csf, sputum, bronchoalveolar lavage, urine and cytological aspirates. several other organs including the skin, heart, testis, prostate and eye have also been affected by c.neoformans there were three reported cases of disseminated cryptococcosis involving the lymph node proven on fnac, of which two were immunocompromised.[79 ] banerjee. in their review highlighted a case similar to ours that had presented with fever, weight loss and cervical lymphadenopathy and diagnosed as tuberculosis. lymph node biopsy revealed abundant c. neoformans cells which were supported by positive testing for cryptococcal antigen in the serum. to conclude, a high index of suspicion is needed to identify mimickers like cryptococcus which in our patient had initially presented as generalized lymphadenopathy.	cryptococcosis is a common opportunistic infection among immunocompromised individuals. some of the commonly affected sites are respiratory and central nervous system. lymph node is an unusual site of involvement which could mimic tuberculosis, as seen in our case. we report a 32-year - old male immunocompromised patient presenting with generalized lymphadenopathy who was clinically suspected to have tuberculous lymphadenitis. he was diagnosed to have disseminated cryptococcosis on fine needle aspiration cytology and fungal isolation on culture.
surgical replacement (aortic valve replacement [avr ]) via sternotomy utilising extracorporeal circulation and cardioplegia still remains the gold standard of therapy for patients with symptomatic stenosis of the aortic valve. excellent long - term clinical results and low mortality, as well as morbidity are achieved in patients with a low perioperative risk. the decision on the type of intervention is taken based on the patient 's age, his/ her overall clinical condition, severity of his/ her problems, disease progression, as well as his/ her social background. interventional cardiology and cardiosurgery units offer minimally invasive, catheter - based avr or transcatheter aortic valve implantation (tavi), as an alternative to avr. in general, high - risk american society of anesthesiologists (asa physical status) iii iv patients are candidates for tavi. the risks and predicted mortality can be calculated using euro score ii, which is most commonly used in cardiac surgery. the success rate of procedures at specialised sites approaches 100%, with perioperative mortality rates reaching 1 - 5%, and are most commonly caused by acute heart failure. compared with classical avr, important benefits offered by the tavi procedure include its mini - invasiveness and the absence of surgical wound, no extracorporeal circulation, no cross - clamping of the aorta, and no need for administration of blood products. tavi is the method of choice in patients with a markedly calcified (porcelain) aorta. however, tavi is also associated with complications, the most common of which are acute left - sided heart failure, occlusion of coronary arteries, bleeding due to aortic injury and groin bleeding. in spite of demonstrably better results of tavi compared with conventional treatment in patients contraindicated for avr and better short - term mortality in high - risk patients after tavi compared with avr, better long - term results (35 years) based on current evidences, tavi appears to be a safe and less complicated method of avr for high - risk patient groups. in 2002, alain cribier performed the first catheter - based avr via the femoral vein. access to the right atrium through the venous circulation and transseptal access to the left - sided compartments have been replaced by the retrograde route via the femoral arteries, which is the most common route of access for tavi today. transapical access is an alternative method in which a mini - thoracotomy is used to puncture the anterolateral wall of the left ventricle and an aortic valve is then inserted. however, this procedure is associated with a relatively higher number of complications including the rupture of the left ventricular apex or even aneurysm formation, injuries to the right ventricle, the inter - ventricular septum and coronary arteries, or damage to the mitral valve. transapical implantation is more suitable in patients with limited femoral blood flow, or other contra indications for its catheterisation. considering the reduction in the sizes of instruments used for transfemoral access, as well as the generally lower complication rates, the transfemoral access has become the preferred access today. patients undergoing tavi procedures are high - risk patients as mentioned already (asa iii iv) and the decision about the anaesthesia management is very important. tavi can be carried out under local anaesthesia, analgosedation, or general anaesthesia (ga). spinal or epidural anaesthesia and analgosedation provide the advantage of a lower overall rates of complications for the patient without affecting cognitive functions and subsequent length of hospital stay. local anaesthesia can be used for tavi when carotid artery is used as route of access. analysis of data from 2326 patients shows that transfemoral tavi performed under local anaesthesia is generally preferred but there is a higher incidence of post - procedural aortic regurgitation. we prefer ga for all tavi procedures at our cardiac centre, including for high - risk patients. the internal jugular vein (most commonly on the right) is cannulated in all patients the evening before the procedure. prophylactic antibiotics are administered and the radial artery is most often cannulated on the left, (based on the results of allen 's test) and a urinary catheter is inserted. the catheterization laboratory is prepared to manage any potential complications [emergency cardiopulmonary bypass (extracorporeal circulation) and extracorporeal membrane oxygenation ]. under ga with tracheal intubation, transvenous right - ventricular stimulation electrode and trans - oesophageal echocardiography probe ga is induced using etomidate 0.20.3 mg / kg or propofol 1% 12 mg / kg ; tracheal intubation is performed with rocuronium 0.6 mg / kg induced relaxation and analgesia provided by sufentanil 0.10.4 g / kg as a single shot. ga is continued with oxygen, air and sevoflurane (expired concentrations not to exceed 1.2%). neuromuscular block is reversed with sugammadex 2 mg / kg and patient shifted to intensive coronary care unit (iccu). ga is needed in order to maintain ventilation and haemodynamic stability of the patient throughout tavi implantation, particularly during the balloon valvuloplasty procedure that precedes the valve implantation. in both the phases of tavi where the flow through the aortic orifice is virtually obliterated, an external pacemaker must be used for some time (usually 2045 s), to reduce the cardiac output by initiating ventricular tachycardia (by overstimulation to approximately 180 beats / min). ga also allows for performing oesophageal echocardiographic assessment at any time during the procedure (to measure the annulus size, determine aortic pathology, determine the left ventricular function, evaluate mitral regurgitation, verify the position of the implanted valve and aortic regurgitation immediately after the procedure). ga is also convenient in the event of a protracted procedure when the patient is expected to be absolutely still. the need for circulatory support using vasopressors (norepinephrine) or inodilators (milrinone, levosimendan) is not uncommon. in the iccu since 2010, medtronics corevalve (medtronics, minneapolis, minnesota, u.s.a.) valves have been used at our unit and till the time of submission of this article, 112 valves have been inserted. our 30-day mortality is at the same level as other large european medical centres. in order to prevent bleeding complications from the puncture site we have not yet encountered post - procedure acute heart failure, adverse cerebrovascular events or coronary ischaemia ; however, two patients needed surgical intervention for pericardial tamponade with successful outcomes. in an effort to avoid difficulties following transfemoral and transapical access and in order to make tavi suitable to patients with compromised femoral and iliac blood flow, some other approaches have been used with success. aortic valve can also be guided through the ascending aorta via a right - sided upper mini - thoracotomy (second or third right intercostal space), in the parasternal region under direct vision. selective ventilation of the left lung throughout the procedure is necessary (performed at our centre) ; it ensures better surgeon comfort. this is done by inserting a double lumen endobronchial tube (insertion of a bronchial blocker is an alternative without the need of reintubating using a regular endotracheal tube at the end of the procedure). selective ventilation of the left lung may, however, precipitate hypoxia in haemodynamically unstable patients with risk of subsequent cardiac failure. this procedure provides the advantage of inserting the instruments in the ascending aorta under visual control. it is not suitable for patients with pleural adhesions as there could be incomplete collapse of the lung during one - lung ventilation. implantation via brachiocephalic artery is suitable in patients with severe atherosclerosis, if the diameter of femoral arteries is less, if there is sepsis in the region of subclavian artery access, prior intrathoracic surgery and in severe calcification of the aorta. implantation via left carotid artery is suitable and safe in patients where the transfemoral, transapical or transaxial access can not be used. implantation via axillary artery is suitable for patients in whom the use of other modes of access is not possible. direct access from the subclavian artery is the method of choice and the most common access. this approach provides the advantages of a smaller surgical wound, lower blood loss without the need for blood transfusion, better healing, fewer infectious complications, and a shorter time of overall hospitalisation. a disadvantage of this procedure is the relatively limited orientation of structures and difficulty in tissue manipulation in the event of sudden massive surgical bleeding. partial upper sternotomy has also been described as an urgent, life - saving approach to handle complications during tavi using transfemoral access. the greatest advantages of upper partial sternotomy for tavi include simpler handling of instruments and the incision of the distal part of the ascending aorta, which is then used to insert the instruments. this approach does not require one - lung ventilation, and therefore it is well tolerated in compromised patients. surgical replacement remains the gold standard of therapy in patients with symptomatic stenosis of the aortic valve. this procedure is not suitable for certain groups of high - risk patients where mini - invasive, catheter - based avr is the method of choice. over the last 12 years each technique has its own merits and demerits ; we prefer ga in our centre.	for symptomatic patients with severe calcified aortic valve stenosis, open heart surgery for aortic valve replacement remains the gold standard. however, elderly patients with an increased risk profile can be treated by using transcatheter approaches (transcatheter aortic valve implantation [tavi ]). the major considerations related to use of general and local anaesthesia for tavi are discussed in this review.
mal de meleda (also known as keratoderma palmoplantaris transgrediens, acroerythrokeratoderma, keratosis extremitatum hereditaria progrediens) is a rare autosomal recessive transgradient keratoderma named after the croatian island of meleda (mljet). this keratoderma begins between the time of birth and 3 years of age, and is characterized by transgradient keratoderma with sharp demarcation, erythema with associated scleroatrophy, nail changes, pseudoainhum around digits and perioral erythema without a tendency for spontaneous resolution. although perioral erythema is well known in mal de meleda, involvement of the lip by keratoderma has not been reported in the english literature. here we present two unrelated cases of mal de meleda who had typical features of the syndrome with unusual lip involvement. a 15-year - old girl presented with thickening of the skin on palms and soles from 6 months of age. there was progressive worsening with slow involvement of the dorsal aspects of the palms and soles with increasing age. she also had pigmented, rough, raised grouped lesions on the elbows and knees from 5 years of age. there was redness of lips since childhood, which developed into mild thickening and dryness recently. two other siblings, parents and relatives, did not have similar complaints. on examination, there was diffuse palmoplantar keratoderma involving bilateral palms and soles and extending proximally up to the wrists and the dorsa of hands and feet with well - defined margins [figures 1 and 2 ]. the skin on the palms and soles was thick, waxy and yellow with loss of dermatoglyphics. multiple well - defined lichenoid papules were present on the extensor aspects of the elbows. well - defined plaques were seen on the extensor aspects of the knee joints [figure 3 ]. a diagnosis of mal de meleda was made based on the above - mentioned features. transgradient palmoplantar keratoderma with sharp margins, conical tapering of the fingers, flexion deformity, and digital constriction of the little finger of the left hand transgradient palmoplantar keratoderma with sharp margins, conical tapering of the fingers, flexion deformity, and digital constriction of the little finger of the left hand scaly plaques on the extensor aspect of the left knee lip involvement with perioral lesions a 24-year - old male presented to our dermatology opd with thickening of skin of palms and soles since birth and with progressive involvement of the dorsa of hands and feet for last 5 years. he also had hyperpigmented, raised, scaly plaques on the extensors of bilateral knees and elbows for last 6 months. he also had redness of lower lip with mildly painful fissuring of bilateral angles of mouth of 6 months duration. no family member was affected by similar illness. on examination, there was diffuse transgradient palmoplantar keratoderma involving bilateral palms and soles extending up to the dorsa of hands and feet with ill - defined irregular margins and prominent knuckle pads [figure 5 ]. skin on the palms and soles was thick, waxy, yellowish, malodorous, with loss of dermatoglyphics. there were multiple erosions and fissuring of varying sizes on both the palms, and pitted keratolysis on the soles. fingers had pseudosclerodermatous appearance with few showing conical tapering and digital constrictions at various places with mild flexion deformity. the patient had multiple hyperkeratotic, scaly, irregular, ill - defined plaques on extensors of bilateral elbows and knees. along with these features the patient also had a dry, erythematous, thickened lower lip with angular cheilitis [figure 6 ]. histopathology of palmar skin showed lining of stratified squamous epithelium with hyperkeratosis, hypergranulosis, and acanthosis. superficial dermis shows mild perivascular lymphocytic infiltrates. a diagnosis of mal de meleda was made. for logistic reasons, genetic analysis for slurp1 transgradient palmoplantar keratoderma with ill - defined margins, conical tapering of the fingers, flexion deformity, and digital constriction of the little finger of the right hand involvement of the lower lip with angular cheilitis a 15-year - old girl presented with thickening of the skin on palms and soles from 6 months of age. there was progressive worsening with slow involvement of the dorsal aspects of the palms and soles with increasing age. she also had pigmented, rough, raised grouped lesions on the elbows and knees from 5 years of age. there was redness of lips since childhood, which developed into mild thickening and dryness recently. two other siblings, parents and relatives, did not have similar complaints. on examination, there was diffuse palmoplantar keratoderma involving bilateral palms and soles and extending proximally up to the wrists and the dorsa of hands and feet with well - defined margins [figures 1 and 2 ]. the skin on the palms and soles was thick, waxy and yellow with loss of dermatoglyphics. multiple well - defined lichenoid papules were present on the extensor aspects of the elbows. well - defined plaques were seen on the extensor aspects of the knee joints [figure 3 ]. a diagnosis of mal de meleda was made based on the above - mentioned features. transgradient palmoplantar keratoderma with sharp margins, conical tapering of the fingers, flexion deformity, and digital constriction of the little finger of the left hand transgradient palmoplantar keratoderma with sharp margins, conical tapering of the fingers, flexion deformity, and digital constriction of the little finger of the left hand scaly plaques on the extensor aspect of the left knee lip involvement with perioral lesions a 24-year - old male presented to our dermatology opd with thickening of skin of palms and soles since birth and with progressive involvement of the dorsa of hands and feet for last 5 years. he also had hyperpigmented, raised, scaly plaques on the extensors of bilateral knees and elbows for last 6 months. he also had redness of lower lip with mildly painful fissuring of bilateral angles of mouth of 6 months duration. no family member was affected by similar illness. on examination, there was diffuse transgradient palmoplantar keratoderma involving bilateral palms and soles extending up to the dorsa of hands and feet with ill - defined irregular margins and prominent knuckle pads [figure 5 ]. skin on the palms and soles was thick, waxy, yellowish, malodorous, with loss of dermatoglyphics. there were multiple erosions and fissuring of varying sizes on both the palms, and pitted keratolysis on the soles. fingers had pseudosclerodermatous appearance with few showing conical tapering and digital constrictions at various places with mild flexion deformity. the patient had multiple hyperkeratotic, scaly, irregular, ill - defined plaques on extensors of bilateral elbows and knees. along with these features the patient also had a dry, erythematous, thickened lower lip with angular cheilitis [figure 6 ]. histopathology of palmar skin showed lining of stratified squamous epithelium with hyperkeratosis, hypergranulosis, and acanthosis. superficial dermis shows mild perivascular lymphocytic infiltrates. a diagnosis of mal de meleda was made. for logistic reasons, genetic analysis for slurp1 transgradient palmoplantar keratoderma with ill - defined margins, conical tapering of the fingers, flexion deformity, and digital constriction of the little finger of the right hand involvement of the lower lip with angular cheilitis the obligatory clinical features of mal de meleda are (1) autosomal recessive inheritance, (2) onset of diffuse keratoderma palmoplantaris soon after birth (or up to 3 years of life), (3) transgressive and progressive nature of the keratoderma which tends to involve the dorsa of hands and fingers, feet and toes, flexor aspect of the wrist (glove and stocking keratoderma) with sharp margin. the facultative clinical features are (1) palmoplantar hyperhidrosis, (2) pitting in the keratoderma palmoplantaris, (3) lichenoid polycyclic plaques on the elbows, knees, and groins, (4) subungual keratosis, koilonychia, dystrophy of the great toenail, (5) progressive conical tapering of the fingertips which may lead to contractures of the fingers, (6) perioral erythema, (7) high arched palate, (8) corneal lesions. digital constrictions other rare features include lingua plicata, syndactyly, left handedness and hair on palms and soles. the involvement of the lips as was seen in both our cases is unique and hitherto unreported in the english literature. there is a great interindividual variation in the clinical manifestations of mal de meleda which should be differentiated from other similar palmoplantar keratoderma syndromes. greither 's disease has several clinical similarities with mal de meleda syndrome, but can be differentiated based on the following features. greither 's disease is autosomal dominant, starts during 8 - 10 years of age with a gradual onset and a tendency to improve with age. mal de meleda is autosomal recessive, appears early after birth and slowly progresses without any tendency for spontaneous involution. both have transgradient keratoderma and hyperkeratotic plaques on the extensor aspects of the knees and elbows. in contrast to mal de meleda syndrome, the palms and soles may be spared in greither 's disease. conical tapering of the fingertips seen in mal de meleda syndrome is not seen in greither 's disease. constrictive bands and nail changes are frequent with mal de meleda, but unusual in greither disease. huriez syndrome (also referred to as sclerotylosis) is an autosomal dominant genodermatosis, characterized by the triad of congenital diffuse scleroatrophy of the distal extremities, mild or lamellar keratoderma of the palms and to a lesser extent the soles, presence from birth and ridging or hypoplastic nail changes. olmsted 's syndrome is a severe, cicatrizing transgradient palmoplantar keratoderma with keratotic plaques around the mouth, nose and anus. onset is in the first year of life, with symmetrical, sharply defined palmar and plantar keratoderma surrounded by erythema, flexion deformities, constriction or spontaneous amputation of the digits, onychodystrophy, follicular keratosis, congenital universal alopecia, hyperextensibility of the joints, absence of premolar teeth and keratosis of the oral mucosa. vohwinkel 's syndrome is an autosomal dominant palmoplantar keratoderma characterized by honeycomb - like keratoderma beginning early in life associated with stellate or starfish keratoses on the dorsa of hands and feet, and the formation of circumferential bands around digits (pseudo - ainhum) and sensorineural deafness. vohwinkel 's syndrome was ruled out in our case by the absence of honey - comb keratoderma and sensorineural hearing loss. camisa 's syndrome is a variant of vohwinkel 's syndrome where sensorineural hearing impairment is absent, but these patients have mild ichthyosis which was not seen in our case. papillon lefevre syndrome (pls) is characterized by palmoplantar hyperkeratosis, severe periodontitis and premature loss of teeth. it manifests by 1 - 4 years of life and patients become edentulous by early teens. pls was easily ruled out in our case by the presence of normal teeth and the absence of periodontitis.	mal de meleda is a rare autosomal recessive transgradient palmoplantar keratoderma characterized by transgradient keratoderma with associated scleroatrophy, nail changes, pseudoainhum around digits and perioral erythema, without a tendency for spontaneous resolution. involvement of the lip by keratoderma has not been reported in the english literature. here we present two cases of mal de meleda with unusual lip involvement. the first case was a 15-year - old girl, born of second - degree consanguineous marriage, who presented with transgradient palmoplantar keratoderma from 6 months of age, with lichenoid papules and plaques on the elbows and knees, conical tapering of the distal digits, flexion deformity of several fingers, digital constriction, knuckle pads and lip involvement. the second case was a 24-year - old male with transgradient palmoplantar keratoderma since birth. he also had scaly plaques on the extensors of bilateral knees and elbows, knuckle pads, pseudosclerodermatous fingers with conical tapering, digital constrictions at various places with mild flexion deformity and lip involvement. both patients were otherwise normal without any family history.
occlusal overload has been given as the primary factor for peri - implant bone loss, as well as loss of implants and of implant - supported prostheses. the transfer of occlusal load is related with several factors such as : a) geometry - position and number of implants, linear or off - set arrangement of the implants, cantilever extension, a displaced occlusal plane, size of the occlusal table, excessive height of the abutment / crown set ; b) occlusion - parafunctional habits, bite force, occlusal contacts ; c) load - bearing capacity of bone - bone density and quality, primary mechanical stability, healing time ; and d) technological - precision of the implant / abutment and abutment / prosthesis interfaces, amount of preload, and type of prosthetic retention. occlusal loads are first introduced to the prosthesis, and are delivered to the bone / implant interface ; hence, the development and maintenance of the bone / implant interface is particularly dependent on the control of biomechanical loads. bones carrying mechanical loads adapt their strength to the load applied on them by bone modeling / remodeling. the response to an increased mechanical stress below a certain threshold will be a strengthening of the bone by increasing the bone density or apposition of bone. on the other hand, fatigue micro - damage resulting in bone resorption may be the result of mechanical stress beyond this threshold. compared to implant - supported total fixed prostheses, implant - supported partial fixed prostheses are more susceptible to the moment generated by occlusal loads, since they lack the benefit of cross - arch stabilization. moreover, the posterior region of the oral cavity presents higher occlusal loading and lower bone quality than the anterior region ; additionally, bone height is limited by the maxillary sinus or the mandibular nerve. in a retrospective clinical analysis of the relation between the fracture of implants and occlusal overload, rangert,. (1997) found that 90% of implant fractures occurred in the posterior segment, supported by one or more implants, in association with cantilever, bruxism or high occlusal loads. recent studies have investigated the stresses caused by implant - supported prosthesis fabrication methods, by varying the type of cylinder. however, these studies observed stresses only during the fixation of implant - supported fixed partial prostheses. strain gauge analysis has been used to evaluate stresses in implant - supported prostheses, both in vitro and in vivo, under static and/or dynamic loads. depending on the site to be evaluated, strain gauges can be bonded close to implants, on the implants, on the abutments, and on the metal structures of the prosthesis. the objective of the present study was to compare the magnitude of peri - implant microdeformation of three - element fixed partial prostheses obtained from prefabricated and plastic cylinders subjected to axial loads. to simulate clinical conditions in a real - life arrangement, three internal hexagon type implants from mesial to distal : labeled 1, 2, and 3 (conect ar ; 3.75-mm diameter, 13-mm depth ; conexo sistemas de prtese, aruj, sp, brazil) were arranged in the middle of a measurement model consisting of a 70x40x30 mm rectangular polyurethane block (polyurethane f16, axson, cergy, france) with known mechanical properties (young 's modulus of 3.6 gpa). the distance between the centers of the implants was set at 7 mm, leaving sufficient space for the strain gauge (sg) (figure 1). positioning of the internal hexagon implants, showing the equidistance and linear configuration microunit abutments (micro unit ; conexo sistemas de prtese) were screwed onto the implants with a 20 ncm torque using the implant manufacturer 's manual torque driver (torque driver ; conexo sistemas de prtese). the patterns were fabricated using a pattern resin (gc pattern resin ; gc europe n.v., leuven, belgium) and wax (kronen wachs ; bego bremer goldschalgerei, bremen, germany). the components were connected to the microunit abutment to eliminate the inevitable dimensional changes originating from impression procedures. the superstructures were fabricated using plastic cylinders (plastic coping ; conexo sistemas de prtese) and pre - machined cobalt - chromium cylinders (machined coping ; conexo sistemas de prtese). the superstructures were sprued, invested, and cast using a cobalt - chromium alloy (wirobond sg, bego bremer goldschalgerei, bremen, germany). to avoid bias resulting from manufacturing conditions, random sets comprising frameworks of different types were put together and cast. the castings were cleaned, finished, and polished, and care was taken not to damage the internal surface of the copings, whose interiors were inspected under a binocular microscope to check for casting imperfections. the abutments received three - unit superstructures, and each group consisted of five superstructures. the superstructures showed satisfactory adaptation which was confirmed by direct vision in conjunction with tactile sensation through an explorer. four strain gauges (sgs) (kfg-02 - 120-c1 - 11n30c2, kyowa electronic instruments co., ltd, tokyo, japan) were bonded onto the surface of each polyurethane block using a thin film of methyl-2-cyanoacrylate adhesive (m - bond 200 ; vishay measurements group, raleigh, nc, usa). sg 01 was placed mesially adjacent to implant 1, sg 02 and sg 03 were placed mesially and distally adjacent to implant 2, respectively, and sg 04 was placed distally adjacent to implant 3. each gauge was wired separately, and the 4 strain gauges were arranged in series to form a full wheatstone bridge. the leads from the strain gauges were connected to a multichannel bridge amplifier to form one leg of the bridge. a computer (intel 775p pentium 4 q6600) was interfaced with the bridge amplifier to record the output signal of polyurethane surface. data - acquisition system software (system 5000 model 5100b ; vishay measurements group, raleigh, nc, usa) was used to record the data. the superstructure 's occlusal screws were tightened onto the microunit abutments with a hand - operated screwdriver until the screws started to engage, based on tactile sensation, while applying a torque of 10 ncm using the manufacture 's manual torque - controlling device. five loading points were selected to apply a static vertical load on the metallic superstructures. point a was located on the hole of the retention screw of implant 1, point b was located centrally between the holes of the retention screws of implants 1 and 2, point c was located on the hole of the retention screw of implant 2, point d was located centrally between the hole of the retention screws of implants 2 and 3, and point e was located on the hole of the retention screw of implant 3. all of the strain gauges were zeroed and calibrated prior to each loading and a vertical load of 30 kg was applied for 10 s, using a universal load - testing machine (dl-1000 ; emic, so jos dos pinhais, pr, brazil). the magnitude of microdeformation on each strain gauge was recorded in units of microdeformation (). this procedure was repeated two more times, making a total of 3 loads per loading point (figure 2). detail of static vertical loading on loading point a in order to compare the magnitude of microdeformation resulting from the type of cylinder and the loading point, the positive and negative strains recorded in the strain gauge analysis were transformed into absolute values, which were used to calculate the mean values of microdeformation of each strain gauge. the experimental variables under study were cylinder (plastic and pre - fabricated) and loading point (a, b, c, d, e). the variable response was the micro - deformation value obtained in the strain - gauge analysis. fifty (50) data obtained were submitted to statistical analysis using the following statistical softwares : graphpad prism version 4.00, 2003 (graphpad software, inc., la jolla, ca, usa), minitab version 14.12, 2004 (minitab corporation, state college, pa, usa) and statix version 8.0, 2003 (analytical software inc. the inferential statistics consisted of analysis of variance of repeated measurements of two factors (cylinder and loading point), in which the variable loading point was considered as a repeated factor. multiple comparisons among the means for the five experimental conditions were made by the tukey 's multiple - comparison test. table 1 lists the descriptive statistical data, analyzing the mean values of microdeformation obtained with each strain gauge (sg) and mean of 4 strain gauges for the plastic prosthetic cylinders and the prefabricated co - cr cylinders at each loading point. mean values and standard deviations of microdeformation () measured by each strain gauge and mean of 4 strain gauges at each loading point for the plastic and prefabricated cylinders table 1 shows the microdeformation values () obtained, analyzing the mean values of microstrain obtained by the four strain gauges (sg) positioned around the implant, for five loading point (a, b, c, d, e), as well as the type of cylinders (plastic and prefabricated). the statistical rm anova indicated that the loading point effect was statistically significant (p=0.0001), while the interaction effect was not statistically significant, demonstrating that the cylinder effect was the same for each loading point. tukey 's multiple - comparison test was then applied to compare the mean values of the 5 levels of the loading point factor (table 2). mean values of the 5 levels of the loading point factor same superscript letters mean no significant difference at 5% (tukey s test). when an occlusal load is applied upon an implant, the load is partially transferred to the bone, with the highest stresses occurring in the implant 's most cervical region. this phenomenon is due to one of the principles of engineering, i.e., when two materials are in contact with each other and one of them is loaded, the stresses will be higher at the materials ' initial point of contact. therefore, the cervical region of the implant is the site where the greatest microdeformations occur, independently of the type of bone and the design of the implant, the configuration of the prosthesis and the load. in the present study, strain gauges were strategically bonded on the polyurethane block, tangentially to the implant platform, to observe the region with the highest concentration of stresses during the application of loads, to correlate it with clinical practice. the flat surface of the polyurethane block made the positioning and bonding of the stain gauges simpler and more precise than in other studies, which opted for bonding on the implants, on the abutments, and on the metal structures of the prosthesis. according to frost (1994) and wiskott and belser (1999), bone homeostasis occurs when the level of microdeformation remains within the range of 100 to 2000 and 50 to 1500 u, respectively. in table 1, most of the microdeformation values obtained for both the plastic and the prefabricated cylinders remained within the level of bone homeostasis, or normal load. however, this ideal clinical situation of occlusal contacts is difficult to achieve with an implant - supported partial fixed prosthesis because the hole of the retention screw makes it impossible to position the occlusal contacts in the center of the implant. nevertheless, based on the results of the current work, it can be inferred that, when a 3-element partial fixed prosthesis supported by 3 implants is loaded axially, with occlusal contacts positioned between the implants and as close as possible to the latter, bone resorption around the implants and occlusal overload can be minimized. if the load were applied around the center of the implants, the torque or moment would be greater, since the torque is directly proportional to the distance between the loading point and the center of the implant. in the present study, it was found that when loads were applied on loading points a, b, d and e, which were positioned on and close to the implants at the extremities, the largest microdeformations occurred in the closest strain gauges, indicating that the amount of load transmitted to the implant and the stresses generated in the bone depend on the location where the load is applied on the prosthesis. in contrast, when loads were applied on loading point c, which was positioned on the central implant, the greatest microdeformations occurred in the most distant strain gauges, indicating that the implants at the extremities were more loaded (table 1). these results suggest that the stresses generated by occlusal contacts located close to the central implant of the screwed fixed partial prosthesis supported on three implants are distributed to the implants at the extremities, while the stresses generated by occlusal contacts positioned close to the implants at the extremities concentrate in those implants. misfit at the abutment / prosthesis interface may lead to significant instability of the implant - supported prosthesis, which increases linearly as a function of the degree of misfit. the precision of the interfaces may also negatively affect the load - bearing ability of the implant - supported prosthesis, affecting the magnitude of the forces in the peri - implantar region. the inaccurate fit of the superstructures can attribute to the impression technique, the control of laboratory analogues, or soldering method. the procedure that attempts to compensate for shrinkage or deformation, the one - piece casting method, waxing was performed directly on the abutment in polyurethane block. studies to evaluate the fit of the abutment / prosthesis interface have demonstrated that the precision of unitary metallic structures obtained with prefabricated cylinders is better than that obtained with plastic cylinders. however, it should be noted that the care involved in handling multiple prostheses is very different from that involved in handling single ones, and the complexity of the laboratory procedures increases proportionally to the number of fixations involved. in this present study, the cylinder effect and the cylinder / this finding suggests that the type of cylinder, plastic or prefabricated, does not affect the magnitude of microdeformation when an implant - supported fixed prosthesis is axially loaded, and that the behavior of both cylinders followed the same pattern at all of the loading points, showing no significant difference. previous strain gauge studies have reported similar results, with fixed partial prostheses screwed onto implants, made from plastic or prefabricated cylinders, producing the same magnitude of microdeformation during tightening of the retention screws, without significant difference between plastic and prefabricated cylinders before and after the application of a dental ceramic. with regard to the loading point effect, a significant difference was found (p=0.0001), suggesting that symmetrical loading points, a versus e and b versus d, did not produce similar magnitudes of microdeformation. the superstructures showed satisfactory adaptation which was confirmed by direct vision in conjunction with tactile sensation through an explorer. therefore, the method used is not able to detect slight distortions of the prosthesis on the implant, probably caused by casting procedures of the implant - supported fixed partial prosthesis. this finding suggests that the fit of the cast metal rods was not homogeneous, i.e., the fit attained by the cast metal rods in implant 3 may have differed from that found in implant 1, which in turn may also have differed from the fit obtained in implant 2. thus, the nonhomogeneous fit may have influenced the distribution of stresses, producing different magnitudes of microdeformation, even when the load was applied on equidistant and symmetrical points. limitations of the present model must be taken into account when interpreting the results of this investigation. this is an in vitro study based on a homogenous model with known mechanical properties instead of bone, which allowed not only proper strain measurements, but also 100% implant - model material contact. in vivo, additional variables like bone density, implant stability, and bone - to - implant contact would have to be considered. the interimplant relationships represented a straight - line configuration of the implants, which seems to be also a simplified situation compared to a curved distribution with a longer segment splinted. the flat occlusal surface of the superstructures did not represent the " real clinical situation ", variables such as cusp inclination, occlusal table and location, direction and magnitude of applied occlusal forces on the superstructures could change the results of this study. based on the obtained results, type of cylinder, plastic or prefabricated, did not affect in the magnitude of microdeformation under axial loading and the location of the applied axial loads affected the magnitude of microdeformation.	objectivesthe present study used strain gauge analysis to perform an in vitro evaluation of the effect of axial loading on 3 elements of implant - supported partial fixed prostheses, varying the type of prosthetic cylinder and the loading points. material and methodsthree internal hexagon implants were linearly embedded in a polyurethane block. microunit abutments were connected to the implants applying a torque of 20 ncm, and prefabricated co - cr cylinders and plastic prosthetic cylinders were screwed onto the abutments, which received standard patterns cast in co - cr alloy (n=5). four strain gauges (sg) were bonded onto the surface of the block tangentially to the implants, sg 01 mesially to implant 1, sg 02 and sg 03 mesially and distally to implant 2, respectively, and sg 04 distally to implant 3. each metallic structure was screwed onto the abutments with a 10 ncm torque and an axial load of 30 kg was applied at five predetermined points (a, b, c, d, e). the data obtained from the strain gauge analyses were analyzed statistically by rm anova and tukey 's test, with a level of significance of p<0.05. resultsthere was a significant difference for the loading point (p=0.0001), with point b generating the smallest microdeformation (239.49) and point d the highest (442.77). no significant difference was found for the cylinder type (p=0.748). conclusionsit was concluded that the type of cylinder did not affect in the magnitude of microdeformation, but the axial loading location influenced this magnitude.
anaphylaxis is a severe acute allergic reaction, which can involve multiple organs and even pose an immediate threat to life. epidemiological studies indicate that the prevalence of organ - specific allergic disorders such as allergic rhinitis, asthma and eczema has increased in recent decades in many western countries. anaphylaxis typically involves the cutaneous, respiratory and cardiovascular systems, presenting with urticaria, dyspnea, wheeze and hypotension. involvement of the skin is reported in 8090% of episodes, the respiratory tract in up to 70%, the gastrointestinal tract in up to 45%, the cardiovascular system in up to 45% and the central nervous system in up to 15%. although gastrointestinal symptoms, including nausea, vomiting, diarrhea and abdominal pain, sometimes occur transiently in some patients with anaphylaxis, persistent abdominal pain is rarely detected. its prevalence is estimated at 0.052%, and the rate of occurrence appears to be increasing, mainly in young people. hospital - based studies suggest a death rate in the order of 1 per 100200 episodes of anaphylaxis treated in an emergency department. death may occur from hypoxemia due to upper airway angioedema, bronchospasm and mucus plugging and/or shock due to massive vasodilation, fluid shift into the extravascular space and depressed myocardial function. factors associated with increased risk of anaphylaxis include intercurrent infection, concomitant medication / foods (particularly -blockers, -blockers, angiotensin - converting enzyme inhibitors, nonsteroidal antiinflammatory drugs, alcohol or spicy food), high ambient temperatures and exercise. regardless of mechanism, the severity and mortality of anaphylaxis are affected by age, concomitant diseases (e.g. asthma, cardiovascular disorders or mastocytosis) and concurrent medication. atherosclerosis is considered one of the risk factors for both cardiovascular events and mesenteric ischemia when anaphylaxis occurs. in general, although chronic atherosclerotic mesenteric ischemia is characterized by frequently unrecognized unspecific symptoms and even though significant degrees of splanchnic artery stenosis usually remain asymptomatic for many years, allergic reactions may cause abdominal symptoms through vasospasms of the splanchnic artery or embolism. allergic reactions can lead to bowel ischemia as a result of arterial contraction in patients with atherosclerosis because the intestine receives 1020% of the cardiac output when fasting and up to 35% after a meal. the hemodynamic explanation is a vascular steal from the intestine to the gastric circulation stimulated by food placed in the stomach. we experienced an elderly case with chronic atherosclerotic mesenteric ischemia who started to develop the symptoms just after anaphylaxis although the association between anaphylaxis and chronic atherosclerotic mesenteric ischemia has been unclear. an 83-year - old woman who had a past history of abdominal migraine in adolescence was referred to our emergency department with acute urticaria and sudden shortness of breath approximately 30 min after taking rectal diclofenac potassium for lumbago. severe hypotension (70/46 mm hg) and an arterial oxygen saturation of 80% were observed upon arrival at the emergency room. after being given 0.3 mg of subcutaneous adrenaline and 500 mg of intravenous corticosteroids, after a few days development of anaphylaxis, the patient experienced epigastric pain especially after meals. the patient attended our hospital 1 week after the onset of anaphylaxis because of repeated epigastric pain. the pain was located in the epigastric area, starting 30 min after meals and lasting 60120 min. she had no change in bowel habits, nausea, diarrhea or fever. typically, the patient had 12 formal stools per day. her medication included amlodipine besilate, olmesartan medoxomil, magnesium oxide, pravastatin sodium, lansoprazole, inhaled tiotropium bromide hydrate and inhaled betamethasone / d - chlorpheniramine maleate. because of postprandial abdominal pain, fear of eating led to a 6 kg weight loss in the 2 months after anaphylaxis. the patient was hospitalized again for further examination and treatment for repeated postprandial abdominal pain. on abdominal examination she had slight tenderness around the umbilicus with no audible bruit, masses or hepatosplenomegaly. initial laboratory investigations (table 1) revealed normal values for complete and differential blood counts, erythrocyte sedimentation rate, serum albumin, liver enzymes and urinalysis. a serum inflammation marker, crp, was not increased and all tumor markers measured were also normal. lipid profile showed increased total cholesterol and triglyceride and normal hdl cholesterol levels (315, 151 and 65 mg / dl, respectively). blood urea nitrogen and creatinine levels were elevated, indication the presence of chronic kidney disease. fecal cytology revealed lots of red - orange - colored droplets which indicated that the feces contained inadequately absorbed lipid that was steatorrhea. the blood levels of d - dimer, fdp and fibrinogen were elevated at 2.1, 3.8 and 332 mg / dl, respectively. abdominal ultrasonography was unable to measure the velocity of the celiac artery due to calcification of the arterial wall (fig. radiographic selective catheter angiography revealed chronic mesenteric ischemia caused by atherosclerosis and abundant collateral arteries between the celiac trunk, the superior mesenteric artery and the inferior mesenteric artery (fig. 3). the patient was prescribed proton pump inhibitor, digestants, anticholinergic agents and prokinetics intermittently, but these had no beneficial effects. since steatorrhea was found by the results of fecal cytology and since blood tests showed elevated serum amylase levels suggesting potential exocrine pancreatic insufficiency, anticholinergic agents, serine protease inhibitors and pancrelipase were administered orally. however, postprandial abdominal pain did not improve even after starting these supplementations, or even under intravenous administration of scopolamine and/or hydroxyzine. based on the finding of celiac atherosclerosis, the additional treatment with secondary preventive drugs including antiplatelet agents and transdermal nitroglycerin had no beneficial effects. it was most probable that this patient with chronic atherosclerotic mesenteric ischemia was suffering from functional abdominal pain syndrome induced by anaphylaxis. since psychiatric disorders were associated with alterations in the processing of visceral sensation in the patient, it was suggested that psychological treatment would be beneficial. two weeks after admission, we facilitated the patient 's understanding of functional abdominal pain syndrome with the psychologist and prescribed gabapentin and lorazepam. postprandial abdominal pain gradually faded after administration of these drugs and the patient left the hospital. acute mesenteric ischemia usually caused by embolism is characterized by a typical acute onset of diffuse abdominal pain, whereas chronic mesenteric ischemia is characterized by frequently unrecognized unspecific symptoms such as postprandial abdominal pain and unintended weight loss. patients with chronic mesenteric ischemia usually present with a clinical syndrome characterized by painful abdominal cramps and colic typically occurring during the postprandial phase. stenosis of visceral vessels, including the celiac artery, is usually well tolerated because of the abundant collateral circulation, leading to a potentially underdiagnosed condition. radiographic selective catheter angiography revealed chronic mesenteric ischemia caused by atherosclerosis, and an elevated blood d - dimer level also suggested chronic mesenteric ischemia in our case. however, the onset of postprandial abdominal pain was clear and symptoms lasted for 2 months. unlike abdominal angina, it has been well known that hypersensitivity reactions associated with underlying coronary artery disease are not rare. allergic angina syndrome was described in 1991 as the coincidental occurrence of chest pain and allergic reactions accompanied by clinical and laboratory findings of classic angina pectoris caused by inflammatory mediators released during the allergic insult. there are currently two variants of kounis syndrome : the first is observed in patients with no cardiovascular risk factors and with healthy coronary arteries in which the inflammatory cascade triggered by the allergic insult causes a coronary vasospasm, the second is observed in patients with pre - existing atheromatous diseases. the existence of mastocytes in heart tissue and their participation in the anaphylactic reaction that triggers tachycardia, coronary vasoconstriction, dysfunctional ventricular contractility and blockade of arterioventricular conduction is well known. mast cells occur in considerably higher amounts in the intima and adventitia of vessels with pre - existing atheromatic lesions, particularly in marginal regions of atherosclerotic plaques. in place of plaque rupture, about 200 times more mast cells are found compared with surrounding vascular segments without atheromatic changes in patients with nonallergic acute myocardial infarction. the histamine concentration and the number of mast cells in the arterial wall increase proportionally with the extent of atherosclerosis. peptidases released from stimulated mast cells activate metalloproteinases which degrade connective tissue covering the atheromatic plaque. moreover, prostaglandin d2, leukotrienes and angiotensin ii influence contracted coronary vessels. applying the pathophysiologic mechanisms of kounis syndrome to chronic mesenteric ischemia to explain the involvement of the gastrointestinal tract in anaphylactic reactions, the onset of postprandial abdominal pain just after anaphylaxis could be understood. acute intestinal ischemia stimulates visceral afferent nerves, but the mechanisms responsible for this excitation are not fully understood. mast cells may participate in this process as they are known to signal to mesenteric afferents during intestinal anaphylaxis. also reported that histamine potentially acts as a mediator in mast cell to afferent nerve communication in the small intestine. histamine has been shown to stimulate nociceptive afferent fibers in a variety of tissues such as skin, joints and muscle. the gastrointestinal tract contains mechanisms that enable differentiation between nutrients indispensable to the organism and antigenic substances that signify a potential threat. hypersensitivity reactions give rise to intestinal sensory and motor events that are designed to dilute and eventually eliminate the antigen from the gut. if activated mast cells are present around nociceptive afferent fibers, persistent postprandial abdominal pain can occur for a long time like in the present case. although severe hemorrhagic gastritis and acute ischemic colitis following anaphylaxis were reported, there was no evidence that the damage to the mucosa or gut was direct or induced by the immune reaction. furthermore, administration of adrenaline to treat anaphylactic shock might induce abdominal angina because adrenaline is capable of causing ventricular fibrillation and severe vasocontraction. the effect of adrenaline ingested was probably secondary to the initial reaction as a joint vasocontraction factor in the vasospasm triggered by anaphylaxis. celiac artery compression syndrome is also known to be a rare cause of postprandial abdominal pain and unintended weight loss. the anatomical abnormalities in which the median arcuate ligament and the origin of the celiac artery are in abnormally close proximity may result in damage to the celiac nerve plexus or partial obstruction of blood flow through the celiac artery due to compression of the celiac artery during respiration. there was no evidence that the median arcuate ligament compressed the celiac artery in computed tomography or angiography in the present case. medical treatment includes all aspects of secondary preventive drug treatment including statins, antiplatelet therapy, blood pressure control, blood glucose control and smoking cessation. during the last decade, although endovascular revascularization has replaced surgical revascularization as the therapy of choice, the patient and her family selected conservative treatment. for lack of confident diagnosis of chronic atherosclerotic mesenteric ischemia, the patient was treated conservatively for functional abdominal pain syndrome with gabapentin and lorazepam. the accepted basis for clinical management of functional abdominal pain syndrome relies on establishing an effective patient - physician relationship for clinical management. we facilitated the patient 's understanding of functional abdominal pain syndrome with the psychologist because factors contributing to an effective patient - physician relationship include empathy toward the patient, patient education, validation of the illness, reassurance, treatment negotiation and establishment of reasonable limits in time and effort. the psychiatrist suggested that her psychiatric symptoms would be diagnosed additionally as having mental disorders due to a medical condition. the present case had asymptomatic chronic atherosclerotic mesenteric ischemia and anaphylaxis that changed to symptomatic one. repeated postprandial abdominal pain caused symptom - specific anxiety, resulting in the need of psychological treatment with anxiolytic agents. in conclusion, developing a satisfactory patient - physician relationship was considered more effective for the management of persistent abdominal pain caused by complicated mechanisms.	an 83-year - old woman was referred to our emergency department with acute urticaria and sudden shortness of breath approximately 30 min after taking rectal diclofenac potassium for lumbago. after treatment with adrenaline and corticosteroids, the patient became hemodynamically stable and left the hospital on the next day. she attended our hospital 1 week after the onset of anaphylaxis because of repeated postprandial epigastric pain. no abnormal lesions were found in endoscopy. radiographic selective catheter angiography revealed chronic mesenteric ischemia caused by atherosclerosis and abundant collateral arteries between the celiac trunk, the superior mesenteric artery and the inferior mesenteric artery. patients with chronic mesenteric ischemia usually present with a clinical syndrome characterized by painful abdominal cramps and colic occurring typically during the postprandial phase. fear of eating resulted in malnutrition. she was prescribed proton pump inhibitor, digestants, anticholinergic agents, serine protease inhibitors, prokinetics, antiplatelet agents and transdermal nitroglycerin intermittently, but these had no beneficial effects. it was most probable that this patient with chronic atherosclerotic mesenteric ischemia was suffering from functional abdominal pain syndrome induced by anaphylaxis. since psychiatric disorders were associated with alterations in the processing of visceral sensation, we facilitated the patient 's understanding of functional abdominal pain syndrome with the psychologist. postprandial abdominal pain gradually faded after administration of these drugs and the patient left the hospital. developing a satisfactory patient - physician relationship was considered more effective for the management of persistent abdominal pain caused by complicated mechanisms.
in the cns, myelin is formed by oligodendrocytes that spirally wrap their plasma membrane around axons. previously, myelin has been regarded as an inert and purely insulating membrane, but it is now clear that myelin is metabolically active, providing support to the underlying axon (fnfschilling., 2012, lee., 2012, in addition, myelin growth in response to neuronal activity has been described, and this may contribute to information processing by modulating velocity and synchronicity of nerve impulses in neuronal networks (fields, 2015, chang., 2016). at first glance, structural dynamics seems to be incompatible with myelin consisting of multilamellar membrane with little cytoplasm (snaidero and simons, 2014). however, most of what we know about myelin ultrastructure is based on electron microscopic studies performed on chemically fixed and dehydrated tissue, often associated with shrinkage and collapse of intracellular spaces. a recent technical advance has been the application of high - pressure freezing electron microscopy to biological tissues leading to an enhanced preservation of tissue and cell architecture, including the cytoplasmic spaces within myelin (mbius., 2010, weil., 2016). with this technique, it is possible to visualize within the developing myelin sheath a system of tube - shaped cytoplasmic expansions residing between the compacted layers of myelin (snaidero., 2014). these channels run through the compacted sheath, connecting the oligodendroglial cell body, the major site of membrane biosynthesis, to the innermost layer of myelin, which is in direct contact with the axon. these cytoplasmic regions are reminiscent of schmidt - lanterman incisures (cytoplasmic incisures of peripheral nervous system myelin) and also comprise the paranodal loops and the outer and inner periaxonal tongues of myelin. the detection of microtubules and vesicular structures within the cytoplasmic regions suggests that they serve as tracks for motor - driven transport processes. to what extent these cytoplasmic channels persist in adult myelin after completed myelination is not known. membrane compaction closes most of the cytoplasmic regions in myelin and is mediated by myelin basic proteins (mbps), the major structural component of myelin. mbp is an intrinsically disordered polypeptide chain with a strong basic character, which is able to bind to the two apposing negatively charged cytoplasmic leaflets of the myelin membrane (harauz., 2009). this interaction neutralizes the positive charge in mbp and triggers self - assembly into a polymeric network (aggarwal., 2013). polymerization of mbp molecules onto and between membranes provides the means to extrude cytoplasm from the myelin sheath (aggarwal., 2011). given the function of myelin in supporting axonal integrity, we now asked how cytoplasmic channels are formed and maintained in the developing and adult nervous system. we identified 2,3-cyclic nucleotide 3-phosphodiesterase (cnp), an oligodendrocyte - specific protein previously implicated in the maintenance of axonal integrity (lappe - siefke., 2003), as an essential factor in the maintenance of intact cytoplasmic regions in the adult myelin sheath. we provide evidence that cnp antagonizes the activity of mbp in compacting myelin membrane layers. we propose that cnp counteracts membrane zippering by associating with and organizing the actin cytoskeleton within the cytoplasmic regions of the myelin sheath, thereby keeping the adjacent cytoplasmic leaflets separated and preventing excessive membrane compaction by mbp. to analyze the role of cnp and mbp in the biogenesis of cytoplasmic channels in the developing myelin sheath, we determined the number of these channels in the optic nerve in mice lacking cnp (cnp - deficient) or a decreased dosage of mbp (heterozygous shiverer) at postnatal day 10 (p10), p14, and p21 (figures 1a1c). we used high - pressure freezing and freeze substitution for electron microscopy to visualize the cytoplasmic regions within the myelin sheath of the developing optic nerve. as shown previously, we find that a large fraction of the cytoplasmic regions disappears with the maturation of the myelin sheath (snaidero., 2014). strikingly, when cnp - deficient animals were analyzed, we observed a decrease of cytoplasmic spaces in myelin. in cnp - deficient mice, the number of cytoplasmic pockets visualized by electron microscopy in cross sections was reduced by 40%, 70%, and 80% compared to wild - type controls at p10, p14, and p21, respectively. in contrast, when heterozygous shiverer (mbp) mice, which are well myelinated, were analyzed and compared to wild - type animals, we observed a transient increase in the number of cytoplasmic regions when compared at p14 (figure 1c). this is reminiscent of increased numbers of schmidt - lanterman incisures in the pns of heterozygous shiverer (mbp) mice (gould., 1995). since the cytoplasmic regions are sparse in thin - caliber axons of the adult optic nerve, we analyzed the spinal cord, which contains thicker myelin sheaths with more cytoplasm (blakemore, 1969). to study the structure of myelin of large - caliber axons, we optimized high - pressure freezing for spinal cord tissue. we found that 5 min of pre - fixation (with paraformaldehyde / glutaraldehyde) of the spinal cord followed by embedding in gelatin and the subsequent cutting of 200-m - thin sections greatly enhances tissue quality for high - pressure freezing. using this protocol, we find that cytoplasmic regions are more frequent in large - caliber axons with thick myelin sheaths (figures 1d1k). when analyzing an earlier time point (p15), more cytoplasmic regions were found in thick myelin sheaths of the spinal cord as compared to p60 and p180 (figure s1). however, contrary to the optic nerve, a large fraction of these cytoplasmic regions within the thick myelin sheaths remained into adulthood (figures 1d1k). when cnp - deficient mice were analyzed and compared to wild - type mice, we observed a striking reduction in the number of cytoplasmic regions within myelin (> 300 nm thickness) both at p60 (figures 1d and 1f) and p180 (figures cytoplasmic regions in myelin sheaths (> 600 nm thickness) were significantly increased. however, contrary to the cnp - deficient mice, in which these abnormalities persist, heterozygous shiverer mice were not significantly different from wild - type mice at p180, showing that the effects of mbp on the cytoplasmic channels are transient (figures 1h1k). mbp is the prototype compact myelin protein, whereas cnp is thought to be enriched in non - compacted regions. we performed immunoelectron microscopy and observed that cnp is indeed highly enriched in the cytoplasmic regions of myelin and almost excluded from compacted myelin (figure s1). our results point to an antagonistic function of cnp and mbp in maintaining cytoplasm within myelin sheaths. one possibility of how cnp could exert such a function is by forming pillars in the cytoplasmic regions of the myelin sheath. such pillars may keep the adjacent cytoplasmic leaflets separated, thereby preventing membrane compaction by mbp. to test this idea, we used our recently established biomimetic in vitro compaction assay (aggarwal., 2013), which examines the interaction of giant unilamellar vesicles (guvs) with supported lipid bilayers (slbs) coated with mbp. in this system, mbp is sandwiched between a slb and guvs, and its adhesive and self - interacting properties induce the spreading of guvs onto the slb (see graphical illustration of the assay in figure 2). different concentrations of recombinant mbp (14-kda isoform) were added onto the slbs before fluorescently labeled guvs were placed on top of mbp - decorated slbs. we found that 0.4 m mbp was necessary to initiate the bursting of the guvs onto the slbs (figure s2). we next tested whether recombinant cnp could prevent the spreading of guvs onto the slbs induced by mbp. since cnp is a lipid - anchored membrane protein, we designed a recombinant variant of cnp with a small stretch of positively charged amino acids at its c terminus to link it to the negatively charged slbs. we found that cnp, but not gfp, was able to antagonize mbp - mediated spreading of the guvs onto the slbs. thus, using a simplified in vitro compaction assay, we have reconstituted the antagonistic role of cnp and mbp (figure 2). previous studies have shown that cnp co - immunoprecipitates with actin from cell lysates (de angelis and braun, 1996), but whether cnp interacts directly with filamentous actin (f - actin) and the relevance of such an interaction for myelin compaction is not known. we used recombinant cnp variants (including full - length cnp, the n - terminal domain alone, the c - terminal catalytic domain alone, a variant of the catalytic domain extending all the way to the c terminus, and an inactive mutant of the catalytic domain) and actin purified from muscle to characterize direct protein - protein interactions in vitro. we carried out in vitro f - actin co - sedimentation assays with high - speed ultracentrifugation and found that cnp pelleted with microfilaments, behaving like a typical f - actin - binding protein (figures 3a and s3). by titrating cnp, we observed that the maximal amount of co - sedimented full - length cnp was close to the amount of actin in the pellet, suggesting a 1:1 stoichiometry for binding (figure s3a). we also tested the n- and c - terminal domains of cnp and found that they both bound to f - actin independently (figures 3a and 3b). adding the c - terminal 22 residues to the catalytic domain (figure 3a), which are believed to be important for membrane anchoring and microtubule interactions, did not affect f - actin co - sedimentation. furthermore, an enzymatically inactive mutant of the catalytic domain, in which both active - site his residues are replaced by gln, also similarly co - sedimented with f - actin, showing that cnpase activity is not required for the interaction (figure s2b). the cnp interaction partner calmodulin (cam) (myllykoski., 2012) prevented cnp co - sedimentation into f - actin pellets, while another ef - hand protein abundant in myelinating glia, s100, did not (figures s3c and s3d). furthermore, a small increase in the actin polymerization rate was observed with cnp (figure s3e). next, we used low - speed centrifugation to analyze the f - actin bundling activity of full - length cnp and its catalytic domain. both constructs showed clear bundling activity, and the effect was already seen at 1 m cnp (figure 3d). immunogold labeling showed f - actin bundles decorated with full - length cnp, while areas devoid of f - actin contained no cnp (figure 3c). to further map the interaction stoichiometry and potential binding surfaces, we carried out covalent crosslinking of cnp and f - actin, followed by mass spectrometric peptide mapping. when both cnp and actin were present in the crosslinked sample, specific patterns of bands were observed on sds - page, indicating protein - protein complex formation. in addition to a 1:1 species, also higher oligomeric states were resolved (figure 3e). the oligomerization pattern was similar between full - length cnp and the catalytic domain, indicating that the c - terminal domain is sufficient to drive an interaction between cnp and f - actin (figure 3e). to determine the interaction sites, all picked crosslinked hybrid bands contained both actin and cnp as shown by matrix - assisted laser desorption - ionization time of flight (maldi - tof). the peptide pattern determined by mass spectrometry was used to predict the interaction sites. for actin, the binding appears to occur near the d - loop in subdomain 2 as well as the long loop of subdomain 3 and for cnp on the surface of the n - terminal pnk - like domain (figure 3f). taken together, the results from co - sedimentation and crosslinking demonstrate that cnp can bind microfilaments directly and induce their bundling. both domains of cnp bind f - actin, and the observed effects are independent of cnp catalytic activity. having demonstrated that cnp is able to bind and bundle f - actin, we used our in vitro compaction assay to determine the effect of f - actin on mbp - mediated membrane spreading. we found that f - actin by itself was not able to block mbp - mediated spreading of guvs onto slbs (figure 3 g). however, when f - actin was added to slbs, which had been pre - coated with cnp, mbp - mediated spreading of the guvs was fully blocked (figure 3h). note that this occurred at a concentration (0.7 m mbp) at which cnp alone did not exert any antagonistic force. thus, cnp and f - actin act synergistically in blocking mbp - mediated membrane spreading (figures 3i and s3). to obtain further evidence for a role of f - actin in stabilizing of cytoplasm - rich areas we have previously shown that these cultures satisfy many of the essential requirements necessary to study the formation of compacted myelin, as they resemble in vivo compact myelin in composition (aggarwal., 2013). cultured oligodendrocytes develop membrane sheets that contain compacted membranes enriched in mbp and cytoplasm - rich regions with f - actin (nawaz., 2015, 2015). to determine the role of f - actin in stabilizing the cytoplasmic regions within the sheets, we depolymerized actin with latrunculin a or cytochalasin b and quantified the area of the sheets covered by mbp after treatment (figure s3). we found that both drugs led to an increased area covered and compacted by mbp in the sheets, indicating that f - actin had blocked membrane compaction as previously shown (dyer and benjamins, 1989). to determine whether increasing f - actin levels reduce the area of the sheets covered by mbp, we analyzed primary cultures from mice that specifically lack cofilin1 and actin depolymerizing factor (adf) in oligodendrocytes (adf ; cnp ; cfl1, also termed adf / cofilin1 double knockout) and as a consequence have elevated levels of f - actin. indeed, when primary cultures of mutant oligodendrocytes were prepared, we found that membrane sheets contained fewer mbp - rich regions (figure s3). to obtain in vivo proof of this finding, we analyzed high - pressure frozen optic nerves of adf / cofilin1 double - knockout mice. we found significantly more cytoplasmic regions in myelin of adf / cofilin1 double - knockout mice (p15) than in controls (figures 3j3l), indicating that f - actin levels contribute to the formation of cytoplasmic regions within myelin sheaths. since our results pointed to an antagonistic role of cnp and mbp in maintaining cytoplasmic channels, we crossed shiverer mice with cnp - deficient mice to create double - mutant mice (cnp ; mbp, also termed cnp1 null / shiverer heterozygotes). we hypothesized that according to our model we should increase the cytosolic space in cnp - deficient myelin by reducing mbp expression. to test this idea, we performed high - pressure freezing on cnp null / shiverer heterozygous spinal cord and analyzed myelin sheath morphology at p60. when the percentage of cytoplasmic regions was determined and compared to that of wild - type and cnp - deficient mice, cnp null / shiverer heterozygous mice were indistinguishable from wild - type animals (figure 4a). to determine whether the rescue persisted into older age, we analyzed the amount of cytoplasmic regions at p180. we found that cytoplasmic regions were maintained into adulthood in cnp null / shiverer heterozygous comparable to wild - type animals (figures 4b and 4c). we hypothesized that intact cytoplasmic channels are necessary for maintaining functional axon - myelin units. since cnp - deficient mice exhibit progressive axonal pathology with amyloid precursor protein (app)-positive swelling and spheroid formation (edgar., 2009, lappe - siefke., 2003), we asked whether the axonal degeneration phenotype is rescued by reducing mbp levels. indeed, when cross sections of the fimbria in cnp null / shiverer heterozygotes (p75) were compared to cnp - deficient mice, we noticed the complete rescue from app+ spheroids (figures 4d4 g). the axonal degeneration in cnp - deficient mice is accompanied by activation of microglial cells, possibly to clear the damaged axons (lappe - siefke. when immunolabeled for the microglial marker mac3, reduced mbp expression was associated with a lower number of microglia in cnp mutants (figure s4). hence, lowering mbp in cnp - deficient mice results in less axonal damage, which is also reflected by a reduced microgliosis. next, we performed electron microscopy and found that at age p60 and p180, when axonal degeneration could be observed in cnp - deficient animals (figures 4h and 4i), the degree of axonal degeneration and pathological myelin outfolding in the spinal cord of cnp null / shiverer heterozygotes was significantly reduced (figure 4i). importantly, when we analyzed the axonal pathology at the later time point, the axon - protective effect of reduced mbp expression on cns axons was maintained at least until p180 (figure 4i). finally, we asked whether the protective effect includes thin - caliber axons of the optic nerve that contain myelin with little cytoplasm. in contrast to the spinal cord, axonal degeneration in the optic nerve was not alleviated in cnp null / shiverer heterozygous mice (figure 4j). taken together, our findings provide evidence for a role of cnp in maintaining cytoplasmic channels in myelin, a function important for maintaining axonal integrity of large - caliber cns axons in mice. in this work, we used high - pressure freezing to improve tissue preservation of cns white matter tracts and to elucidate myelin structure close to its native state. using this technique, we identified cnp as an essential protein in setting up and maintaining normal cytoplasmic regions within myelin sheaths. at the molecular level, we find that cnp acts together with f - actin to antagonize the membrane adhesive forces exerted by polymerizing mbp molecules. one model of how cnp could exert such a function is by forming pillars anchored to the membrane by the actin cytoskeleton in the cytoplasmic space of the myelin sheath. such cnp struts could keep the cytoplasmic leaflets separated, thereby preventing membrane compaction by mbp. keeping these spaces open is likely to allow a more efficient diffusion of metabolites and enable the motor - driven transport of vesicular cargo. thus, two antagonistic molecular forces appear to operate in myelin : one depending on mbp and the other based on cnp and the actin cytoskeleton. it is possible that there is a tug - of - war-type regulation of a cytoplasmic compartment within myelin in which the actin cytoskeleton in association with cnp prevents mbp from compacting the membrane multilayer (figure 4k). this model is in good agreement with the observation of more non - compacted myelin in transgenic mice overexpressing cnp (gravel., 1996). our analysis now shows that cytosolic channels remain a prominent compartment of (non - compact) myelin around large - caliber axons in the adult. these findings are consistent with previous studies, which have been able to visualize cytoplasmic channels in thick myelinated fibers of adult spinal cord using dye injections (velumian., 2011). however, cytoplasmic channels were also identified after myelination (p30) in oligodendrocytes of rat optic nerve (butt and ransom, 1993) and in cortical myelin of adult mice (murtie., 2007). in the peripheral nervous system, microtubules, actin, and mitochondria have been documented in the cytoplasmic pockets (or schmidt - lanterman incisures) within compacted myelin (hall and williams, 1970). it is therefore tempting to speculate that they are required for the transport of molecules across the myelin sheath to the axon and for providing plasticity to the myelin sheath. consistent with this concept, cnp - deficient mice have not only a reduced number of cytoplasmic regions but also ongoing axonal pathology, with axonal swelling and spheroid formation (edgar., 2009, lappe - siefke., 2003). here, the frequently observed enlargement of inner adaxonal tongues, filled with granular material (lappe - siefke., 2003), can be explained by the traffic block within cytoplasmic channels and the backlog of cytosolic cargo that leads to secondary swelling of the inner tongue and paranodal abnormalities (rasband., 2005). importantly, we observed that by reducing mbp levels in cnp - deficient mice, cytoplasmic channels became more prominent again and axonal pathology was rescued in large - caliber axons. the rescue of axonal integrity was not seen in thin - caliber axons, possibly because oligodendrocytes that generate myelin around thin - caliber axons have shorter internodes (bechler., 2015). in summary, we can propose a model for a molecular mechanism by which the cytoplasmic compartment is regulated in size and maintained in myelin sheaths. alive and metabolically active and how oligodendrocytes remain functionally connected to the axonal compartment. we hypothesize that a system of cytoplasm - rich channels, bidirectionally connecting the oligodendroglial cell body with the inner adaxonal tongue of myelin, are necessary to provide metabolic support, maintain functional axon - glial units over a long period of time, and regulate myelin thickness within active neuronal circuits. mice were killed by cervical dislocation, and freshly extracted optic nerves and spinal cords were cryo - fixed using a high - pressure freezer hpm100 (leica) and further processed by freeze substitution and epon - embedding following the tannic acid - oso4 protocol described in (mbius., 2010). prior to the freezing of the cervical spinal cord samples were immersion fixed for 5 min in 4% pfa and 2.5% ga followed by vibratome sectioning (vt 1200, leica) in 200-m slices. ultrathin cross sections were obtained with a ultracut s ultramicrotome (leica) and contrasted as described previously (mbius., 2010). sections were imaged using a leo 912 omega electron microscope (zeiss) equipped with an on - axis 2k charge - coupled device (ccd) camera (trs). 515 randomly selected areas of 150 m were imaged per animal in which 100300 myelinated axon profiles with four or more myelin wraps were assessed. for immunohistochemistry, antibodies specific for amyloid precursor protein (app ; 1:1,500, chemicon), glial fibrillary acidic protein (gfap ; 1:200, novocastra), and mac3 (1:400, bd pharmingen) were used. four or five male mice per genotype (blinded to the genotype) were analyzed at p75. per marker and mouse, one histological section comprising both fimbriae were analyzed, and the mean of both fimbriae was used for statistical assessment. statistical analysis was performed using excel (microsoft) and graphpad prism (graphpad software). a one - way anova followed by a tukey post hoc test was performed for comparison of three or more groups. to analyze and compare the bursting of guvs over time for multiple conditions, n.s. and c.v. designed and performed experiments, analyzed the data, and wrote the manuscript	summarythe myelin sheath is a multilamellar plasma membrane extension of highly specialized glial cells laid down in regularly spaced segments along axons. recent studies indicate that myelin is metabolically active and capable of communicating with the underlying axon. to be functionally connected to the neuron, oligodendrocytes maintain non - compacted myelin as cytoplasmic nanochannels. here, we used high - pressure freezing for electron microscopy to study these cytoplasmic regions within myelin close to their native state. we identified 2,3-cyclic nucleotide 3-phosphodiesterase (cnp), an oligodendrocyte - specific protein previously implicated in the maintenance of axonal integrity, as an essential factor in generating and maintaining cytoplasm within the myelin compartment. we provide evidence that cnp directly associates with and organizes the actin cytoskeleton, thereby providing an intracellular strut that counteracts membrane compaction by myelin basic protein (mbp). our study provides a molecular and structural framework for understanding how myelin maintains its cytoplasm to function as an active axon - glial unit.
hemodialyzed and peritoneal dialysis patients are currently evaluated in order to detect structural and functional alterations in the arterial tree. since it has been demonstrated that renal function replacement therapy significantly improves survival rates of end - stage renal disease (esrd) patients, the improvement of biochemical and hemodynamic parameters is desirable aims. nevertheless, the underlying mechanism by which these improvements occur is not entirely understood, and not all cardiovascular parameters tend to reach their normal values. pulse wave velocity (pwv) is a well - known independent predictor of cardiovascular disease that is currently obtained by using a simple, noninvasive technique. this method to quantify arterial stiffness has been used during the last decades, and a good number of analyses have been reported, including longitudinal studies. furthermore, cross - sectional studies were performed, including the comparative analysis between hemodialysis and peritoneal dialysis patients. nevertheless, the relationship between renal replacement therapy and arterial stiffness remains unclear, since the research results are controversial ; sometimes they are inconsistent and other times it is not possible to compare them. the purpose of this work was to analyze the literature in which longitudinal or transversal studies were performed in hemodialysis and/or peritoneal dialysis patients and (1) to summarize the effects of the dialysis modality on arterial stiffness and (2) to comment on the relationship of arterial stiffness impairment with the etiological factors mentioned in the revised literature. for practical reasons, the text was organized taking into account the dialysis modality and the nature of the research : whether it is a transversal or a longitudinal study. when comparisons among the reported researches were feasible in recent years, great emphasis has been placed on the role of arterial stiffness in the development of cardiovascular diseases. aortic stiffness, which results in increased pulse pressure (pp), cardiac overload, and left ventricular hypertrophy, is an established predictor for cardiovascular morbidity and mortality in several disease (i.e., chronic kidney disease, ckd). indeed, the assessment of arterial stiffness is increasingly used in the clinical assessment of patients. basically, arterial stiffness can be measured systemically (the cardiovascular system as a whole), regionally (large arterial segments or pathways), or locally (arterial rings or short arterial segments) [1, 2 ]. in contrast to systemic arterial stiffness, which can only be estimated from models of the circulation (i.e., windkessel model), regional and local arterial stiffness can be measured directly and noninvasively, at various sites along the arterial tree. a major advantage of the regional and local evaluations of arterial stiffness is that they are based on direct measurements of parameters strongly linked to wall stiffness [1, 2 ]. local arterial stiffness is directly determined, from the change in pressure driving the change in arterial diameter (distension or volume). however, because it requires a high degree of technical expertise and different techniques (i.e., pressure measurement using applanation tonometry and diameter measurement using ultrasound) and takes longer than measuring regional stiffness, local measurement of arterial stiffness is only really indicated for mechanistic analyses in pathophysiology, pharmacology, and therapeutics, rather than for epidemiological or clinical studies [1, 2 ]. in contrast, the measurement of arterial pulse wave velocity (pwv : a regional stiffness marker) is generally accepted as the most simple, noninvasive, robust, and reproducible method to determine arterial regional stiffness. measured along the aortic and aortoiliac pathway, it is the most clinically relevant, since the aorta and its first branches are what the left ventricle sees and are thus responsible for most of the pathophysiological effects of arterial stiffness. carotid - femoral pwv has been used in the epidemiological studies demonstrating the predictive value of aortic stiffness for cardiovascular events. in contrast, pwv measured outside the aortic pathway (i.e., at the upper or lower limb) allows determining the arterial stiffness in those regions but has no predictive value. the moens - korteweg equation states that pwv is proportional to the square root of the incremental elastic modulus (einc) of the vessel wall given constant ratio of wall thickness, h, to vessel radius, r, and blood density,, assuming that that the artery wall is isotropic and experiences isovolumetric change with pulse pressure [1, 2]:(1)pwv = einch2r. epidemiological studies in almost all populations have clearly shown that aging is the most determinant risk factor for cardiovascular diseases. this age - associated risk for the development of cardiovascular complications is associated with numerous normal or physiological deleterious changes in the structure and function of the arterial system (i.e., increase in aortic pwv). among the most characteristic changes associated with aging are central arteries stiffening and remodeling (i.e., intima and/or media layers thickness increase, increased length, and tortuousness) of large arteries. the degeneration of elastic (elastine) fibers is associated with an increase in collagen fibers and ground substance and depositions of calcium. in this context, in end - stage renal disease (esrd) patients the arterial structural and functional changes are characterized by an accelerated influence of aging. premature or early vascular aging (eva syndrome) are observed with progression of ckd and in esrd. this accelerated aging is associated with outward remodeling of large and medium arteries, characterized by an increased arterial diameter not totally compensated for by artery wall thickness increase. in addition, despite the fact that deposition of calcium salts in the walls of human arteries is a normal or physiologically inevitable consequence of aging, the extent of calcifications is more pronounced in esrd. as will be discussed, arterial stiffening in ckd and esrd patients is of multifactorial origin. as was mentioned, the role of vascular calcifications has been pointed out as a determinant factor associated to arterial aging. in 2013, london. reported that the incremental elastic modulus (einc, i.e., the arterial wall stiffness) of the carotid artery was increased in hemodialyzed patients. this research included 155 hemodialyzed patients whose age (abscissa) versus arterial stiffness (ordinate) relationship (x / y graph) was higher (a significantly steeper slope) than that obtained in 105 healthy control subjects. curiously, in the same cohort of hemodialyzed patients, the age versus arterial stiffness relationship of patients without arterial calcifications was lower than that obtained in healthy subjects. this finding is coincident with those reported (by our group) in an animal model of arterial calcinosis. on the contrary, since the age versus arterial stiffness relationship of the noncalcified hemodialyzed patients is lower than that obtained in the healthy cohort, the beneficial role of renal replacement therapy could be ensured. calcinosis of the media in hemodialysis patients has been linked to increases of arterial stiffness by several authors [6, 7 ]. on the other hand, an interesting research by shinohara and coworkers, published a decade ago, demonstrated that hemodialysis has no adverse effects on renal replacement therapy when compared to uremic predialysis patients. the authors emphasized on the active role of the control of metabolic alterations patients have in esrd (table 1). the probably most important finding of this research was that hemodialysis patients (n = 144) showed significantly lower pwv values (p 10%) and nonresponders (increased or decreased by < 10%) ; interestingly the responders significantly decreased pwv at the end of the follow - up period (from a baseline value of 11.37 2.80 m / s to 10.18 2.57 m / s ; p < 0.001). this distinct response to hemodialysis was not observed in a five - year follow - up of 25 hemodialyzed patients carried out by our group (argentina and uruguay), in which a significant decrease of carotid - femoral pulse wave velocity was demonstrated. brachial - ankle pwv (a parameter influenced for the aortic and lower - limb arterial stiffness levels) has been used to analyze the correlation between arterial stiffness and several different parameters in a sample of peritoneal dialysis patients in macao, china. the patients included in the study (n = 96) were divided into two groups : (a) low brachial - ankle pwv group including those patients with values below the mean value of the entire population and (b) high brachial - ankle pwv group including those patients with values higher than the mean value of the entire population. the latter were significantly older than the former and included a high proportion of females (p < 0.004). the whole population of peritoneal dialysis patients showed that brachial - ankle pwv is independently correlated with age, serum albumin level c - reactive protein, and residual renal creatinine clearance. szeto and coworkers affirmed, in a very simple way, that peritoneal dialysis patients significantly increased carotid - femoral pwv in 24 months. in contrast, tang and coworkers showed that a pwv improvement found in peritoneal dialysis patients is associated with modifiable risk factors. the authors found that 23% of patients undergo a decrease of carotid - femoral pwv values after a six - month follow - up, compared to the baseline data (table 2). the outcomes in pwv obtained in these peritoneal dialysis patients were divided into progressors (increases higher than 1 m / s or 15% between baselines to 6-month follow - up) and regressors (decreases lower than 1 m / s or 15% between baselines to 6-month follow - up). coincidentally with the above - mentioned publication of tang., another study in which the cohort outcome of peritoneal dialysis patients was divided was reported by demirci and coworkers. they analyzed carotid - femoral pwv in 89 peritoneal dialysis patients at baseline and after a nine - month follow - up. patients with an aortic pwv variation higher than 5% were considered progressors, and, on the contrary, patients with equal or lower values than 5% of variation were considered nonprogressors (table 2). patients included in the nonprogressor group showed a significant decrease of carotid - femoral pwv (from 8.56 2.73 m / s in baseline to 7.30 2.02 m / s after 9 months ; p < 0.0001). a polish group reported an interesting research in which peritoneal dialysis and hemodialyzed patients were compared. the carotid - femoral pwv of 35 ambulatory patients receiving peritoneal dialysis was 9.9 1.2 m / s, while aortic pwv of hemodialyzed patients was 10.0 1.4 m / s. the comparison between pwv of nondiabetic and that of diabetic patients showed significant differences (p < 0.01) in both peritoneal dialysis and hemodialyzed patients (table 3). on the contrary, hemodialysis and peritoneal dialysis have been shown to improve arterial stiffness comparatively with predialysis subjects. this study included ambulatory peritoneal dialysis patients (n = 62), hemodialyzed subjects (n = 56), a cohort of uremic patients (n = 128), and a control group (n = 40) of healthy subjects. the comparative analysis included control, peritoneal dialysis, hemodialysis, and nondialyzed patients in esrd. the authors concluded that dialysis improved arterial stiffness evaluated through pwv and augmentation index (table 3). it has been pointed out that the dialysis modality (peritoneal or hemodialysis) may impact arterial stiffness in different ways. in an interesting in vivo and in vitro work of chung and coworkers, it has been reported that the duration of renal replacement therapy did not correlate with arterial stiffness. the most important finding of this work perhaps is the difference between the in vivo and in vitro (measured in the) determination of arterial pwv. no differences in noninvasive pwv were found among nondialysis, peritoneal dialysis, and hemodialysis patients (table 3). on the contrary, the in vitro determination of pwv showed that peritoneal dialysis and hemodialysis patients exhibit higher values than nondialyzed patients (p < 0.01). pwv has been found to be higher in hemodialyzed patients than in those on continuous ambulatory peritoneal dialysis after 12 months of renal replacement therapy (table 3). aortic stiffening has been demonstrated in early stages of kidney disease and, as mentioned above, is increased in both hemodialyzed and peritoneal dialysis patients. furthermore, it has been pointed out that continuous peritoneal dialysis could contribute to determine the most important increases of arterial stiffness. peritoneal - dialysis and hemodialyzed patients with abnormal increases of aortic stiffness showed a direct correlation between pwv and blood pressure (systolic, diastolic, and mean) values [8, 17, 19 ]. nevertheless, the lowering of blood pressure levels is not always followed by decreases of aortic stiffness evaluated through pwv measurements. this fact obviously shows the multifactorial nature of the arterial stiffening observed in uremic patients. in 2010, gao and coworkers reported a follow - up in 107 peritoneal dialyzed patients in which carotid - femoral and carotid - radial pwv were measured and the fluid overload was determined. the authors pointed out that the resulting mean values of carotid - femoral pwv were lower than those obtained in a similar population in which salt intake was higher. recently, a report by sipahioglu. showed that the aortic stiffness index (; a local marker of the ascending aorta stiffness) measured in a cohort of peritoneal dialysis patients showed higher values than that obtained in healthy subjects. moreover, in this 2-year longitudinal follow - up, 156 patients showed a significant correlation of the index with age (p < 0.001) and kt / v (p < 0.023). in patients with esrd there is an increase of degenerative processes that affect the structural integrity of the arterial wall, which involve the deposit of abnormal product derived from proteins or lipids. several reports mentioned that the reduction of ages (advanced glycation end products) improves arterial stiffening. alteration of calcium and phosphate metabolism has been found to be associated with structural and functional changes of the arterial wall. particularly, the development of mediacalcinosis has been linked to increases of elastic arterial stiffening. this pathological process which involves the extracellular matrix is accompanied by cellular changes of the arterial wall. as previously reported, uremic toxins resulted in the transformation of vascular smooth muscle cells in osteoblast - like cell ; considering this fact together with the increase of arterial stiffness in patients in which the dialysate calcium was high, the association between vascular calcification and arterial stiffening is clear. furthermore, carotid - radial pwv was found to be significantly associated with vascular calcification in a study that included three groups : chronic kidney disease in stage 5, hemodialyzed, and peritoneal dialysis patients. carotid - femoral pwv was found to inversely correlate with serum albumin levels, obtained in a cohort of peritoneal dialysis patients. additionally, increases in brachial - ankle pwv have been associated with proteinuria and low creatinine clearance in nondialyzed patients participating in a health - check japanese program. phosphate levels have been found to be significantly correlated with arterial wall stiffness in uremic patients. endothelial dysfunction has been observed in uremic patients and their reversibility was reported in hemodialyzed patients. hemodialysis has no acute effects on endothelial function and aortic pwv in patients who are on continuous renal replacement function therapy. arterial stiffness has demonstrated to be correlated with c - reactive protein serum levels [4, 12, 16 ]. very few authors performed carotid - radial pwv measurements in patients under renal replacement therapy (table 4). for instance, utescu and coworkers concluded that after a 1.2-year follow - up hemodialysis determined a significant decrease of pwv in upper limbs (arterial stiffness reduction) simultaneous to aortic stiffness significant increases. on the other hand, szeto and coworkers demonstrated significant increases of carotid - radial pwv after a 2-year follow - up of peritoneal dialysis patients (from 10.05 1.44 m / s to 10.80 1.90 m / s). mourad and coworkers concluded that radial arteries showed an increase of arterial stiffness (evaluated through einc) in hemodialyzed patients, compared to hypertensive and normotensive control subjects. see table 4. according to a previous research reported by our group in hemodialysis patients, a significative reduction in the carotid - brachial pwv moreover, this reduction was greater in patients with the arteriovenous fistula performed in the upper arm than in those with the shunt in the forearm. the outcome examination of the reports in which a follow - up of arterial stiffness in hemodialyzed patients was performed shows differences, perhaps determined by the lack of a unique data collection methodology. so, the effects of renal replacement therapy on arterial stiffness are a controversial topic. the nature of the differences in terms of arterial stiffness outcomes observed among the above - mentioned reports could be attributed todifferences among pharmacological drug resources used to control comorbidities and metabolic alterations secondary to renal failure in the analyzed population, the use of higher dialysate calcium, which would result in impairment of aortic stiffness, the presence or absence of calcinosis of the arterial wall, which was found to be associated with changes in vascular stiffness, differences in data collection and type of analysis (transversal and or longitudinal studies). differences among pharmacological drug resources used to control comorbidities and metabolic alterations secondary to renal failure in the analyzed population, the use of higher dialysate calcium, which would result in impairment of aortic stiffness, the presence or absence of calcinosis of the arterial wall, which was found to be associated with changes in vascular stiffness, differences in data collection and type of analysis (transversal and or longitudinal studies). the list of the factors involved in the source of arterial stiffness alterations at present seems to be incomplete. nevertheless, the therapeutically connotations of the commented findings are very important, since the stiffening process evidences a high risk for both peritoneal dialysis and hemodialyzed patients. finally, it is important to mention that several authors demonstrated a positive relationship between pwv and body volume overload parameters [37, 38 ]. furthermore, the arterial stiffness changes could be originated in variations in the exchangeable sodium pool involving the arterial wall, as suggested by hogas and coworkers. consequently, it is hypothetically possible that the origin of the observed discrepancies among the analyzed reports in this review could be explained by tissue hydration changes. the renal replacement therapy effects on arterial stiffness remain controversial.the origin of the arterial wall stiffening found in hemodialyzed and peritoneal dialysis patients is multifactorial.arterial stiffness increases and decreases seem to be part of a generalized process that involves territories different from the aortic territory, such as the carotid - radial pathway.contradictory results in arterial stiffness should be analyzed considering all of the variables involved in the determinants of the vascular dynamic behavior, both in elastic and in muscular arteries.arterial stiffening of elastic and muscular arteries should be evaluated in order to check preventive and therapeutic options that were capable of stopping the impairment of the arterial wall stiffness in hemodialyzed and peritoneal dialysis patients. the origin of the arterial wall stiffening found in hemodialyzed and peritoneal dialysis patients is multifactorial. arterial stiffness increases and decreases seem to be part of a generalized process that involves territories different from the aortic territory, such as the carotid - radial pathway. contradictory results in arterial stiffness should be analyzed considering all of the variables involved in the determinants of the vascular dynamic behavior, both in elastic and in muscular arteries. arterial stiffening of elastic and muscular arteries should be evaluated in order to check preventive and therapeutic options that were capable of stopping the impairment of the arterial wall stiffness in hemodialyzed and peritoneal dialysis patients.	the increase of arterial stiffness has been to have a significant impact on predicting mortality in end - stage renal disease patients. pulse wave velocity (pwv) is a noninvasive, reliable parameter of regional arterial stiffness that integrates the vascular geometry and arterial wall intrinsic elasticity and is capable of predicting cardiovascular mortality in this patient population. nevertheless, reports on pwv in dialyzed patients are contradictory and sometimes inconsistent : some reports claim the arterial wall stiffness increases (i.e., pwv increase), others claim that it is reduced, and some even state that it augments in the aorta while it simultaneously decreases in the brachial artery pathway. the purpose of this study was to analyze the literature in which longitudinal or transversal studies were performed in hemodialysis and/or peritoneal dialysis patients, in order to characterize arterial stiffness and the responsiveness to renal replacement therapy.
gingival enlargement produces adverse esthetic challenges, functional impairment and clinical symptoms which includes gingival pain, tenderness, bleeding, speech disturbances, abnormal tooth movement and dental malocclusion. this condition is ascertained to be caused by medications like antiepileptic drugs (aeds), calcium channel blockers and immunosuppressants, genetic abnormalities, such as hereditary gingival fibromatosis, proliferative lesions, etc. epilepsies are a group of disorders characterized by paroxysmal cerebral dysrhythmia, manifesting as brief periods of loss or disturbance of consciousness. it was correctly recognized as the disease of lightening by jh jackson a century ago and is the most common chronic neurological disorder in human. a number of aeds have been used successfully to treat this disease but like any other drugs these antiepileptic medications have various side effects, one of which is gingival enlargement. drug - induced gingival enlargement was first observed in patients who were taking phenytoin for epilepsy, with approximately 50% of incidence. three significant factors which are important in the expression of these gingival changes comprises : drug variables, plaque - induced inflammatory changes in the gingival tissues and genetic factors - the latter determining the heterogeneity of the gingival fibroblasts. this article focuses on a case of more commonly used aed phenytoin and rarely used phenobarbitone induced gingival enlargement which was successfully managed with appropriate periodontal therapy and platelet rich fibrin (prf), a biological bandage. a 35-year - old male patient came to the department of periodontics with the chief complaint of difficulty in speech, mastication, pain and bleeding, while brushing due to swollen gums. the medical history revealed that the patient was epileptic and was under a combination of aeds which encompassed phenytoin, carbamazepine and eslicarbazepine for 8 years and phenobarbitone for the last 6 months. the history of present illness demonstrated that the patient noticed the enlargement for the past 6 months which was painless and progressive in nature. on intraoral clinical examination, there was generalized gingival enlargement with few areas covering the occlusal aspect of the posterior teeth [figure 1 ]. the enlargement was pale pink in color, firm, resilient with minutely lobulated surface except in areas superimposed with inflammation. due to the enlargement, the treatment plan was put forward which started with the extraction of hopeless teeth (# 11 and # 25) followed by phase i therapy to reduce the inflammation and phase ii gingivectomy procedure to reduce the bulk tissue. prior to the surgery, the patient underwent extraction of # 11 and # 25. this was followed by phase i therapy along with topical application of 10% metronidazole gel. the patient was advised to take folic acid supplements (1 mg 1 - 3 times a day) as phenytoin decreases folate absorption and increases its excretion. once the inflammation was reduced the patient was taken for surgery. after obtaining the physician 's consultation regarding patient 's medical condition for the surgical procedure the gingivectomy procedure was planned for one quadrant every appointment at an interval of 1 week. after achieving adequate anesthesia using 2% xylocaine hcl with adrenaline (1:2,00,000), precise identification of the pockets on all the surfaces of all teeth within the surgical field was done with the help of a pocket marker at three points / tooth (mid radicular, mesial and distal line angles) on the facial / buccal and lingual / palatal aspects. an external bevel incision was given using no 15 bard parker blade and kirkland surgical periodontal knife [figure 3 ]. interdental incisions were made using orbans knife and the bulk of tissue was removed with the help of curettes. prf was placed [figure 4 ] and protected with a tin foil in areas where bone exposure was encountered over which periodontal dressing was placed. the patient was given post - operative instructions and was instructed to take analgesics (combination of paracetamol-500 mg and diclofenac-50 mg) thrice daily for 3 days. patient was kept under regular follow - up and oral hygiene instructions were reinforced at every appointment. kirkland knife for gingivectomy placement of platelet rich fibrin the excised specimen was sent for histopathology report. specimen showed dense fibrous connective tissue consisting of numerous collagen bundles with moderate to severe chronic inflammatory cell infiltrate predominantly consisting of lymphocytes and plasma cells. the epithelium was nonkeratinized in some areas and parakeratinized in other areas with long thin rete ridges in few areas and blunt short rete ridges in the rest of the areas [figure 5 ]. the american academy of periodontology 2001 defines drug - induced gingival enlargement as an overgrowth or increase in size of gingiva resulting in whole or part from systemic drug use. currently, there are over 20 medications from three pharmaceutical categories including anticonvulsants, calcium channel blockers and immunosuppressants that are associated with gingival enlargement. gingival enlargement is one of the most frequent adverse effects associated with the administration of phenytoin. incidence rates have ranged from 3%-93%, but about 50% of patients on long - term phenytoin therapy develop gingival enlargement. phenobarbitone induced gingival enlargement is poorly documented and its prevalence is < 5%. in our case, the patient was under phenytoin medication for 8 years and phenobarbitone for the past 6 months. there are several proposed hypotheses, some of which are controversial ; however, the cause is likely multifactorial. it was proposed that phenobarbitone induced gingival enlargement results in the formation of pseudoepitheliomatous hyperplasia due to alteration in the epidermal growth factor / transforming growth factor (tgf)- ratio. there is an inconclusive relationship between the severity of the gingival enlargement and drug dosage, duration of therapy and drug concentrations in serum, saliva and gingival crevicular fluid. it has been found out that the incidence and severity of phenytoin induced overgrowth is greatest on the labial surfaces of maxillary and mandibular anterior teeth whereas for phenobarbitone, severity is more in the posterior regions compared with anterior regions. in the present case, there was severe gingival enlargement in the maxillary posterior and mandibular anterior region when compared to maxillary anterior and mandibular posteriors respectively which was unique for this case. moreover, enlargement was more on the left side when compared to right. one of the foundations for treatment of all drug - induced gingival overgrowths is discontinuation of the medication along with drug substitution. however, in the present case, it was not possible as those drugs were lifesaving drugs for the patient according to the patient 's physician. hence we proceeded with the best possible treatment in order to enable the patient to maintain his oral hygiene. given the significance of plaque and calculus as risk factors for exacerbation of gingival overgrowth, initial periodontal therapy was aimed at non - surgical periodontal treatment which included scaling and root planing, usage of 0.12% chlorhexidine mouthwash and regular application of metronidazole gel. in our case, patient was asked to use metronidazole gel as various studies have evidenced its effectiveness in treating gingivitis. patient was also instructed to take folic acid on regular basis for 30 days as numerous literatures have shown that phenytoin may precipitate folic acid deficiency and megaloblastic anemia. unfortunately, these measures failed to resolve the overgrowth to a satisfactory level, as the gingival lesions have been long - standing. hence, surgical intervention was taken into consideration. simple gingivectomy procedure along with adequate plaque control was opted over periodontal flap surgery due to generalized moderate bone loss and adequate width of keratinized gingiva. reconstruction of exposed raw wound surfaces after gingivectomy procedures allegedly considered challenging earlier is now overcome in the modern era of tissue engineering by using prf, an autologous blood borne growth factor. in the present case, futhermore, the presence of denuded bone has been related to increased incidence of infection which can further complicate the wound. hence, to fasten the wound healing process we decided to use autologous prf to shield the open wound which acts as a matrix for neoangiogenesis and epithelial cell migration. prf also consists cytokines such as interleukin 1 beta (il-1) -4, and -6, and growth factors such as tgf- 1, platelet derived growth factor (pdgf), vascular endothelial growth factor and connective tissue growth factor. thus, prf has been substantiated in protecting open wounds and accelerating the healing process. relapse may occur 3 - 6 months after surgical treatment, but in the present case 12 months recall visits showed no recurrence of the enlargement [figure 6 ]. while, it may be prevented through scrupulous periodontal maintenance and home care, it is essential for the periodontist to work together with the patient 's physician in order to productively treat this condition once it occurs. one of the main goals of the clinician is to satisfy the patient 's needs. in the present case, the patient was satisfied with the treatment as all his grievances like difficulty in speech, mastication, pain, and bleeding while brushing due to swollen gums were resolved.	medication - related gingival enlargement is a common reactionary phenomenon that occurs with the use of several types of therapeutic agents, including antiepileptic drugs (aeds). this disorder has been documented since 1939, shortly after the introduction of phenytoin. in the present case, a concise review of literature concerning the etiopathogenesis and management of aeds (phenobarbitone and phenytoin) induced gingival enlargement has been described. it is vital that not only the periodontist, but also dental surgeons and medical practitioners should become aware of the potential etiologic agents, characteristic features, and the differential diagnosis of drug induced gingival enlargement in order to be able to prevent, diagnose and successfully manage the condition.
immunoglobulin g4-related sclerosing cholangitis (igg4-sc) is characterized by lymphoplasmacytic tissue infiltration with a predominance of igg4-positive plasma cells, increased igg4 production, leading to bile duct wall thickening and good response to steroid therapy. it was regarded as the most frequent extrapancreatic manifestation of type 1 autoimmune pancreatitis, present in over 70 percent of such patients. now igg4-sc belongs to the spectrum of immunoglobulin g4-related disease (igg4-rd), which encompasses a large number of medical conditions that share similar histopathological features. as an independent type of igg4-rd the most often affected organs are pancreas and biliary duct. likewise, many igg4-sc patients have other organs involved, such as pancreas and major salivary glands (submandibular gland, parotid). the clinical and pathological characteristics of isolate igg4-sc had been described ; however, rare investigations investigated the differences of clinical characteristics of igg4-sc between with and without other organ affected. does the igg4-sc patient without other organ involved has a mild manifestation, good response to steroid therapy ? it is still unclear. considering the susceptibility of pancreas and salivary glands and the screening facility, we focused on its differential characteristics between igg4-sc patients with or without these glands affected. a series of patients with igg4-sc in the period from january 2006 to december 2015 at our hospital and who did not receive any therapy before were included. the diagnosis were based on recognized international criteria, including the hisort criteria and the japan pancreas society criteria. each patient accepted magnetic resonance cholangiopancreatography (mrcp) and endoscopic retrograde cholangiopancreatography (ercp) for biliary and pancreatic ducts, ultrasound and thin - layer computed tomography for pancreas and major salivary glands. for mass or diffusely enlarged glands, biopsy was performed. the features of pancreas, submandibular gland, and parotid affected included a focal mass or a diffusely enlarged gland (focal or diffuse duct stricture), a lymphoplasmacytic infiltrated within the tissues and a subsequent prompt response to steroid therapy. aimed to investigate the differential characteristics of igg4-sc with or without these glands affected, these patients were divided into 3 groups : single lesion group (without extra - biliary involved), double lesions group (1 extra - biliary organ involved), and multiple lesions group. the initial corticosteroid therapy, which was prednisone 40 mg / day orally for 4 to 6 weeks then taper by 5 mg / week for total of 13 weeks of treatment with regular monitoring of biochemistry and repeated imaging were given to all included patients. there was no steroid - sparing agents (azathioprine, methotremate, cyclophosphamide) for these patients, and no use of maintenance therapy in the initial therapy in the study. (1) disease response was defined as symptomatic, biochemical and radiologic improvement after the commencement of treatment. (2) disease remission referred to the maintenance of the improvements after cessation of treatment. (3) disease relapse was defined as recurrences of disease activity after achievement of remission and cessation of treatment. (4) failed weaning was defined as an inability to wean steroids completely because of a flare of disease activity. clinical information was collected and analyzed including demographics, clinical presentations, igg4 serology levels, imaging features and treatment outcomes. the statistic difference was performed by using anova and chi - square (spss 19, spss, inc, chicago). meier curves were used to assess differences in relapse - free survival rates between groups. all procedural protocols were approved and supervised by the ethics committee of our hospital, and informed consent was signed by each patient. the study identified 72 igg4-sc patients, including 60 males and 12 females (the ratio is 5:1). the initial presentation included obstructive jaundice in 59 of 72 patients (81.9%), whereas 9 (12.5%) with abdominal pain alone. in 4 patients 56 patients (77.8%) had undergone surgery eventually, the other had puncture biopsy. among all the igg4-sc patients, 10 patients had only bile duct involved, and the other 62 patients had pancreas involved. in total, 36 patients had a focal pancreatic mass at presentation, and 26 patients had diffuse pancreatic enlargement. pancreatic ductal disease was seen on mrcp and/or in 39 of 62 (62.9%) patients. focal pancreatic duct strictures were found in 22 of 62 (35.5%) patients, whereas the diffuse structuring was seen in 17 of 62 (27.4%) patients. besides the bile duct and pancreas, 12 patients had submandibular gland involved, 9 patients had parotid involved, and 1 patient had both (table 1). however, 10 patients had submandibular gland mass and 8 patients had parotid gland mass ; the other patients only had diffusely enlarged glands. comparison of clinical features between single and multiple lesions in immunoglobulin g4-related sclerosing cholangitis patients. to compared the differential characteristics between igg4-sc patients with or without other organs affected. the patients were divided into single lesion group, double lesions group, and multiple lesions group. as to the manifestation, the complaint was not more serious with more organs involved, but more complaints were given. the mean number of complaint was 2.9 kinds in the multiple lesions group, whereas the complaint was 1.4 kinds in the single lesion group (p 135 mg / dl in serum, as a cut - off value, demonstrated a sensitivity of 97% and a specificity of 79.6% in diagnosing igg4-rd. measurements of the serum igg4 concentration remain important for screening and evaluation of the disease. the more organs the disease involved, the higher serum igg4 level in our study. igg4-related sc displays segmental and long strictures of bile duct tree. in our cohort, the ratio of type 1 cholangiographic classification was 50% in triple or more lesions patients. that might indicate that the inflammatory igg4-positive plasma cells infiltrated more lesions of bile duct tree with more extra - biliary organs involved. systemic glucocorticoids, which are well known to induce nonselective apoptosis of lymphocytes, are the first - line approach for the most patients with igg4-rd. the majority of igg4-rd patients respond to glucocorticoids, particularly in early stages of disease. in some subsets of organ disease (e.g., pancreatitis), although the initial steroid response is excellent, relapses are common after early withdrawal of steroids for igg4-sc patients. considering too many interference factors in the treatment of igg4-sc, we only compared the initial response of glucocorticoids. the dosage and taper scenario were referred to the previous consensus and accordingly given to each patient. in our study, the igg4-sc patients relapse rate was 66.1%. in addition, our results reveal that the more extra - biliary organs involved or the more segments of bile duct involved, the higher rate of recurrence and the shorter relapse - free survival time. this indicates that the response of igg4-sc with multiple organs affected is poorer than that with the single lesions group. the study of hart had also shown that bile duct involvement were independent risk factors of igg4-rd disease relapse. initial experience suggested that steroid - sparing agents and maintenance therapy can maintain long - term remission. azathioprine, mycophenolate mofetil, 6-mercaptopurine, methotrexate, and tacrolimus have all been used as steroid - sparing agents. maintenance therapy refers to continuous low - dose glucocorticoids or any of the steroid - sparing agents following the achievement of remission. our study indicates that if more complaints, higher serum igg4 level and more stricture lesions of biliary tract were found, more organ lesions should be mentioned. moreover, considering the high relapse rate and short relapse - free survival, when more lesions are certified, more aggressive treatment should be given. in conclusion, the igg4-sc patients with multiple organs affected had more complaints, higher serum igg4 levels, and poor response to initial steroids.	abstractigg4-related sclerosing cholangitis (igg4-sc) is a rare biliary manifestation in which many other organs might be affected. the purpose of our study was to investigate the different clinical characteristics and initial steroid response between igg4-sc patients with and without other organs affected.a series of patients with igg4-sc in the period from january 2006 to december 2015 at our hospital were included. the pancreas and major salivary glands were screened, and the initial corticosteroid therapy was given. clinical information was collected and analyzed including demographics, clinical presentation, igg4 serology, imaging features, and treatment outcomes.the study identified 72 igg4-sc patients, including 60 males and 12 females. the mean age was 59.8 years old. among these igg4-sc patients, 10 patients had only bile duct involved, 42 patients had 2 organs involved and 20 patients had multiple organs involved. in patients with multiple organs involved, more complaints were given (mean 2.9 kinds), higher serum igg4 levels were found (23458 19402.7 mg / l), and more stricture lesions of biliary tract were shown. all 72 patients exhibited a disease response within 4 to 6 weeks of starting steroids. the remission rate in the multiple lesions group was lower (60%), and the recurrence rate is higher (83.3%). the relapse - free survival was 20.0 months in the single lesion group, which is longer than that in the multiple lesions group (3.1 months, p < 0.05).the igg4-sc patients with multiple organs affected had more complaints, higher serum igg4 levels, and poor response to initial steroids.
all study conduct adhered to the tenets of the declaration of helsinki, and the study was approved by the institutional review board at kim 's eye hospital. we conducted a retrospective review of the medical records of patients who were diagnosed with macular edema secondary to rvo between january 2010 and december 2012. the inclusion criteria were as follows : (1) initially treated with two or more consecutive intravitreal bevacizumab injections (1.25 mg/0.05 ml), (2) refractory to bevacizumab therapy (300 m), (3) underwent intravitreal ta injection (4 mg/0.1 ml) within 8 weeks of last bevacizumab injection, (4) followed - up for at least one month after ta injection. exclusion criteria included severe media opacity, previous vitreoretinal surgery, intraocular inflammation, and other disorders that may have influenced macular function (e.g. exudative age - related macular degeneration, proliferative diabetic retinopathy, epiretinal membrane). all subjects underwent a comprehensive ophthalmologic examination, including best - corrected visual acuity (bcva) measurement, 90-diopter lens slit - lamp biomicroscopy, fundus photography, fluorescein angiography, and spectral domain optical coherence tomography (sd - oct ; spectral oct / slo ; oti ophthalmic technologies inc., miami, fl, usa). because the evaluation of macular volume was not routinely performed as part of sd - oct testing, cft measurements were used in analyses. the vertical distance between the internal limiting membrane and the retinal pigment epithelium at the foveal center the mean of oct parameters measured on the horizontal and vertical scans were used in analyses. visual acuity measurements were converted to the logarithm of the minimal angle of resolution for analyses. the bcva and cft, one month after the last bevacizumab injection, was compared with those measured one month after ta injection. eyes exhibiting > 150 m of a decrease in cft or a cft 250 m after ta injection were classified into the responsive group. bcva and cft after bevacizumab injection were compared to measurements made one month and three months after ta injection. patient age, diagnosis, bcva, and cft before ta injection were compared between groups, as was the time between symptom onset and ta injection. comparisons of values between different time points within the same group were performed using a paired t - test, repeated measures analysis of variances, or friedman test. comparisons between the responsive and nonresponsive groups were performed using a mann whitney u - test or a fisher 's exact test. statistical analyses were performed with a commercially available software package (spss version 12.0 for windows, spss sciences, chicago, il, usa). a p 300 m), (3) underwent intravitreal ta injection (4 mg/0.1 ml) within 8 weeks of last bevacizumab injection, (4) followed - up for at least one month after ta injection. exclusion criteria included severe media opacity, previous vitreoretinal surgery, intraocular inflammation, and other disorders that may have influenced macular function (e.g. exudative age - related macular degeneration, proliferative diabetic retinopathy, epiretinal membrane). all subjects underwent a comprehensive ophthalmologic examination, including best - corrected visual acuity (bcva) measurement, 90-diopter lens slit - lamp biomicroscopy, fundus photography, fluorescein angiography, and spectral domain optical coherence tomography (sd - oct ; spectral oct / slo ; oti ophthalmic technologies inc., miami, fl, usa). because the evaluation of macular volume was not routinely performed as part of sd - oct testing, cft measurements were used in analyses. the vertical distance between the internal limiting membrane and the retinal pigment epithelium at the foveal center was measured based on horizontal and vertical foveal - centered sd - oct images. the mean of oct parameters measured on the horizontal and vertical scans were used in analyses. visual acuity measurements were converted to the logarithm of the minimal angle of resolution for analyses. the bcva and cft, one month after the last bevacizumab injection, was compared with those measured one month after ta injection. eyes exhibiting > 150 m of a decrease in cft or a cft 250 m after ta injection were classified into the responsive group. bcva and cft after bevacizumab injection were compared to measurements made one month and three months after ta injection. patient age, diagnosis, bcva, and cft before ta injection were compared between groups, as was the time between symptom onset and ta injection. comparisons of values between different time points within the same group were performed using a paired t - test, repeated measures analysis of variances, or friedman test. comparisons between the responsive and nonresponsive groups were performed using a mann whitney u - test or a fisher 's exact test. statistical analyses were performed with a commercially available software package (spss version 12.0 for windows, spss sciences, chicago, il, usa). twenty - seven eyes from 27 patients satisfied eligibility criteria. among these, 4 eyes were excluded because sd - oct had not been performed after intravitreal ta injection. ultimately, 23 eyes from 23 patients (12 male [52.2% ], 11 female [47.8% ]) were included in study analyses [table 1 ]. central rvo (crvo) and branch rvo (brvo) were diagnosed 15 (65.2%) and eight eyes (34.8%), respectively. eighteen eyes (78.3%) were phakic, and five eyes (21.7%) were pseudophakic. at the time of rvo diagnosis, mean bcva was 0.67 0.34 (snellen equivalent = 20/93, range : 20/40020/30) and mean cft was 523.5 120.9 m (range : 357804 m). baseline characteristics of patients with macular edema secondary to retinal vein occlusion that was refractory to intravitreal bevacizumab therapy (n=23 eyes) patients were initially treated with an average of 2.4 0.6 (range : 24) monthly injections of intravitreal bevacizumab. mean bcva and cft measured at 1 month after the last bevacizumab injection were 0.72 0.34 and 516.6 112.4 m, respectively. the ta injection was performed an average of 5.8 1.4 weeks after the last bevacizumab injection and the mean duration between symptom onset, and ta injection was 19.8 4.6 weeks. one month after ta injection, mean bcva and cft had changed to 0.67 0.35 and 402.3 159.7 m, respectively [fig. 2 ]. visual acuity had improved by one to two lines in 3 eyes (13.0%) and by two lines or greater in four eyes (17.4%). when compared with values measured before the injection, cft had significantly decreased (p 150 m decrease in cft or a cft 250 m after ta injection (responsive group, n = 9). the remaining eyes were included in the nonresponsive group (dashed line, closed square, n = 14) patient age (p = 0.926), diagnosis distribution (p = 0.179), bcva (p = 0.898), and cft (p = 0.219) before ta injection were not significantly different between the two groups [table 2 ]. in addition, the time between symptom onset and ta injection was not significantly different between the two groups (p = 0.477). comparison of characteristics of patients between the responsive group (n=9) and the nonresponsive group (n=14) eight eyes of the responsive group completed 3 months follow - up. in these eyes, the bcva before ta injection and at 1 month and 3 months after the injection was 0.75 0.32, 0.55 0.30, and 0.65 0.28, respectively. the cft was 491.9 102.5 m, 238.4 36.9 m, and 351.6 159.8 m, respectively. the bcva and cft measured after ta injection was significantly decreased when compared with the value before the injection (p = 0.017 and p = 0.011, respectively). when compared to the values before the injection, bcva had improved by one to two lines in two eyes (25.0%) and by two lines or greater in two eyes (25.0%). nine eyes of the nonresponsive group completed 3 months follow - up. in these eyes, the bcva before ta injection and at 1 month and 3 months after the injection was 0.73 0.29, 0.77 0.27, and 0.79 0.26, respectively. the cft was 547.0 106.7 m, 522.4 108.3 m, and 511.2 94.0 m, respectively. the bcva and cft measured after ta injection was not significantly changed when compared with the value before the injection (p = 0.137 and p = 0.074, respectively). an increase in intraocular pressure was noted in four eyes (17.4%), which were subsequently treated with topical anti - glaucoma medication. patients were initially treated with an average of 2.4 0.6 (range : 24) monthly injections of intravitreal bevacizumab. mean bcva and cft measured at 1 month after the last bevacizumab injection were 0.72 0.34 and 516.6 112.4 m, respectively. the ta injection was performed an average of 5.8 1.4 weeks after the last bevacizumab injection and the mean duration between symptom onset, and ta injection was 19.8 4.6 weeks. one month after ta injection, mean bcva and cft had changed to 0.67 0.35 and 402.3 159.7 m, respectively [fig. 2 ]. visual acuity had improved by one to two lines in 3 eyes (13.0%) and by two lines or greater in four eyes (17.4%). when compared with values measured before the injection, cft had significantly decreased (p 150 m decrease in cft or a cft 250 m after ta injection (responsive group, n = 9). the remaining eyes were included in the nonresponsive group (dashed line, closed square, n = 14) patient age (p = 0.926), diagnosis distribution (p = 0.179), bcva (p = 0.898), and cft (p = 0.219) before ta injection were not significantly different between the two groups [table 2 ]. in addition, the time between symptom onset and ta injection was not significantly different between the two groups (p = 0.477). comparison of characteristics of patients between the responsive group (n=9) and the nonresponsive group (n=14) eight eyes of the responsive group completed 3 months follow - up. in these eyes, the bcva before ta injection and at 1 month and 3 months after the injection was 0.75 0.32, 0.55 0.30, and 0.65 0.28, respectively. the cft was 491.9 102.5 m, 238.4 36.9 m, and 351.6 159.8 m, respectively. the bcva and cft measured after ta injection was significantly decreased when compared with the value before the injection (p = 0.017 and p = 0.011, respectively). when compared to the values before the injection, bcva had improved by one to two lines in two eyes (25.0%) and by two lines or greater in two eyes (25.0%). nine eyes of the nonresponsive group completed 3 months follow - up. in these eyes, the bcva before ta injection and at 1 month and 3 months after the injection was 0.73 0.29, 0.77 0.27, and 0.79 0.26, respectively. the cft was 547.0 106.7 m, 522.4 108.3 m, and 511.2 94.0 m, respectively. the bcva and cft measured after ta injection was not significantly changed when compared with the value before the injection (p = 0.137 and p = 0.074, respectively). an increase in intraocular pressure was noted in four eyes (17.4%), which were subsequently treated with topical anti - glaucoma medication. in the present study, a significant decrease in macular edema was noted after intravitreal ta injection in eyes with macular edema secondary to rvo that was refractory to intravitreal bevacizumab therapy. one month after ta injection, a cft decrease > 150 m or a cft value < 250 m was noted in approximately 40% of eyes. although overall improvement in visual acuity was not significant, visual acuity improvements were significant in eyes with a marked decrease in macular edema following ta therapy. visual acuity improved by at least one line in approximately 33% of eyes at 1 month and 24% at 3 months, suggesting that ta therapy is effective in some patients. the marked decrease in macular edema in some patients likely resulted from the distinct effect of ta, which is very different from the effects of anti - vegf agents. the excellent efficacy of anti - vegf therapy strongly indicates that vegf plays an important causative role in the development of macular edema. however, various cytokines, including interleukin-6 (il-6) and il-8, have also been associated with macular edema in eyes with rvo. it is possible that the macular edema in our patients, who were refractory to intravitreal anti - vegf therapy, had developed with mechanisms more closely associated to pathologic changes unrelated to vegf. the ta injection can reduce il-6 and il-8 that can not be modulated by anti - vegf therapy. another possible explanation is the time lag between anti - vegf and ta injection. in the present study, an average of 5.8 weeks separated the last anti - vegf injection and the ta injection. it is well - known that a spontaneous decrease in macular edema and an improvement in visual acuity can occur without intervention in eyes with rvo. to minimize the effect of this spontaneous improvement, patients with an interval between anti - vegf and ta injections longer than 8 weeks were excluded from analyses. however, it is possible that some patients did have this spontaneous improvement in visual acuity in the 5.8 week interval period. jonas. previously reported the efficacy of intravitreal ta injection in treating the macular edema refractory to bevacizumab injection. they observed a marked decrease in macular edema and a subsequent increase in visual acuity in 2 of 2 patients, who were administered 20 mg of intravitreal ta. in the present study, we used a much smaller ta (4 mg), which may explain, at least in part, our much more limited efficacy. in this study, several analyses were performed to determine factors predictive of 1 month cft decrease after ta injection. although we failed to verify any significant factors, this may have been because of our small sample size. one notable finding was the marked difference in the proportion of brvo between the responsive group (56%) and the nonresponsive group (21.4%). the proportion of brvo in the responsive group was almost twice as great as in the nonresponsive group, suggesting that ta injection may be more beneficial for patients with brvo. this study has several limitations, mainly due to its retrospective design and small sample size. intravitreal ta injection was found to be beneficial in some patients with macular edema secondary to rvo refractory to bevacizumab therapy. at 1 month after ta injection lead to a marked reduction in macular edema in approximately 40% of patients, and 33% patients had an improvement in visual acuity. therefore, this study suggests that intravitreal ta injection may be a treatment option for macular edema secondary to rvo refractory to intravitreal anti - vegf therapy. however, deterioration in visual acuity and increase in retinal thickness between the 13 months follow - up period suggests the limited long - term efficacy of this therapy. further studies with a larger study population and a longer follow - up are needed.	aims : to evaluate the 1-month efficacy of intravitreal triamcinolone acetonide (ta) in treating macular edema secondary to retinal vein occlusion (rvo) that was refractory to intravitreal bevacizumab.materials and methods : this retrospective, observational study included 23 eyes from 23 patients with macular edema secondary to rvo. macular edema that did not respond to two or more consecutive intravitreal bevacizumab injections was treated with intravitreal ta. central foveal thickness (cft) and best - corrected visual acuity (bcva) were compared before and one month after ta injection.results:fifteen eyes were diagnosed with central rvo, and eight eyes were diagnosed with branch rvo. all patients were previously treated with 2.4 0.6 intravitreal bevacizumab injections. the ta injection was performed, on average, 5.8 1.4 weeks after the last bevacizumab injection. the cft before ta injection was 516.6 112.4 m and significantly decreased to 402.3 159.7 m after ta therapy (p < 0.001). the logarithm of the minimal angle of resolution bcva was 0.72 0.34 before ta therapy and was not significantly improved by the treatment (0.67 0.35, p = 0.119), despite a decrease in cft. however, seven eyes (30.4%) had a bcva gain of one or more lines.conclusions:intravitreal ta therapy was beneficial in some patients with macular edema secondary to rvo that was refractory to intravitreal bevacizumab therapy. this study suggests that intravitreal ta should be considered as a treatment option for refractory macular edema.
black men of african descent have variously been reported to be at especially high - risk for developing prostate cancer.1 while the incidence and mortality rates are ostensibly higher in african - americans than americans of other racial groups,2,3 african- caribbean men are reported to have the highest rate of prostate cancer in the world.1 even worse is the discovery that these african descendants are most likely to present at a younger age with more advanced disease, and historically have a poorer disease prognosis.4 this supports autopsy reports showing that african - american men 20 years or older who died of other causes had a higher incidence and higher grade of intraepithelial prostate neoplasia as compared with age - adjusted cohorts of caucasian men.5 in addition, numerous studies of the pathologic characteristics of prostate tumors show that african - american patients have higher tumor volumes than caucasian patients with a similar stage of disease.6 all these observations suggest that biological differences in tumorigenesis may be a factor in the course of disease among races.6 indeed, numerous studies have characterized several genes, the expression patterns and variants7,8 of which determine the tumoral heterogeneity9 which characterizes the disparity in incidence and aggressiveness of disease found among african - american and caucasian patients.10,11 currently, there is a good deal of literature claiming that lifestyle factors impact the risk of prostate cancer in african- americans. accordingly, the wide variations in cancer incidence among populations living in different regions of the world echo the suggestion that the majority of malignant tumors are triggered by environmental factors.12 epidemiological and population studies indicate that lifestyle variables seldom account for the disparities in prostate cancer risk among different groups,13 but rather suggest that interaction of specific genetic and lifestyle factors critically predisposes populations to most cancers.1416 regrettably, there has been no consensus as to whether the higher incidence of prostate cancer or the disturbing two - fold higher mortality rate from this disease among african - americans17 is driven by behavior, biology, or both. historically, all individuals in this diaspora are people of african descent, and it is logical that men of african ancestry share common genetic or familial factors that may increase their vulnerability to prostate cancer.18 although the contribution of hereditary factors in cancer has been considered minor, the estimated effects of genetic factors for five tumors vary considerably, with the highest effect observed in prostate cancer.16 this has been corroborated by recent studies showing that african - american patients with gender - specific malignancies like prostate cancer have worse survival than white patients, despite controlling for prognostic, treatment, and socioeconomic factors.19 this has led to the conclusion that unrecognized interactions of host biological, hormonal, and/or inherited factors may contribute to differential survival outcomes by race in gender - specific malignancies like prostate cancer. the mechanism of inherited genetic predisposition, which is responsible for the varied interethnic response to environmental factors, could be obscured in the genetic framework of ancestral populations in the african diaspora. mining of such data could reveal trends in the incidence of prostate cancer in these populations. consistency in incidence trends in such ancestral populations as the african diaspora could encourage the search for an inherited genetic predisposition that responds to behavioral triggers for the disease. in this paper, we present emergent trends in the reported incidence of prostate cancer in modern day nigeria, a country that accounts for a highly significant proportion of the african diaspora.20 correlation in features of the disease in these two populations could guide the search for the biology of the disease in these populations. we grouped together prospective and retrospective data previously obtained from histologic and clinical records at the tertiary referral or university teaching hospitals serving the six nigerian geopolitical zones (figure 1), each of which covers a core state and several peripheral states. the data provided by the teaching hospitals in each of the geopolitical zones and their peripheral states are shown in table 1. our study population was broadened because we examined data from two or more such tertiary health institutions in a zone. because our data embraced all of the geopolitical zones in nigeria, the information it provides may constitute the most extensive hospital - based prostate cancer incidence report for nigeria so far. nonetheless, the present data compensate for the lack of satisfactory prostate cancer incidence data that ought to be coordinated and collated by the ibadan university college hospital (uch) national headquarters for cancer registries in nigeria. such data ought to be generated by population - based screening for prostate and other cancers in the presently existing university teaching hospital - based registries at maiduguri, zaria, jos, kano, ilorin, calabar, enugu, lagos, and ife - ijesha. the data presented in this report were retrieved by researchers from these teaching hospital - based registries, except for the usman danfodiyo uch records, which were obtained from a cancer patient register in the department of histopathology. the histology and cytology specimens21,22 used for some of these studies were obtained from tertiary, secondary, and private hospitals located close to the core tertiary hospital and the surrounding peripheral states, towns, and villages23 within that particular geopolitical zone. in a few surveys of prostate cancer risk from a seldom - screened cohort of the rural population, participants were invited for a health survey after alerting them to the procedures involved, including a blood test and digital rectal examination (dre) by a local nurse.24 in many of the retrospective studies, information on the age of patients at presentation,21,2527 histopathological grading, mode of presentation, clinical and biochemical response to chemotherapy, their relative frequencies,28 and the level of prevailing tumor biomarkers were obtained from patient files.2931 in prospective studies, data were obtained from patients presenting with histologically diagnosed carcinoma of the prostate during the study period.32 again, in the prospective studies, various patient information was documented including, but not limited to, prevailing biomarker levels such as serum acid phosphatase, dre, blood electrolyte profile, prostate biopsy, transabdominal ultrasonography, and a survey of the skeleton.32,33 in some of the more recent cases, detection of prostate cancer by prostate - specific antigen (psa) was preceded by dre. in most cases, the diagnosis was made on the basis of dre, an ultrasound scan (which when combined with artificial neural network classification tools enabled encouraging differentiation between cancerous and noncancerous tissue),34 and confirmed by tru - cut (uk medical limited, sheffield, uk) prostatic biopsy.21 in certain cases too, the clinical staging of the disease was determined using the tumor, node, metastasis (tnm) system at the time of dre.29 in others, the prostate was carefully assessed for size, hardness, nodularity fixation, and discomfort.33 staff at the registries was centrally trained through programs run by the national headquarters for cancer registries in nigeria and delivered as instructed data items abstracted from pathology reports, medical charts, and questionnaires to treating physicians in order to obtain complete data on therapies. to avoid duplication, all retrieved records were reported to have been double - checked by name and matched with hospital numbers. cases presented by the reports ranged from 125 to 4686, where large number of cases represented retrospective studies of all malignant diseases studied over a longer period. age - standardized rates were calculated using nigerian census figures for the appropriate study periods. the mean sample size for assessment of prostate tumors and benign prostatic hyperplasia was 327 280.12. studies were variously approved by the research and ethics committees of the tertiary health institutions involved.24 many statistical measures have been developed to compare the incidence rates of particular cancers for different age groups in two or more populations. a long list of references and sources of information indicate extensive discussion on the subject.3537 we used some of the accepted standards to calculate different statistics for estimating and comparing the incidence rate of prostate cancer for different age groups in different zones of nigeria. in order to delineate the different age structures of the populations in each geopolitical region, the direct method of standardization the primary statistics in the direct method are age - specific rates, ai,38 calculated as the incidence rate per 100,000 by the formula : where di is the number of cases in the ith age group and yi is the person - years of observation in the ith age group. the above formula indicates the age incidence rate of prostate cancer per 100,000 of each age group for the population covered by a particular hospital calculated by dividing the number of new prostate cancer cases by the number of person - years at risk for every age group and multiplying that by 100,000.38 person - years at risk were found by multiplying the number of males in different age groups in the nigerian population (obtained from population pyramids of nigeria)39 during the observation period. then, in order to compare the age - specific rates between different age groups within a geopolitical region and between geopolitical regions, the observed age - specific rates were standardized against the reference population, ie, the standard world population for each age group. this became the hypothetical value for age - specific rates, which would have occurred in the ith age class of the reference population. standardization in the ith age class was found by the product aiwi, where wi was the reference population for the age group i. the total of all the incidence rates (ai, wi) gave the age - standardized incidence rate per 100,000 world population for prostate cancer among nigerians. finally, the most important statistic is the mean annual age - standardized incidence rate (asr) of prostate cancer per 100,000 of males in nigeria, and is calculated as : variance is given by the formula : and the 95% confidence interval by : for data in tables 4 and 5, the asr, the standard error (se = var(asr)), and 95% confidence interval were calculated for each region and provided summary comparison statistics. two more statistics were also included in the data, ie, firstly, the crude (all - ages) rates per 100,000 person - years for all regions, which were used to amplify the effect of age and the geopolitical zone on the average incidence rate of prostate cancer and, secondly, the cumulative rate and the corresponding measure for cumulative risk. the cumulative rate was the total ai, ti where it was the observation period of i age class of each geopolitical zone, and cumulative risk was the percentage risk a nigerian residing in one of the geopolitical regions could have of developing prostate cancer (if no other causes of death were present). we grouped together prospective and retrospective data previously obtained from histologic and clinical records at the tertiary referral or university teaching hospitals serving the six nigerian geopolitical zones (figure 1), each of which covers a core state and several peripheral states. the data provided by the teaching hospitals in each of the geopolitical zones and their peripheral states are shown in table 1. our study population was broadened because we examined data from two or more such tertiary health institutions in a zone. because our data embraced all of the geopolitical zones in nigeria, the information it provides may constitute the most extensive hospital - based prostate cancer incidence report for nigeria so far. nonetheless, the present data compensate for the lack of satisfactory prostate cancer incidence data that ought to be coordinated and collated by the ibadan university college hospital (uch) national headquarters for cancer registries in nigeria. such data ought to be generated by population - based screening for prostate and other cancers in the presently existing university teaching hospital - based registries at maiduguri, zaria, jos, kano, ilorin, calabar, enugu, lagos, and ife - ijesha. the data presented in this report were retrieved by researchers from these teaching hospital - based registries, except for the usman danfodiyo uch records, which were obtained from a cancer patient register in the department of histopathology. the histology and cytology specimens21,22 used for some of these studies were obtained from tertiary, secondary, and private hospitals located close to the core tertiary hospital and the surrounding peripheral states, towns, and villages23 within that particular geopolitical zone. in a few surveys of prostate cancer risk from a seldom - screened cohort of the rural population, participants were invited for a health survey after alerting them to the procedures involved, including a blood test and digital rectal examination (dre) by a local nurse.24 in many of the retrospective studies, information on the age of patients at presentation,21,2527 histopathological grading, mode of presentation, clinical and biochemical response to chemotherapy, their relative frequencies,28 and the level of prevailing tumor biomarkers were obtained from patient files.2931 in prospective studies, data were obtained from patients presenting with histologically diagnosed carcinoma of the prostate during the study period.32 again, in the prospective studies, various patient information was documented including, but not limited to, prevailing biomarker levels such as serum acid phosphatase, dre, blood electrolyte profile, prostate biopsy, transabdominal ultrasonography, and a survey of the skeleton.32,33 in some of the more recent cases, detection of prostate cancer by prostate - specific antigen (psa) was preceded by dre. in most cases, the diagnosis was made on the basis of dre, an ultrasound scan (which when combined with artificial neural network classification tools enabled encouraging differentiation between cancerous and noncancerous tissue),34 and confirmed by tru - cut (uk medical limited, sheffield, uk) prostatic biopsy.21 in certain cases too, the clinical staging of the disease was determined using the tumor, node, metastasis (tnm) system at the time of dre.29 in others, the prostate was carefully assessed for size, hardness, nodularity fixation, and discomfort.33 staff at the registries was centrally trained through programs run by the national headquarters for cancer registries in nigeria and delivered as instructed data items abstracted from pathology reports, medical charts, and questionnaires to treating physicians in order to obtain complete data on therapies. to avoid duplication, all retrieved records were reported to have been double - checked by name and matched with hospital numbers. cases presented by the reports ranged from 125 to 4686, where large number of cases represented retrospective studies of all malignant diseases studied over a longer period. age - standardized rates were calculated using nigerian census figures for the appropriate study periods. the mean sample size for assessment of prostate tumors and benign prostatic hyperplasia was 327 280.12. studies were variously approved by the research and ethics committees of the tertiary health institutions involved.24 many statistical measures have been developed to compare the incidence rates of particular cancers for different age groups in two or more populations. a long list of references and sources of information indicate extensive discussion on the subject.3537 we used some of the accepted standards to calculate different statistics for estimating and comparing the incidence rate of prostate cancer for different age groups in different zones of nigeria. in order to delineate the different age structures of the populations in each geopolitical region, the direct method of standardization the primary statistics in the direct method are age - specific rates, ai,38 calculated as the incidence rate per 100,000 by the formula : where di is the number of cases in the ith age group and yi is the person - years of observation in the ith age group. the above formula indicates the age incidence rate of prostate cancer per 100,000 of each age group for the population covered by a particular hospital calculated by dividing the number of new prostate cancer cases by the number of person - years at risk for every age group and multiplying that by 100,000.38 person - years at risk were found by multiplying the number of males in different age groups in the nigerian population (obtained from population pyramids of nigeria)39 during the observation period. then, in order to compare the age - specific rates between different age groups within a geopolitical region and between geopolitical regions, the observed age - specific rates were standardized against the reference population, ie, the standard world population for each age group. this became the hypothetical value for age - specific rates, which would have occurred in the ith age class of the reference population. standardization in the ith age class was found by the product aiwi, where wi was the reference population for the age group i. the total of all the incidence rates (ai, wi) gave the age - standardized incidence rate per 100,000 world population for prostate cancer among nigerians. finally, the most important statistic is the mean annual age - standardized incidence rate (asr) of prostate cancer per 100,000 of males in nigeria, and is calculated as : variance is given by the formula : and the 95% confidence interval by : for data in tables 4 and 5, the asr, the standard error (se = var(asr)), and 95% confidence interval were calculated for each region and provided summary comparison statistics. two more statistics were also included in the data, ie, firstly, the crude (all - ages) rates per 100,000 person - years for all regions, which were used to amplify the effect of age and the geopolitical zone on the average incidence rate of prostate cancer and, secondly, the cumulative rate and the corresponding measure for cumulative risk. the cumulative rate was the total ai, ti where it was the observation period of i age class of each geopolitical zone, and cumulative risk was the percentage risk a nigerian residing in one of the geopolitical regions could have of developing prostate cancer (if no other causes of death were present). demographic information from the registries was divided into the pre - psa and psa eras. pre - psa era comprised data recorded between 1970 and 1999, and only included reports in the 1990s that did not detect prostate cancer by any of the psa assays. psa era encompassed cases that were detected between 1991 and 2007, and considered mostly prostate cancer cases that were detected by any of the psa assays. this classification considered the late 1980s as the period when psa became the diagnostic tool for prostate cancer in the western world, while its countrywide application in nigeria was only very recent. data from the pre - psa era comprised various prospective and retrospective hospital - based records of prostate cancer across the geopolitical zones, except for the inaccessible figures from the north - east (ne), arranged according to age and incidence (table 2). representative data obtained from countrywide registries revealed that approximately 98% of the prostate cancer cases were adenocarcinoma.23,29,30,50 nonetheless, two patients (1.4% and 1.1%) presented with squamous cell carcinoma or adenosquamous carcinoma in the south - south (ss) and south - west (sw), respectively.29,43 however, the report from the ne indicated that most of these patients presented with symptoms similar to those of benign prostatic hyperplasia.21 apart from specifying the differentiated types of adenocarcinomas, identification of specific histologic subtypes as, eg, small cell, ductal, and mucinous, was not reported. data from these zonal registries showed that the highest incidence rate for prostate cancer was among patients in the 6069 year and 7079 year age brackets. in contrast, the age cohorts below 59 years presented with the lowest incidence of prostate cancer in all the zones during the study period. despite the sparse tertiary hospital - based incidence data obtained for each geographic population, we computed a crude and age - standardized incidence rate of prostate cancer for the different age groups (tables 4 and 5). furthermore, even in the psa era, we observed a trend similar to the age - specific incidence of disease found in the pre - psa data (table 3). however, in most data from the psa era, age - specific incidence rates of disease in cohorts below 59 years were surprisingly high, especially in the ss1, sw2, and north - west (nw) zones. the detection of some concealed cases of prostate cancer in the ss and a section of the se by psa tests is presented in tables 6 and 7. distribution of psa values according to patient age (table 6) shows that the mean values for prospective, cross - sectional, and prospective hospital - based studies ranged between 1.17 ng / ml and 18.0 ng / ml. furthermore, the complementarity of psa and dre in predicting prostate cancer was also established in these populations (table 7). here, between 41.4% and 46.15% of prostatic cases detected by a psa cutoff 4.0 were confirmed as symptomatic and palpable by dre tests. in contrast, between 23.08% and 96% of prostatic cases detected by a psa cutoff 4.0 were shown to be enlarged but asymptomatic by dre. irreconcilable data from the zones for the detection of prostate cancer by psa measurements were omitted. figures 2 and 3, respectively, show our calculated crude pre - psa and psa era annual incidence rates for prostate cancer per 100,000 of defined age groups for the estimated census populations serving the specific zonal tertiary health institutions. these curves show an exponential annual incidence rate of disease in the age group 5079 years, with a peak annual incidence at 7079 years in most cases except in the cross - sectional studies from ss1 (figure 2) where the incidence rate reached a peak at 80 years. in both eras, the calculated annual incidence rate for all zonal populations dropped beyond the age of 79 years, except for the reported rise in incidence in ss1. overall, the annual incidence rate recorded for the sw geopolitical zone in both eras was dramatically different from that in the other zones. the crude rate and the average annual age - standardized rate for prostate cancer by region in the period before and during the psa era are shown in tables 4 and 5, respectively. based on these data, further cumulative risk (%) ranking conferred the sw and north - central (nc) zones with the highest crude and age - standardized rates in the pre - psa era (table 4). this cumulative risk ranking was surpassed by values obtained from the ss zone during the psa era (table 5). the few reports that described the extent of prostate cancer spread were based on tnm classification (table 8) and whitmore - jewett stages (table 9). from the tnm classification, 81.5% of the tumors in hospital - based cases from the se zone had spread through the prostatic capsule, as opposed to 19.3% of such cases reported in the ss zone. worse still, within the ss zone, most patients reporting to hospitals had tumors that had invaded nearby structures, as observed by t4 clinical staging (62.1%). approximately one - third or more of the patients whose tumors were scored by whitmore - jewett staging presented with prostate tumors extending through the capsule at the time of reporting to hospital (28.6%, 42.9%, and 27.2% reported in the se, sw1, and sw2, respectively). expectedly, whitmore - jewett staging also showed that not less than half of the patients seen in tertiary hospitals in all the reported zones presented with tumors that had spread to other organs (59.7%, 51.3%, and 59.2% in the se, sw1 and sw2, respectively, table 9). this trend is thus similar to the picture presented by previous grading of patients on the tnm classification. finally, from the histologic subtypes (table 10), 20%49.5% of tumors recorded in the northern zones were considered to be high grade or poorly differentiated. again, using this scoring system, 51%80% of tumors were classified as generally low grade tumors. generally, in this case, a little less than a third or more of the tumors seen were defined as high grade, which is close to the proportion seen for tnm and whitmore - jewett staging reported earlier. no study to date has evaluated the nationwide annual incidence rate and clinical characteristics of prostate cancer in nigeria. in the present study, data from all university teaching hospital - based registries in the geopolitical zones of nigeria except for the ne zone showed that patients aged 6069 years and 7079 years had the highest rate of prostate cancer. the only available information on prostate cancer demographics21 from the ne zone is a pubmed abstract indicating that the majority (56%) of affected patients were 65 years of age or younger. the report also showed that most patients in this group presented with symptoms similar to benign prostatic hyperplasia. however, details such as histologic tumor grade and age distribution of patients were inaccessible for our study. in addition, the high incidence of the disease observed in men aged 60 years and older in all geopolitical zones of nigeria is consistent with the observation that age - specific incidence rates of prostate cancer rise steadily with advancing age worldwide.41 these results corroborate those of previous studies identifying the 6570 year age group in males from ibadan and washington, dc, as presenting with the peak incidence of carcinoma of the prostate.42 the average peak incidence according to age in most of the reports predated the seventh decade of life. this is slightly lower than the mid years of the seventh decade reported for caucasians in europe and the us,4345 leading us to suspect that prostate cancer occurs at a relatively younger age among nigerians.43 equally, the available data from nigeria show lower prostate cancer incidence rates (2%16% and 4.2%37.1% for the pre - psa and post - psa era, respectively) for patients aged younger than 50 years. the observation that a subset of patients with prostate cancer present at a younger age is consistent with the universal pattern whereby only few prostate cancer cases in the population have a genetic predisposition leading to early onset of the disease. 29,46,47 close scrutiny of the age distribution of prostate cancer cases from the zonal registries in nigeria revealed the disease burden in a number of patients whose ages ranged from (slightly less than) 50 years and above. this justifies the sensitivity of psa as a tumor marker, and also demonstrates its benefit in detecting previously undiagnosed cases that might only present in old age. data obtained from prospective cross - sectional studies on the age distribution of prostate cancer in the psa era reveal a narrowing of the incidence of the disease from the age group 6079 years to that > 50 years or 5059 years.24 this finding strengthens the evidence for an earlier age of disease prevalence in the nigerian population, but weakens the argument for use of hospital - based registry reports to demonstrate the incidence of prostate cancer in nigeria. given that hospital - based incidence records do not reflect the true nature of the disease in nigeria, the observation of advanced disease in these patients points strongly to the need for cross - sectional or population - based surveys that would uncover latent and asymptomatic cases, especially in the middle - aged population. currently, the influence of the environment, including dietary risk factors, in early onset of disease is being emphasized.48,49 nonetheless, the reliability and significance of the different ages quoted in these data have previously been questioned considering the literacy level of the parents of some of these patients and unreliability of some of their birth records.50 this concern seems genuine, considering that reliable birth recording was not mandatory in some remote parts of the country until 50 years ago. indeed, the sources of data for this report made calculation of a nationwide age - standardized incidence of prostate cancer almost impossible. a reason for this is that the exact population sizes encompassed by the different tertiary hospitals within the geopolitical zones are indeterminate. this is associated with the imprecise and overlapping character of the surrounding states and towns served by these hospitals. in such cases, census figures for calculating persons at risk in a defined area are based on guesswork. these figures could be so large as to reduce the expected age standardized incidence rates of the disease following a measure of the quotient obtained from the number of reported cases versus the product of the standard world population of persons at risk. furthermore, the futility of calculating an age - standardized incidence rate is also associated with the reliance on hospital - based data in a society with little health education, poor health care, and inadequate screening programs. overall, inadequacy in health education and the lack of effective screening programs could be a factor in the presentation of very severe cases in hospitals, therefore skewing the actual prevalence of the disease against the age - standardized incidence rates. the overall impact of this shortcoming could be inaccuracy of locally obtained hospital - based data on age - standardized incidence rates when compared with data obtained around the world. notwithstanding these shortcomings, the crude and average annual age - standardized incidence rates for prostate cancer in all the zones were calculated to draw attention to the exact nationwide character of the disease. the pre - psa data for the sw and nc zones showed the highest crude and age - standardized rates, but were surpassed by the ss zonal studies in the psa period. in addition to the disparity in health care that occasionally causes regional differences in the incidence of the disease, other factors like lower screening rates, cultural factors, and less aggressive therapy, could account for the differences observed in the regional crude and age - standardized rates obtained in this study. the reliability of data from such settings should be investigated in either a series of cross - sectional studies or, better still, a countrywide population - based screening program. it is to be expected that the age - specific incidence of prostate cancer among nigerian men aged 59 years or younger increased during the psa period as opposed to the pre - psa era. a reason for this, as shown by reports from the ss1,24 sw2,43 and nw25 zones is adherence to existing methods for diagnosis of prostate cancer and risk assessment. annual dre and measurement of serum psa are currently recommended beginning at age 50 years and advocated to start at age 45 years in those whose first - degree relatives had the disease.51,52 the increase in the number of diagnosed prostate cancer cases among nigerian age cohorts that previously showed moderate cases confirm that, most men are now diagnosed with the disease in its early stages (localized disease) because of the detection of elevated or rising serum psa detected during screening.51 a parallel may be drawn with the younger age at onset of prostate cancer among nigerian and african - american patients as opposed to caucasians.53 reasons for such differences have been suggested to be genetic, pathologic, molecular, and/or socioeconomic. 53 nevertheless, age is still universally accepted as an important risk factor for prostate cancer, which is rarely seen in men younger than 40 years of age. the increased use of psa as a screening test for nigerian men, in addition to the increase in cross - sectional or population - based surveys, has led to detection of a significant number of latent cases, as has been observed in other places, which may potentially lead to a dramatic reduction in the number of patients with metastatic disease at the time of diagnosis.51 the rapidity with which the calculated annual incidence of prostate cancer per 100,000 for defined age groups in the corresponding zonal populations progressed beyond 50 years of age is similar to previously reported us data on age - specific incidence curves.54 however, this upsurge in incidence of prostate cancer from 50 years of age onwards contrasts with the steady rise observed in china and the middle east.55 the upsurge in incidence after 50 years of age in the us had been attributed to its increased detection in these age cohorts.55 also, higher life expectancy in the us could have caused the sharp rise in incidence rate in men aged over 50 years, because most men in the us live long enough to develop this disease of the elderly. in contrast, the sharp rise in prostate cancer among nigerian men aged 5079 years could be attributed to the survival of these men into the period of life when prostate cancer manifests clinically and is detected. the peak annual incidence at 7079 years accounted for a third of all hospital - based prostate cancer cases observed in nigeria. there may be a number of reasons for the decline in annual incidence rate for men aged 80 years and older in all the zones. to begin with, life expectancy in nigeria is far less than 80 years, and as such, very few men live to this age,56 thus skewing the number of hospital cases. this could be compounded by the fact that 50%81% of nigerian patients present with clinically obvious and advanced disease,29,56 ie, higher rates of palpable disease with adverse prognostic features. the influence of life expectancy on the incidence of prostate cancer in the elderly is well known from data generated in the western world.57,58 again, the decline in incidence rate after 80 years of age in nigeria could relate directly to the low socioeconomic status of the majority of the population, poor access to health care, and poor health awareness nationwide. in such a scenario, these men are unlikely to undergo prostate cancer screening, and could have impalpable and undetectable prostate cancer, which might only be detected as an incidental finding or on biopsy. lastly, the dramatic deviation in annual incidence of prostate cancer recorded in the sw26,43 in favor of much higher incidence rates as compared with other geopolitical zones could be explained by the historic location of the cancer registries serving the tertiary university teaching hospitals at ibadan and ile - ife. the main cancer registry at ibadan is the only population - based registry in nigeria, serving a population of 1.22 million (1991 census) or more (2001 census) with a defined area of 70 square kilometers in ibadan, oyo state, sw nigeria.59 data for uch ibadan is regularly obtained from all the hospitals and health facilities (public, mission, and private) in the local government counties subserving the registry. 59 in addition, the uch is the national headquarters for cancer registries in nigeria, which coordinates the training programs and establishment of other zonal registries throughout the country. in this respect, data from this registry could be more precise, better managed, and its specific population boundaries better defined to enhance its apparent recording of higher incidence rates. traditionally, uch ibadan, which is the nation s leading tertiary health institution, remains the last referral option for most nigerians, irrespective of their regions of domicile, and this may have contributed to the higher than average incidence of prostate cancer recorded by its registry. on the other hand, the urbanized nature of ibadan, its environs, and its outer reaches, like ile - ife, could have been ideal for an increased culture of cancer screening, and health care access provided by the tertiary hospitals in the sw zone of nigeria. the highly skewed incidence rates of disease in this region could thus be explained, at least in part, by its early adoption of the psa test, and the level of health awareness of its population. prostate cancer stage of t3, c or greater, recorded for the vast majority of patients from one of the tertiary hospitals in the se zone of nigeria, has been attributed to the late presentation of prostate cancer among these patients.60 the greater than 80% presentation of advanced disease in these patients may not have appropriately represented the demographic characteristics of the disease in this zone. one reason for the poor demographic representation of data in this zone was the very low number of cases seen (n = 27) over a 5 year period, indicating that these were hospital - referred symptomatic cases at the time of presentation. again, the presence of a major tertiary hospital (ie, university of nigeria teaching hospital, enugu) within 200 km of the source of these data suggests that case loads might have been lifted off the smaller registries. whereas a lower percentage (19.3%) of advanced disease was reported in a ss zonal study, a much worse staging of t4 (62.1%) was reported for patients in this zone at the time of presentation to hospital. although there were only two comprehensive zonal representative reports available on tnm staging of prostate cancer in nigeria, the uncovering of advanced disease here does not deviate from countrywide reports of the advanced stages of disease seen in hospitals.21,43 the staging of close to one third or more of tumors in the southern zones, such as those extending through the capsule (stage c) using whitmore - jewett criteria agrees closely with the tnm staging scheme reported earlier. tumor staging in the geopolitical zones by the whitmore - jewett criteria also revealed that more than half of the patients had tumors that had spread to other organs (stage d). this also closely resembled the tumor grading found in the ss zone by the tnm scheme. to estimate the destructive potential and ultimate prognosis of the disease, pathologists from the ss, nc, and nw zones used the histologic scoring system to show the profile of surgical and biopsy specimens.25,31,61 based on histologic scoring of nc and nw specimens, 20% and 49%, respectively, were considered to be high - grade or poorly differentiated. this suggests that these patients had an increased tumor burden that was associated with a higher risk of metastatic disease, an increased chance of post - treatment failure, and a worse overall prognosis.6264 using this prostatic adenocarcinoma classification scheme, between 13% and 73% of tumor specimens were considered moderately differentiated or generally low - grade tumors. the 73% outlier value reported from the nw1 derives from the summing of well and moderately differentiated carcinoma as a group.25 furthermore, 38%67% of tumor specimens analyzed from these zones were well differentiated or low grade tumors. based on the gleason grading system, regarded as the gold standard for classifying prostatic adenocarcinoma,65 an average of one third of the northern zonal cases were high grade, corroborating the tnm and the whitmore - jewett staging reported earlier from the southern zones. evaluation of the significance of race on prostate cancer presentation and progression revealed a correspondence between the 52% rate of stage d disease found in african - american men66 and the observation that more than 50% of patients in this study presented with the disease in the southern zones of nigeria. the 52% rate of stage d disease observed in african - american men was far higher than the 26% rate observed in caucasians with access to the same military health care system.66 the biological difference that could account for the different stages of prostate cancer among african - americans and caucasians provided with the same standard of health care is yet to be elucidated.53 in addition, african - american men have been reported to present with a higher incidence (16.1%) of advanced disease or distant metastasis (from combined categories t3/ t4, n1, or m1) than caucasians (3.8%).53 more disturbing is the observation of a worse scenario in which 62.1% of nigerian men from the ss zone presented with t4 disease. on the gleason grading system, a comparable number of african - american men (35%) and nigerian men (30%) from the northern geopolitical zones had high grade tumor scores when viewed against data for caucasians (25%).53 the reasons for presentation of advanced disease by most inner city african - americans and most likely nigerians, in contrast with caucasians, could be attributed to rare screening or hospital visitation by the former, except during symptomatic and aggressive disease.67 limitations of this study include the dominance of its data by hospital - based registry reports and its substantially retrospective nature, both leading to an underestimation of the definite number of prostate cancer cases in the studied populations. the reasons for these low cases could be associated with exclusion of latent and symptom - free subjects from hospital reports, and the incomplete documentation and retrieval systems used by most of the registries. another limitation of this study is the discordant research strategy and data presentation, causing overlap and divergence of the study periods, leading to great difficulty in harmonizing the data. finally, most of the data presented are limited by the exclusion of specific histologic subtyping of cases, which would have had significant prognostic and therapeutic implications. this study suggests that the incidence of prostate cancer among nigerian men is higher at a younger age than in western countries. reasons for such early disease occurrence may include behavior, biology, or both. this early age of disease prevalence in nigeria weakens the rationale for use of hospital - based registry data to demonstrate the incidence of prostate cancer in nigeria. the finding of advanced disease in more than one - third of hospital - based cases seen in all the zones, regardless of the tumor grading scheme used, is an indication that the reported cases are mostly for symptomatic patients. this justifies funding of quality, controlled, population - based screening programs in all the regions. the data also suggest the need to investigate closeness of incidence of disease in the nigerian and african - american populations as opposed to caucasian populations. this is particularly relevant, given that our study population accounted for a highly significant proportion of the african diaspora, indicating that both populations may harbor similar inherited genetic predispositions that trigger alterations associated with increased risk of prostate cancer. considering the inconsistent pattern of reported findings for each study, we recommend the formation of a national study group on prostate cancer, which would formulate national guidelines for a research strategy and presentation of study protocols. such an arrangement would enhance the harmonization of these diverse investigations into coherent demographic and health reports of prostate cancer. we also recommend the formation of collaborative study programs on population - based screening of nigerian men and african - american men, in addition to examining genetic variants predisposing to prostate cancer that may be common in these populations. elucidation of such common genetic variants may unmask common pathways in prostate carcinogenesis and reveal a universal etiology of prostate cancer. moreover, identification of specific histologic subtypes of cancer in pathology reports may lead to significant prognosis of the cases and enable development of suitable therapeutic interventions.	backgroundto date there has not been any nationwide age - standardized incidence data reported for prostate cancer in nigeria. we examined and integrated diverse trends in the age - specific incidence of prostate cancer into a comprehensive trend for nigeria, and examined how best the existing data could generate a countrywide age - standardized incidence rate for the disease.methodsdata were obtained from studies undertaken between 1970 and 2007 in referral hospital - based cancer registries. records from at least one tertiary hospital in each of the six geopolitical zones of nigeria were examined retrospectively. data were also reported for the rural population in cross - sectional prospective studies. age - standardized incidence rates and the annual incidence of disease were calculated.resultshigher incidence rates for prostate cancer during this period were recorded for patients aged 6069 years and 7079 years, with a lower incidence rate for patients aged younger than 50 years. an exponential annual incidence rate of disease was observed in the 5079 year age group and peaked at 7079 years before dropping again at age 80 years. the results showed metastasis in more than half of these hospital - based prostate tumors.conclusionour results suggest that prostate cancer occurs at a relatively young age in nigerians and that hospital - based registry reports may not appropriately reflect the incidence of the disease in nigeria. a countrywide screening program is urgently needed. finally, the difference in reported stages of disease found in nigerians and african - americans versus caucasians suggests biological differences in the prognosis. nigeria may thus typify one of the ancestral populations that harbor inherited genes predisposing african - americans to high - risk prostate cancer.
chronic myeloid (myelogenous) leukaemia (cml) is a clonal myeloproliferative neoplasm characterised by a reciprocal rearrangement and fusion of the bcr genes on chromosome 22 and abl gene on chromosome 9. this mutation leads to the productions of an oncoprotein that can be p190, p210 or p230 depending on the breakpoints of the bcr - abl. this oncoprotein is constitutively active leading to uninhibited cell proliferation that is the main driving factor for the progression of the disease.12 chronic myeloid leukaemia is said to have a uniform worldwide incidence of 1 - 2/100,000.3 in the united states of america, the incidence is 1.1 and 2 per 100,000 population in female and males, repectively.2 the mean age at diagnosis of cml is put at 60 - 65 years even though about 30% of new diagnosis are less than 45 years old.24 the median age at diagnosis of cml in nigeria is 38 years and very uncommon by age 60.35 janus kinases (jak) are a group of proteins involved in normal cell signalling.6 however, reports have shown that the v617f somatic mutation of jak2 gene on chromosome 9 and the finding that it is found in all philadelphia - negative (ph) myeloproliferative diseases such as polycythaemia vera (pv) and essential thrombocythaemia (et) but absent in philadelphia - positive (ph+) cml.789 recent reports however indicate that some cases of cml may also paradoxically present with both ph+ and jak2v617f mutations.1011 this case report is to highlight the occurrence of both ph+ and jak2v617f mutations in a patients in our centre. a 60-year - old male retired soldier presented with a 4-month history of recurrent low - grade fever, cough, abdominal distention and remarkable weight loss. he was chronically ill looking, wasted, moderately pale, splenomegaly of 16 cm, and hepatomegaly of 6 cm. haematological investigation showed packed cell volume (pcv) of 21.7% (37.0 - 53.0%), total white blood cell (wbc) count of 30.5 10/l (3.0 - 13.2 10/l) with differentials of granulocyte of 36.3%, lymphocyte 53.6%, medium sized (mid) 10.1%. other investigations revealed erythrocyte sedimentation rate (esr) 80 mm in the first hour. chest radiograph (hilar opacities), mantoux test (negative) and sputum acid bacilli (negative). the patient was then classified as smear - negative tuberculosis. he was then placed on empirical anti tuberculosis therapy according to the nigerian national guideline for tuberculosis therapy. however, there was no clinical response after 4 weeks. on haematological review he was found to be emaciated, mildly pale, mildly dehydrated without significant peripheral lymphadenopathy. repeat haematological investigations revealed a pcv of 20.8%, wbc count of 48.8 10/l differentials were neutrophils 70%, lymphocyte 8%, monocyte 4%, band forms 2%, metamyelocyte 10% and myelocyte 6%. the peripheral blood film showed 416 nucleated red blood cells (nrbc) per 100 wbc, macrothrombocyte, leuco - erythroblastosis, tear drop poikilocytes, target cells, fragment cells and hypogranular granulocyte with bizarre segmentation. these laboratory and clinical findings led to a diagnosis of cml (accelerated phase) to rule out cellular phase of myelofibrosis (mf) ; thus, cytoreductive therapy was started with hydroxyurea (hu) for 3 months while awaiting results of molecular biologic test. however, response to imatinib was sub - optimal ; thus, jak2 analysis was requested on the same sample, and it turned out to be positive for the jak2 mutation (exon14v617f). he was then maintained on imatinib, blood transfusion support, erythropoietin and regular follow - up, but the patient died in a peripheral hospital about 10 months after diagnosis. it was initially hypothesised that occurrence of both bcr - abl and jak2v617f mutation could be mutually exclusive in cml or exist in independent clones.12131415 however, the possibility of a coexistence mutation has so far been shown to occur on a background of a previous treatment with a tyrosine kinase inhibitor (tki).101316 other studies show that simultaneous occurrence of the two is possible without tki therapy.1015161718 the presentation of this case although atypical warrants that a diagnosis of cml in acceleration be entertained. also, due to the thrombocytosis, leucoerythroblastosis and marrow fibrosis thus myelofibrosis (mf) was a strong differential diagnosis. the brief response to empirical anti - tuberculosis agents may be due to recrudescence of latent tb due immunosuppresion that accompanies malignancies. in this patient, simultaneous occurrence of bcr - abl and jak2v617f gene mutation in the major exons e13/14 and exon 14, respectively, is noted. literature is still controversial as to the possibility of a single clone carrying both mutation, or they are independently derived from different susceptible polyclonal stem cells.1315161718 this does not exclude the possibility that the bcr - abl - positive clone might have represented a sub - clone of jak2v617f mutated cells, which had growth advantage either by direct suppression of bcr - abl by the jak2v617f cells or as a result of treatment with tki.101315161718 this case represents the appearance of bcr - abl and jak2v617f in a patient before tki therapy in our centre for the first time. this case report underscores the need for haematologist and other physicians alike to be vigilant, especially if myeloproliferative neoplasm (mpn) patients present with atypical features.	the world health organisation (who) classifies myeloproliferative neoplasm (mpn) into bcr - abl positive chronic myeloid leukaemia (cml ph+) and ph mpn. the jak2 v617f mutation is specific for ph mpn and occurs in approximately 50% of primary myelofibrosis. earlier reports suggest that the occurrence of jak2 and bcr - abl mutations are mutually exclusive. however, recent reports have documented the coexistence of bcr - abl and jak2 mutation in the same patient mostly following treatment with tyrosine kinase inhibitors (tkis). we thus report a 60-year - old male with atypical clinical and laboratory features of mpn and the presence of both bcr - abl and jak2 mutations.
are obligate intracellular protozoan parasites of humans and animals, and are the causative agents of malaria. transmission of these parasites to humans occurs via the anopheles mosquito vector and the geographic distribution of endemic regions puts almost half of the world 's population at risk to contracting malaria. this disease is a major source of morbidity and mortality worldwide, which results in 300500 million clinical cases and 12 million deaths annually (1,2). while several species of plasmodium cause disease in humans (including p. vivax, p. malariae, p. ovale and p. knowlesi), p. falciparum is by far the deadliest (1,3). the life cycle of the plasmodium parasite takes it through multiple cell types (in the vertebrate host and arthropod vector) during which the parasite undergoes multiple developmental changes (both sexual and asexual). the different life - cycle stages are marked by specific genomic, transcriptomic, proteomic and metabolomic states. understanding how these changes are triggered and orchestrated requires mechanisms to view and interrogate genomic and functional genomic data in a powerful and intuitive manner. over the past 10 years, plasmodb has evolved into a venue that integrates such data and allows the user to perform complex queries tailored to their specific needs and interests. the data available in plasmodb has expanded to include genomic and functional data from eight plasmodium species and is summarized in table 1 (4). the current release (plasmodb 5.5) contains fully sequenced and annotated genomes of p. falciparum, p. vivax, p. yoelii, p. berghei, p. chabaudi and p. knowlesi. importantly, plasmodb 5.5 contains results of annotation efforts from multiple sources including the recent systematic effort to update the p. falciparum genome that is an ongoing project started at a workshop in late 2007 co - organized by the wellcome trust sanger institute (wtsi) and eupathdb (formerly apidb) teams. reannotation data have been released in incremental steps (snapshots) in order to provide timely information to users of plasmodb and to solicit user comments regarding the reannotations. table 1.types of data available in plasmodb and example queriestype of dataspecies for which this data is availableexample querygenomic data full sequence and annotationp. falciparum, p. vivax, p. yoelii, p. berghei, p. chabaudi, p. knowlesisearch annotations for specific keyword (see figure 1c). falciparum, p. berghei, p. yoeliiidentify genes expressed at specific life - cycle stages. falciparum, p. vivax, p. berghei, p. yoeliiconfirm gene models and alternative gene models. falciparum, p. berghei, p. yoeliiidentify genes with protein expression evidence at specific life - cycle stages.population biology snp microsatellite isolate datap. falciparumfind highly polymorphic genes or distinguish isolates based on their snp profile.protein interaction yeast two hybrid interactome mapp. falciparumidentify possible interaction partners of a gene of interest.putative function go annotationp. falciparum, p. vivax, p. yoelii, p. berghei, p. chabaudi, p. knowlesiidentify genes that have go annotations. falciparum, p. yoelii, p. knowlesiidentify genes with enzymatic annotations. metabolic pathwaysp. falciparum, p. vivax, p. yoelii, p. berghei, p. chabaudi, p. knowlesiidentify genes specific to apicomplexa. falciparum and p. yoeliiidentify homologs of a gene or list of genes of interest.protein features protein motifs interpro / pfam domains molecular weight isoelectric point protein structure immune epitopesp. falciparum, p. vivax, p. yoelii, p. berghei, p. chabaudi, p. knowlesiidentify genes with specific protein attributes.protein localization signal peptide transmembrane domains targeting to the rbcp. falciparum, p. vivax, p. yoelii, p. berghei, p. chabaudi, p. knowlesiidentify genes targeted to the host cell. types of data available in plasmodb and example queries transcript expression data [microarray, expressed sequence tags (ests) and serial analysis of gene expression (sage) ] available through plasmodb has expanded dramatically over the past few releases to include microarray data from multiple life - cycle stages, gene knock - out mutants of p. falciparum and p. berghei (512) and multiple stages of p. yoelii (mosquito, erythrocytic and liver stages) (13). also included are est data from over 130 libraries (p. falciparum, p. vivax, p. berghei and p. yoelii) (14,15) [dbest (http://www.ncbi.nlm.nih.gov/dbest/) ] and sage data (p. falciparum only) (1618). protein expression evidence includes data from various life - cycle stages (p. falciparum, p. berghei and p. yoelii) (11,13,1921 ; leiden malaria group, unpublished data). population biology evidence (p. falciparum only) includes mapping of microsatellite data (22) onto the genome (available as a genome browser track), single nucleotide polymorphism (snp) data from resequencing efforts of more than 20 p. falciparum strains (p. reichenowi is included as an out - group for comparison purposes) and data from nearly 100 p. falciparum isolates (2325). orthomcl analyses provide ortholog determinations between the different species facilitating discovery of shared genes between lineages (26). protein function assignments are aided by a number of additional functional data types available through plasmodb 5.5 including evidence of protein protein interaction (yeast two hybrid and predicted interactome) (27,28), genome ontology (go) (29) and interpro domain (30) annotations for p. falciparum, p. vivax, p. berghei, p. yoelii, p. knowlesi and p. chabaudi, enzyme commission (ec) number (29) annotation for p. falciparum, p. yoelii and p. knowlesi (31) and metabolic pathway assignments for p. falciparum (31). in addition, subcellular localization of proteins is available through signal peptide (32) and transmembrane domain predictions (33) for p. falciparum, p. vivax, p. berghei, p. yoelii, p. knowlesi and p. chabaudi, and parasite - specific predictions (p. falciparum only) for apicoplast localization (34) and export to the host cell (3537). a visitor to plasmodb can use the database in two general ways : (i) to retrieve all available information associated with a particular gene of interest using a search for an exact gene i d, gene name or gene product name. (ii) to ask single questions (table 1) and/or conduct a series of searches followed by refining the results by combining them or subtracting them from one another. starting with the plasmodb home page (figure 1a), a user can perform a quick search by entering an identifier or test term, or select a specific query from a number of drop - down menus (data not shown). alternatively, queries may be accessed by visiting the queries and tools section of plasmodb (figure 1a), which includes a grid displaying all available queries / searches. by using the queries and tools, a user can interrogate data in plasmodb the third column of table 1 includes example data - specific questions that are available. (a) the top of the screenshot shows the plasmodb logo. on the left side are links to various sections of plasmodb and a point for logging in or registering as a user (not required for using the site but useful for storing search histories. the query grid is in the center and provides an access point to all searchable data in plasmodb. (b) this is a scheme of a workflow that a user may follow when building a set of queries. beginning at the left, queries can be performed starting from the query grid and the results can be joined using operations available through the query history page. (c) screen shots of a key word search page, an example gene query history and a gene results page. note the add column feature in the results page that allows the addition of columns with additional data and the ability to sort results. (a) the top of the screenshot shows the plasmodb logo. on the left side are links to various sections of plasmodb and a point for logging in or registering as a user (not required for using the site but useful for storing search histories. the query grid is in the center and provides an access point to all searchable data in plasmodb. (b) this is a scheme of a workflow that a user may follow when building a set of queries. beginning at the left, queries can be performed starting from the query grid and the results can be joined using operations available through the query history page. (c) screen shots of a key word search page, an example gene query history and a gene results page. note the add column feature in the results page that allows the addition of columns with additional data and the ability to sort results. when conducting queries with the purpose of combining results it may be useful to visualize the searches in a workflow environment where nodes are connected using different criteria (and, this would be accomplished by performing a number of queries and subsequently combining the results in the query history section (figure 1c, middle screen shot). for example, one may be interested in identifying a short list of possible vaccine candidates. one possible way of accomplishing this would be by identifying all proteins predicted to be exported to the host cell in p. falciparum. there are three exported protein datasets in plasmodb and a union (or function) of all three results retrieves 405 genes (figure 1b, steps 1 and 2). to restrict this list further, intersecting (and function) these results with genes that have no orthologs in mammals reduces the results to 321 genes (figure 1b, step 3). next a user may further prune this list by intersecting the results with other queries, such as genes that are nonpolymorphic between a chloroquine sensitive (3d7) and resistant strain (dd2). this cuts the number of candidates to 32 genes (figure 1b, step 4 and figure 1c, right screen shot). alternatively, one may be interested in the genes that have protein expression evidence in a particular stage in the parasite 's life cycle (the results of an intersection with genes that have proteomic evidence in gametocyte yields 27 genes). finally, examination of the list reveals several genes encoding for rifins (a family of clonally variant proteins expressed on the surface of infected red blood cells) (38), and a user may wish to investigate genes other than rifins this can be accomplished by excluding (not operation) results of a keyword query using the term rifin (figure 1b, step 5 and figure 1c, left most panel). a user may examine the specific gene pages for more gene - specific details, download results with their associated data or log in (if they have not done so already) to ensure that their search strategy is saved for future examination. it is expected that plasmodb will continue its data content and tool expansion as user needs require. we anticipate the incorporation of multiple new data sets including microarray, proteomic and specific parasite isolate data. additionally, over the next few years we look forward to incorporating sequence data from a dramatically expanded plasmodium spp., we will also release a new user interface that will include a workflow - based search strategy page, similar to what is shown in figure 1b, which we anticipate will provide a more biologically intuitive and dynamic experience for scientists accessing plasmodb and other eupathdb sites. federal funds from the national institute of allergy and infectious diseases ; national institutes of health ; department of health and human services, under contract no. funding to pay the open access publication charges for this article was provided by this contract.	plasmodb (http://plasmodb.org) is a functional genomic database for plasmodium spp. that provides a resource for data analysis and visualization in a gene - by - gene or genome - wide scale. plasmodb belongs to a family of genomic resources that are housed under the eupathdb (http://eupathdb.org) bioinformatics resource center (brc) umbrella. the latest release, plasmodb 5.5, contains numerous new data types from several broad categories annotated genomes, evidence of transcription, proteomics evidence, protein function evidence, population biology and evolution. data in plasmodb can be queried by selecting the data of interest from a query grid or drop down menus. various results can then be combined with each other on the query history page. search results can be downloaded with associated functional data and registered users can store their query history for future retrieval or analysis.
the posterior fossa is the part of the cranial cavity containing brainstem and cerebellum and is located between foramen magnum and tentorium cerebelli. tumors of the posterior fossa account for 5055% of the intracranial tumors in infants, children and adolescents. congenital torticollis results mostly after birth trauma or intrauterine malpositioning with injury to the sternocleidomastoid muscle and subsequent soft tissue swelling over the muscle. congenital torticollis is characterized by shortening and fibrosis of the sternocleidomastoid muscle detected at birth or shortly after birth. most patients are successfully treated with physiotherapy. on the other hand, acquired torticollis occurs later in childhood and can be caused by various underlying pathologies including ligamentous, muscular, osseous, ocular, psychiatric and neurologic disorders,. there is strong evidence of internuclear connections between cranial nerves ii, v and vii acting as important mechanisms in the association with epiphora and photophobia. a posterior fossa tumor can activate and irritate the trigeminal and/or facial cranial nerves,. a 7-month - old caucasian girl presented with severe photophobia and epiphora at the right eye since birth and from the age of 2 months, a progressive torticollis was seen. despite physiotherapeutic treatment, ocular, general and family history were unremarkable. physical examination revealed a torticollis with a head turn to the right and a chin tilt downwards. the degree of torticollis varied between 60 up to 90 (figure 1 (fig. 1)). there was a severe photophobia that was rated by the parents as 9 on a scale of 10. ocular alignment on light reflex was normal, there was no fixation movement on cover test and alternate prism cover test. bielschowsky s head tilt test could not be performed because of limited cooperation and extreme torticollis. she sees card g with the preferential looking test of cardiff, which is within normal limits for her age. intraocular pressure, measured with tonopen was 17 mmhg at the right eye and 18 mmhg at the left eye. neuro - pediatric examination showed a child with an alert behaviour and well - developed fine motor skills. however, there was an asymmetric development with reduced grasping with the right hand and a pronounced torticollis. she grasped objects, transfered toys from one hand to another and to her mouth. she spontaneously rolled over fluently from supine to prone position over her left side but she needed extra stimulation to roll over the right side. full spine there was a sinistroconvex cervico - thoracic scoliosis, a dextroconvex thoraco - lumbar scoliosis and flattening of the thoracic kyphosis and lumbar lordosis. a magnetic resonance imaging (mri) of the brain with and without gadolinium was performed. the mri revealed a broad - based exophytic mass at the right posterolateral aspect of the medulla oblongata, obstructing the right foramen of lushka and with a mass effect on the right cerebellar hemisphere (figure 2 (fig. 2)). surgical resection of the tumor was performed (figure 2 (fig. 2)). unfortunately, complete resection of the mass was impossible because of the risk of damaging adjacent structures. there was a good post - operative evolution with improvement of the right torticollis. at 6 and 42 months post - operatively, there was a residual torticollis of respectively 30 and 10 (figure 1 (fig. photophobia was rated 7 and 2 on a scale of 10 respectively 6 and 42 months post - operatively. epiphora improved much faster and was rated 2 and 0 at the follow - up visits respectively at 6 and 42 months. although their clinical behaviour may vary, the majority of low - grade gliomas are indolent and do not undergo malignant transformation. this is in contrast with low - grade gliomas in adults that have a more aggressive phenotype. tumors are classified according the who criteria, which provides a grading or malignancy scale. the most common histological subtypes in children are the pilocytic (grade 1) and the diffuse fibrillary astrocytoma (grade 2). the peak incidence is in the 5- to 9-year - old age range. in this case, the patient is exceptionally young to present with a low - grade glioma grade 1. since torticollis was noticed shortly after birth, it could be interpreted as a congenital torticollis but the association with photophobia and epiphora indicated an underlying pathology. who grade 2 tumors are more commonly seen in younger children with a mean age of 18 months. evaluation of tumor size and relation to other structures remain primary imaging endpoints in the evaluation for most pediatric patients with central nervous system (cns) neoplasms,. series, complete resection was associated with a 10-year overall survival rate of more than 90%. correlation between posterior fossa and cervical spinal cord tumors and secondary torticollis is well known and described in literature. torticollis originating from the cns, is most commonly associated with lesions of the corpus striatum, thalamus and brain stem / mesencephalon. the importance of the cerebellum, particularly the vermis and fastigial nucleus, in the control of head position suggests that the cerebellum may play a role in secondary torticollis. the patient in our case shows a clear mass effect of the tumor on the right hemisphere of the cerebellum, explaining the right - sided torticollis but also the asymmetric development of coordination. lesions in the cervical spinal cord may cause torticollis due to increased excitability of the spinal motor neuron, because of dysfunction of the inhibitory descending paths. the primary ophthalmologic signs in a posterior fossa tumor are extra - ocular muscle paresis, nystagmus and papilledema. there is strong evidence of internuclear connections between cranial nerves ii, v and vii acting as important mechanisms in this association. a posterior fossa tumor can activate and irritate the trigeminal and/or facial cranial nerves. facial pain, itching or a decreased blink reflex have been reported in posterior fossa tumors. a decreased blink reflex has been seen in patients with trigeminal or facial cranial nerve dysfunction and cerebellopontine angle tumors. central lesions of the thalamus with activation of the trigeminal system have also been associated with photophobia,. ocular sensory innervation is served by the first division of the trigeminal nerve (v1) through the subnucleus in the brain stem. lacrimation is served by the lacrimal nerve, a branch of the facial nerve (vii). fibres originate in the superior salivatory nucleus (ssn), which serves as a major parasympathetic flow to the eye. recently, nociceptive neurons were identified in the superficial laminae of trigeminal nucleus caudalis (vc / c1). these nociceptive neurons are activated by bright light through an intraocular mechanism driven by a luminance - responsive circuit and increased parasympathetic outflow. microinjection of lidocaine into the ssn diminished light - evoked vc / v1 activity and lacrimation suggesting that increased parasympathetic outflow was critical for light - evoked responses. the importance of trigeminal sensory nerves in the perception of photophobia was confirmed by the observation that intra - vitreal or intra - trigeminal ganglion (trg) micro - injection of lidocaine completely blocked light evoked vc / c1 neural activity. the ssn is a major source of parasympathetic preganglionic neurons to the eye, especially to the choroidal blood vessels. direct activation of the ssn increases blood flow to the anterior choroid more than 3-fold, whereas inhibition of ssn prevented light - evoked increase in tear volume, confirming a decrease in parasympathetic activity. pathological increase in parasympathetic flow by pressure of a posterior fossa tumor on ssn for example, may lead to increased tearing and dilation of choroidal blood vessels. subsequently, intraocular trg neurons can be activated by transmitters released from parasympathetic postganglionic neurons or, for those fibers apposed to dilated choroidal blood vessels, by mechanical deformation of these blood vessels. these potent vasoconstrictive agents prevented light - evoked vc / c1 neural activation by inhibiting dilatation of choroidal blood vessels and release of neurotransmitters activating intraocular trg neurons. furthermore, the olivary pretectal nucleus (opn) also plays a role in these autonomic pathways. opn receives a dense direct input from retinal ganglion cells and is involved in several photic - induced responses such as pupillary light reflex, eye blink and circadian rhythms. opn has extensive efferent projections to hypothalamic and brain stem regions associated with autonomic control. activation of vc / c1 neurons also requires a relay of luminance information through accessory visual pathways since inhibition of opn completely blocks light - evoked vc / c1 neural activity and lacrimation (figure 3 (fig. although their clinical behaviour may vary, the majority of low - grade gliomas are indolent and do not undergo malignant transformation. this is in contrast with low - grade gliomas in adults that have a more aggressive phenotype. tumors are classified according the who criteria, which provides a grading or malignancy scale. the most common histological subtypes in children are the pilocytic (grade 1) and the diffuse fibrillary astrocytoma (grade 2). the peak incidence is in the 5- to 9-year - old age range. in this case, the patient is exceptionally young to present with a low - grade glioma grade 1. since torticollis was noticed shortly after birth, it could be interpreted as a congenital torticollis but the association with photophobia and epiphora indicated an underlying pathology. who grade 2 tumors are more commonly seen in younger children with a mean age of 18 months. evaluation of tumor size and relation to other structures remain primary imaging endpoints in the evaluation for most pediatric patients with central nervous system (cns) neoplasms,. series, complete resection was associated with a 10-year overall survival rate of more than 90%. correlation between posterior fossa and cervical spinal cord tumors and secondary torticollis is well known and described in literature. torticollis originating from the cns, is most commonly associated with lesions of the corpus striatum, thalamus and brain stem / mesencephalon. the importance of the cerebellum, particularly the vermis and fastigial nucleus, in the control of head position suggests that the cerebellum may play a role in secondary torticollis. the patient in our case shows a clear mass effect of the tumor on the right hemisphere of the cerebellum, explaining the right - sided torticollis but also the asymmetric development of coordination. lesions in the cervical spinal cord may cause torticollis due to increased excitability of the spinal motor neuron, because of dysfunction of the inhibitory descending paths. the primary ophthalmologic signs in a posterior fossa tumor are extra - ocular muscle paresis, nystagmus and papilledema. our patient presented with epiphora and photophobia without other neuro - ophthalmologic signs. there is strong evidence of internuclear connections between cranial nerves ii, v and vii acting as important mechanisms in this association. facial pain, itching or a decreased blink reflex have been reported in posterior fossa tumors. a decreased blink reflex has been seen in patients with trigeminal or facial cranial nerve dysfunction and cerebellopontine angle tumors. central lesions of the thalamus with activation of the trigeminal system have also been associated with photophobia,. ocular sensory innervation is served by the first division of the trigeminal nerve (v1) through the subnucleus in the brain stem. lacrimation is served by the lacrimal nerve, a branch of the facial nerve (vii). fibres originate in the superior salivatory nucleus (ssn), which serves as a major parasympathetic flow to the eye. recently, nociceptive neurons were identified in the superficial laminae of trigeminal nucleus caudalis (vc / c1). these nociceptive neurons are activated by bright light through an intraocular mechanism driven by a luminance - responsive circuit and increased parasympathetic outflow. microinjection of lidocaine into the ssn diminished light - evoked vc / v1 activity and lacrimation suggesting that increased parasympathetic outflow was critical for light - evoked responses. the importance of trigeminal sensory nerves in the perception of photophobia was confirmed by the observation that intra - vitreal or intra - trigeminal ganglion (trg) micro - injection of lidocaine completely blocked light evoked vc / c1 neural activity. the ssn is a major source of parasympathetic preganglionic neurons to the eye, especially to the choroidal blood vessels. direct activation of the ssn increases blood flow to the anterior choroid more than 3-fold, whereas inhibition of ssn prevented light - evoked increase in tear volume, confirming a decrease in parasympathetic activity. pathological increase in parasympathetic flow by pressure of a posterior fossa tumor on ssn for example, may lead to increased tearing and dilation of choroidal blood vessels. subsequently, intraocular trg neurons can be activated by transmitters released from parasympathetic postganglionic neurons or, for those fibers apposed to dilated choroidal blood vessels, by mechanical deformation of these blood vessels. these potent vasoconstrictive agents prevented light - evoked vc / c1 neural activation by inhibiting dilatation of choroidal blood vessels and release of neurotransmitters activating intraocular trg neurons. furthermore, the olivary pretectal nucleus (opn) also plays a role in these autonomic pathways. opn receives a dense direct input from retinal ganglion cells and is involved in several photic - induced responses such as pupillary light reflex, eye blink and circadian rhythms. opn has extensive efferent projections to hypothalamic and brain stem regions associated with autonomic control. activation of vc / c1 neurons also requires a relay of luminance information through accessory visual pathways since inhibition of opn completely blocks light - evoked vc / c1 neural activity and lacrimation (figure 3 (fig. the etiologies of acquired torticollis in children are numerous, but posterior fossa and cervical spine cord tumors should always be considered. children with epiphora and photophobia may first present to the ophthalmologist s office. this case report shows the importance of recognizing this clinical triad of torticollis, epiphora and photophobia and to perform neuro - imaging in order not to delay diagnosis and treatment of posterior fossa lesions in these patients. there is strong evidence of internuclear connections between cranial nerves ii, v and vii acting as important mechanisms in this association. furthermore, though pilocytic astrocytoma occurs mostly in children of 59 years old, it can occur in younger children, as illustrated in this case.	a 7-month - old caucasian girl presented with an acquired, spasmodic torticollis to the right side with the head tilted downwards, photophobia and epiphora. diagnostic work - out revealed a posterior fossa pilocytic astrocytoma. the symptoms improved after surgical resection. there is evidence of internuclear connections between cranial nerves ii, v and vii acting as important mechanisms in this triad (okamoto. 2010).
diabetes mellitus is a major health problem worldwide that increases morbidity and mortality ratios because of the development of various complications mostly related to the cardiovascular system1. type 2 diabetes mellitus (t2 dm) is characterized by insulin resistance and deterioration of -cell function2. the prevalence of t2 dm is increasing in relation to the rapidly changing lifestyles in developed and developing countries3. t2 dm affects 366 billion people, and this number is estimated to increase to about 522 billion by the year 20304. physical activity and regular exercise play an important role in glycemic control, which is considered essential in t2 dm treatment. regular physical activity improves blood glucose control, may prevent or delay t2 dm, and enables better and more effective glucose utilization by reducing insulin resistance. furthermore, it affects blood lipids, blood pressure, cardiovascular risk factors, mortality, and quality of life in a positive way5,6,7,8,9,10,11,12. coronary heart disease, cerebrovascular disease, and peripheral vascular disease may occur at an earlier age and may progress more aggressively when comorbid with diabetes mellitus. this may lead to decreased life expectancy in addition to decreased quality of life13. according to the world health organization, the quality - of - life concept is how individuals perceive their lives in relation to their personal goals, expectations, standards, and worries in the system of culture and values that they live in14. individuals with diabetes expend additional effort on a daily basis in using their medication or insulin, monitoring their dietary routine, and controlling their blood glucose levels throughout the day. this is a difficult psychosocial situation that affects their quality of life negatively15, 16. the literature indicates a lower level of quality of life in individuals with diabetes mellitus than in controls16, 17. decreased quality of life affects not only the happiness and satisfaction of individuals, but also their labor force participation rate, social functions, and treatment compliance15. it is well known that the addition of an exercise regimen to treatment programs in order to increase and pursue quality of life is of utmost importance15, 18, 19. physical activity might be effective in increasing quality of life and healthylife expectancy in the treatment of diabetes6, 7, 13, 20. this study aimed to investigate physical activity level, and its relationship with quality of life in patients with t2 dm. patients with t2 dm who attended the diabetes and internal medicine outpatient clinics at haydarpaa education and research hospital between december 2013 and june 2014 participated in this study. the study was approved by the ethics committee of istanbul medipol university and conducted according to the principles of the declaration of helsinki (1975, revised 1983). the study sample consisted of 129 subjects aged 18 years or older who were diagnosed with t2 dm at least 1 year prior to this study. patients who met any of the following criteria were excluded from the study : mental, communication, and behavioral disorders that may cause problems in understanding or answering the questions ; psychiatric disorders ; severe visual and hearing impairments ; malignant neoplasms ; and pregnancy. demographic data, diabetes symptoms, time of initial diagnosis, and treatment approaches were recorded on an assessment form prepared by the authors. physical activity level was assessed by using the short version of the international physical activity questionnaire (ipaq - sf). diabetes - related quality of life was assessed by using the diabetes-39 (d-39) questionnaire. subjects who were not literate were assisted by a researcher, and completed the questionnaires at a face - to - face interview. the ipaq - sf was developed to assess physical activity during the past week among adults21, 22. the ipaq - sf contains 7 items and assesses the frequency of activity (days) and duration (minutes and/or hours) in vigorous- and moderate - intensity activities, walking, and sitting activities. metabolic equivalent (met)-minute is computed by multiplying the met score of an activity duration (minute). the following values were used for scoring : walking 3.3 mets, moderate physical activity 4.0 mets, vigorous physical activity 8.0 mets, and total physical activity met - min / wk = sum of walking + moderate + high met - min / wk scores21, 22. the diabetes-39 (d-39) instrument is specific to diabetes mellitus types i and ii, includes dimensions to assess diabetes control (12 items), anxiety / worry (4 items), social burden (5 items), sexual function (3 items), energy / mobility (15 items), and overall qol. the answers for each item ranged from 1 (not affected at all) to 7 (extremely affected). the possible score ranges at a scale of 39 to 273, and a high score represents poor quality of life23. data analysis was performed using the statistical package for social science (spss) version 16. in all the statistical analyses, descriptive statistical parameters (mean, standard deviation, range, min, and max) were calculated and spearman s correlation analysis was performed. all demographic and clinical findings of the patients are presented (table 1table 1.the demographic and clinical characteristics of the patientsvariablesmean sdrange or n (%) age (years)56.4 11.1 (2079)duration of diabetes (years)10.3 6.5 ((130)genderfemale83 (64%)male46 (35%)use of insulin66 (51.1%)use of a glucometer at home89 (68.9%)regular exercise28 (21.7%)comorbiditieshypertension 49 (37.9%)hyperlipidemia35 (27.1%)heart failure 10 (7.7%)vascular disease2 (1.5%)menopause42 (50.6%)educationilliterate3 (2.3%)primary education94 (72.8%)high school 24 (18.6%)university8 (6.2%)). the mean age of the cases was 57.7 years, and the mean duration of diabetes was 10.3 years. in this study, 66 patients (51.1%) were using insulin as medical therapy and 89 (68.9%) were monitoring their blood glucose levels with a glucometer at home. of the subjects, 28 (21.7%) regularly performed exercise based on their own perception of performing exercise, that is, regardless of whether exercise was a regular habit for them. ninety - six patients (74.4%) reported the presence of comorbidities (table 1). the results of the ipaq - sf and d-39 questionnaire surveys are presented in table 2table 2.the ipaq - sf and d-39 scores (mean of the total and subgroup scores)variablesmean sdmedian (range)ipaq - sf (met - min / week)total score1,186.4 1,372.5714 (06,237)ipaq - sflevels of physical activity(met - min / week)low activity187.2 122148.5 (0448.5)moderate activity 1,320.3 625.21,115 (4622,844)high activity4,560.1 1,448.34,812 (1,5886,237)ipaq - sfsitting time (min)302.3 244.1(01,200)d-39total score154.4 4.7151 (60266)d-39 subgroup scoresdiabetescontrol48.9 16.350 (15129)anxiety16.2 517 (427)social life19 6.918 (534)sexual life9.21 5.77 (320)energy mobility61 18.2363 (2299) as total and subgroup scores. of the patients, 51 (39.5%) had low, 67 had moderate (51.9%), and 11 (8.5%) had high activity levels. the correlation analysis of quality of life with total and subgroup scores of physical activity level showed that the total and all the subgroup (except for diabetes control) scores of quality of life had a statistically significant negative correlation with physical activity level (table 3table 3.correlation analysis between the ipaq - sf and d-39 scoresipaq - sfd-39total scorediabetes controlanxiety social lifesexual lifeenergy mobility total score0.2910.1810.2240.3190.2290.336low activity0.1270.0380.1380.1250.0700.184moderate activity0.0780.1170.0330.0550.0340.053high activity0.1750.1140.1330.2370.1230.179sitting time (min)0.0560.0130.1010.0720.1830.019spearman correlation test, p < 0.05, p < 0.001). when the physical activity intensity and sitting duration were related with the quality - of - life scores, a statistically significant negative correlation was obtained only between the intense activity and social life subgroups. in this study, the physical activity levels and quality of life of the patients with t2 dm were evaluated. a statistically significant negative correlation was obtained between the total ipaq - sf and d-39 scores. lower d-39 scores indicate better quality of life that could explain the negative correlation. our results show that physical activity has a positive impact on quality of life. the physical activity level of the 129 patients with t2 dm was evaluated with the ipaq - sf in this study, and the ipaq - sf scores showed that 51 patients (39.5%) were inactive and only 11 patients (8.5%) had high physical activity level. reported that in their study, 62% of 100 patients with t2 dm had low physical activity level and only 4% had high activity level25. another study reported that high activity level decreased in diabetes mellitus patients26. in the present study, the mean weekly total energy was 1,186.4 1,372.5 met - min / week. moderate activity represents moderate- or vigorous - intensity activities, achieving a minimum of at least 600 met - min / week, and high activity represents achieving a minimum of at least 3,000 met - min / week (21). reported the total energy expenditure of the declared weekly physical activity level in 31 t2 dm patients was calculated as 2,513 1,349 metmin / week for males and 2,428 1,348 met - min / week for females24. in our study, the mean weekly walking time was 231.7 minutes. in the research by gibson and colleagues, the mean walking time of the intervention group was reported as 182.9 minutes and that of the control group was 203.5 minutes before the treatment27. physical activity and regular exercise are important elements that should be considered for delaying the onset of and treating t2 dm, and for improving the quality of life and long - term life expectancy of t2 dm patients5,6,7,8,9,10,11, 18, 28. a previous study found that t2 dm patients with low activity level had shorter life expectancy by about 0.10.5 years compared to patients with moderate - to - high activity levels18. awareness on increasing physical activity and lifestyle modifications should be raised in patients in the early phase of diabetes mellitus and in individuals with risk of diabetes mellitus. patients with chronic diseases have to make important and various lifestyle modifications to control the disease. the literature indicates that the quality of life of t2 dm patients is lower than that of controls15, 17. rubin and peyrot reported that patients with diabetes mellitus had worse quality of life than people with no chronic illnesses, but had better quality of life than patients with most other serious chronic diseases16. this questionnaire is sensitive to changes in health status23, 29, 30. in the present study, health - related quality of life the mean d-39 score was 154.4 and estimated to range from 60 to 266. lower scores indicate better quality of life23. the results of the present study showed an association between physical activity level and health - related quality of life. statistically significant negative correlations were found between the ipaq - sf total score and the d-39 total and subgroup scores, except for the diabetes control subgroup. a significant negative correlation was found between the high - activity and social - life subgroup scores. in the literature, adding an exercise program to patients diet plan was reported to provide better quality of life15, 18. two hundred patients with t2 dm were evaluated by daniele. by using another quality - of - life questionnaire and the ipaq - sf31. they reported that patients with sedentary lifestyles had low quality of life, and that functional capacity and general state of health subscales of the quality - of - life questionnaire were independently associated with physical activity31. increasing the consciousness about this subject in patients diagnosed with diabetes or with the risk of diabetes is highly important. patients with chronic diseases who received sufficient information and education are known to have better quality of life and physical activity level. planned exercise educational program and making exercise a part of lifestyle can improve the quality of life in patients with t2 dm. due to the cross - sectional nature of the study design, our results can not accurately reflect the characteristics of all patients with diabetes mellitus in the population. this can be considered as a limitation of our study. however, the age our study sample ranged from 20 to 79 years, and the diabetes duration ranged from 1 to 30 years. thus, we believe that our results may provide additional information in the literature. we observed no statistically significant relationship between the physical activity level classifications and d-39 total and subgroup scores. studies with larger sample sizes and various assessment methods and that include educational and exercise programs to improve the quality of life should be addressed in the future research studies. the efficacies of different types, duration, and frequency of exercises in protecting and promoting the quality of life of t2 dm patients need to be investigated as well.	[purpose ] physical activity and regular exercise play an important role in glycemic control, which is considered an important part of the treatment of type 2 diabetes mellitus. this study evaluated physical activity level and its relationship with quality of life in patients with type 2 diabetes mellitus. [subjects and methods ] we evaluated 129 subjects with type 2 diabetes mellitus through a face - to - face interview using the short version of the international physical activity questionnaire and diabetes-39. demographic data, diabetes symptoms, time of initial diagnosis, and treatment procedure / approaches were recorded. [results ] of the study subjects, 51 (39.5%) had low, 67 had moderate (51.9%), and 11 (8.5%) had high activity levels. the mean weekly sitting duration was 302 minutes. the mean weekly walking time was 231.7 minutes. except for the diabetes control domain, scores for all the subgroups and the total score in the quality - of - life assessment had a statistically significant negative correlation with physical activity level. [discussion ] physical inactivity negatively affects the quality of life of diabetic patients. a planned exercise education program and incorporation of exercise into the lifestyle can improve the quality of life of patients with type 2 diabetes mellitus.
this case refers to a shearing type osteochondral humeral head fracture following a traumatic posterior shoulder subluxation. the spectrum of the pathoanatomical findings after posterior shoulder subluxations or dislocations have already been described in the literature. this is the first reported case to our knowledge where an osteochondral fracture is associated with posterior labral lesion after a traumatic posterior subluxation. this important case presents a trauma of the shoulder which has potential to be misdiagnosed. posterior glenohumeral dislocations represent only 2% of all shoulder dislocations, and their initial diagnosis is missed in more than 60% of cases. posterior shoulder dislocations result from axial loading of the adducted and internally rotated shoulder, violent muscle contractions resulting from seizers or electrocution, or a direct posterior force applied to the anterior shoulder. a 26-year - old male patient was referred to our hospital presenting with a traumatic posterior subluxation on his left shoulder. the patient reported that he sustained a direct impact injury on his left shoulder in which he felt severe shoulder pain and a popping sensation. after clinical and radiological work - up he was diagnosed with an osteochondral shearing type fracture of the humeral head [figure 1 ]. the patient suffered no other injuries. also, the patient reported no history of shoulder dislocation or subluxation or any other chronic or acute diseases. preoperative anteroposterior x - ray of the shoulder showing the osteochondral fracture in the axillary pouch the patient was managed operatively under general anesthesia in a beach chair position. the delto - pectoral approach was performed with maximum deltoid muscle preservation and tenotomy of the upper third of the subscapularis. with the arm in abduction and external rotation the osteochondral defect was evident at the antero - medial aspect of the humeral head [figure 2 ]. the osteochondral fragment missing from the humeral head was fished out of the posterior - inferior axillary pouch [figure 3 ]. following inspection of the glenohumeral joint, a posterior slap (superior labral anteroposterior) type ii lesion with a posterior extension till the 1.00 o'clock position was found. the osteochondral fragment was anatomically reduced using two k - wires and finally fixed in place with two headless 16 mm screws (herbert, wemmel, belgium). the slap lesion and the posterior labral capsular detachment were repaired with two absorbable suture - anchors (panalock, depuy mitek, inc. the subscapularis tendon was anatomically repaired with non - absorbable ultra - braided sutures (orthocord, depuy mitek, inc., a johnson & johnson co, ma). the patient was fitted with a 15 abduction sling with the arm in neutral rotation for 5 weeks. one year postoperatively the patients ' range of motion was comparable to the non - affected shoulder. the radiological work - up showed full fragment integration, without any absorption or necrosis. intraoperative osteochondral defect of the humeral head free osteochondral fragment of the humeral head anatomical provisional reduction of osteochondral fragment with k - wires and final with herbert screws final postoperative radiograph a traumatic posterior subluxation or dislocation are relatively rare injuries and account for approximately 2% of all shoulder dislocations. in the literature, lesions commonly associated with traumatic posterior subluxation / dislocation are the reverse hill - sachs (or mclaughlin lesion), a posterior labral detachment, glenohumeral ligament lesions, rotator cuff tears or posterior bony fractures. after such an accident and type of blunt force trauma on the shoulder we would expect to see a reverse hill - sachs lesion, an impression fracture on the anteromedial humeral articular surface as first described by mclaughlin. this is the first case to our knowledge in which a shearing type osteochondral fracture was observed in a traumatic posterior subluxation. the intraoperative pathoanatomic findings of the posterior labrocapsular detachment, the anteromedial aspect of the fracture of the humeral head, the patient 's description with the direct impact of the shoulder toward posterior direction and the sensation of his shoulder popping enforces our diagnosis for the posterior shoulder subluxation. yun sun choi. (2005) reported a shearing osteochondral fracture of the humeral head in a 12-year - old right - handed - dominant boy injured while playing football following an anterior dislocation. to the best of our knowledge, our patient represents the first reported case of an osteochondral shearing fracture of the humeral head. this fracture resulted from a traumatic posterior subluxation of the shoulder. because imaging techniques may be unreliable in such fractures	traumatic posterior shoulder subluxations are rare entities which require clinical suspicion upon presentation. although literature presents many sequels of posterior shoulder subluxations, we have not come across any shearing type osteochondral fracture in the literature. in this case report we present diagnosis, treatment and follow - up results of this rare fracture in a 26-year - old male following a fall from a motorcycle.
diabetes mellitus is the most common cause of end - stage renal disease, non - traumatic lower - limb amputation and blindness. furthermore, diabetic retinopathy (dr) is the most important cause of visual loss worldwide. the prevalence of dr increases with duration of diabetes. some other risk factors of dr development include poor glycemic control, type of diabetes, and the presence of associated disorders such as dyslipidemia, hypertension, smoking, pregnancy, and nephropathy. dr could present as non - proliferative retinopathy, proliferative retinopathy or macular edema, however, the most important cause of visual loss among these patients is macular edema. retinopathy is essential for the diagnosis of diabetic nephropathy, but it presents in 90% and 60% of type 1 and 2 diabetic patients, respectively. because the rate of progression of retinopathy may be rapid, and treatment can be beneficial for reduction of disease progression, it is important to screen diabetic patients regularly for the development of retinopathy, so in type 1 diabetes, after 5 years and in type 2 diabetes, at the time of diagnosis and then annually, retinopathy should be evaluated. one of the other complications of diabetes is generalized atherosclerosis which can be presented as ischemic heart disease, cerebrovascular accident or peripheral vascular disease. diabetic atherosclerosis can be detected by measurement of intima - media thickness (imt) of common or internal carotid artery (cimt). the cimt was used to predict of cardiovascular outcomes in diabetic patients. the easy applicability and the noninvasive nature of b - mode ultrasonography make it suitable for using as a surrogate endpoint for measuring the atherosclerotic burden in people with cardiovascular risk factors. the mean cimt values by the different incidences were reported as 1.26 0.6 mm (transversal), 1.17 0.54 mm (longitudinal anterolateral), and 1.18 0.58 mm (longitudinal posterolateral). increases in the thickness of the carotid imt may be associated with an increased risk of myocardial infarction and stroke in old patients without a history of cardiovascular disease. mean cimt was reported a reliable marker of risk of ischemic stroke in type 2 diabetic patients and could be used as a simple noninvasive screening test for the assessment of atherosclerosis in these patients. mean cimt may be associated with age, systolic blood pressure (sbp), smoking, the ratio of low - density lipoprotein to high - density lipoprotein cholesterol, mean glycosylated hemoglobin value (hba1c), and urinary albumin excretion rate. in type 2 diabetic patients, significant predictors of imt progression included albuminuria, advanced age, male sex, smoking, and higher sbp. in diabetic patients without a history of clinical cvd, the presence of advanced stage of dr is associated with subclinical coronary artery disease. dr was shown as subclinical atherosclerosis marker, so retinopathy may warrant a more careful cardiovascular assessment even in the early stages of diabetes. intima - media thickness is a sensitive marker of early carotid atherosclerosis, so ultrasound cimt measurement can be used to assess the cardiovascular risk and to determine indications for intensified diabetic treatment ; on the other hand, dr is an early and reliable marker of microvascular disease, and probably diabetic nephropathy, so the aim of our study was evaluation of relationship between retinopathy and cimt as two valuable noninvasive methods for early detection of micro and macrovascular complication of diabetes. in a cross - sectional study (2012), 154 diabetic patients who had a medical history and follow - up in imam ali clinic of shahrekord were enrolled in the study. they were divided into two equal groups of 77 patients that were case group (with retinopathy) and control group (without retinopathy). diabetes is defined as fasting blood sugar equal or more than 126 mg / dl. dr is defined as a microvascular complication of diabetes that affect retinal arterioles and it has two forms as nonproliferative and proliferative retinopathy. exclusion criteria were : age lesser than 40, presence of cataract in the ophthalmologic exam that prevents retinopathy evaluation, noncooperative patients during the study. it has been explained to the participants that all information will be confidential and a written consent form were filled in by all patients. common carotid intima - media thickness (cimt) is defined as the largest distance between the luminal intima interface and the medial adventitia interface that is located at 1 cm of the initiation of the common carotid artery that was measured by one sonographist in recombinant position and using doppler sonography devices (siemens, g50, germany). demographic criteria such as age, duration of diabetes, body mass index (bmi), sbp and diastolic blood pressure (dbp), and laboratory results including fasting blood sugar (fbs), glycated hemoglobin (hba1c), blood urea nitrogen (bun), and creatinine (cr) were checked. bmi was measured by formula (body weight [kg]/length [m ]) and laboratory tests were conducted by biotecnica instruments (bt 3000). collected data were entered to spss (statistical package for the social sciences, version 19.0, spss inc, chicago, ill, usa) and analysis was done by t - test, chi - square test, and pearson correlation. this study was the result of research project number of 986, which approved by the research committee of shahrekord university of medical sciences. mean age of the patients with retinopathy (group 1) was 62.5 9.75 years and in the patients without retinopathy (group 2) was 58 10 years (p = 0.006). mean cimt of all patients was 0.84 0.18 and in the female and male patients were 0.82 0.16 mm and 0.88 0.22 mm respectively (p = 0.03). in all of the patients, cimt was associated with age, duration of diabetes (based on history), sbp, serum bun, and cr [table 1 ]. the number of male and female in group 1 was 34 (44.2%) and 43 (55.8%) and in group 2 were 21 (27.3%) and 56 (72.7%), respectively. cimt of the patients of group 1 was significantly greater than group 2 (p < 0.001). mean hba1c in group 1 ad 2 were 7.20 1.21 and7.04 1.33, respectively (p = 0.39). table 2 showed that in patients of group 1, cimt was correlated only with sbp (p = 0.01) ; however in the patients of group 2, cimt was correlated with dbp. to remove confounding bias effect, comparison of different variables in two groups of the patients association of cimt with different variables in the patients prevalence of dr in type 2 diabetic patients was reported 34.6% in yau. study with no difference in male and female, whereas zhang. showed slightly more common in male patients. besides, harris. found a greater prevalence and severity of dr in non - hispanic and mexican americans with type 2 diabetes. there are a few studies about correlation of cimt and retinopathy in diabetic patients, for example, miyamoto in evaluation of 102 diabetic patients showed the significant correlation between retinopathy and common carotid artery thickness, also torres. in a study on 173 patients with hypertension showed, significant and independent association of carotid intima - media thickness with arteriolar caliber of retina. in our study cimt was associated with age, duration of diabetes, sbp, serum bun, and cr whereas cardoso. found the correlation of cimt with age, male sex, smoking status, and ambulatory blood pressure. ogawa. in a study on 634 type 2 diabetic patients reported the positive correlation of cimt and patients bmi, also he found correlation between maximum bmi and retinopathy, but we did nt find this association in the patients. the reason of this discrepancy may be due to different number of patients in two studies. correlation of cimt and htn was reported in some studies, also in our study cimt was correlated with sbp in patients with retinopathy and with dbp in patients without retinopathy, however, alizadeh. in a study on study also cimt had not correlation with control of blood sugar. about association of hba1c and cimt furthermore similar result was found by choi. in the study on 370 type 2 diabetic patients. small sample size is a limitation in our study, so we suggest more studies to be carried out using larger sample size. therefore, the relationship between cimt and other complications of diabetes such as microalbuminuria, macroalbuminuria or neuropathy need to be evaluated. in diabetic patients, cimt is a marker of atherosclerosis and macrovascular damage which has had a correlation with dr (as a potential reliable marker of microvascular damage), so we may use sonographic measurement of cimt as a simple, available, and noninvasive method for screening of macro and microvascular complications among diabetic patients. all authors have assisted in preparation of the first draft of the manuscript or revising it critically for important intellectual content. all authors have read and approved the content of the manuscript and confirmed the accuracy or integrity of any part of the work.	background : this study was carried out in order to evaluate the relationship between retinopathy and carotid intima - media thickness (cimt).materials and methods : in a cross - sectional study, 154 diabetic patients who had a history of diabetic disease were evaluated in two equal groups of 77 patients with and without retinopathy, respectively. cimt was evaluated in all of the patients.results:mean age of the patients was 59.65 9.37 years. mean cimt of all patients was 0.84 0.18. cimt of patients with retinopathy was significantly greater than patients without retinopathy (p < 0.001). cimt also correlated with age, duration of diabetes, systolic blood pressure, blood urea nitrogen, and serum creatinine.conclusion:cimt may be used as a simple, available and noninvasive method for screening of macro and microvascular complication of diabetic patients.
kinetoplastidae are flagellated protozoan parasites, including serious human pathogens that are transmitted by different insect vectors. diseases caused by kinetoplastids include human african trypanosomiasis (hat, also known as african sleeping sickness), which is due to infection with trypanosoma brucei gambiense or t. brucei rhodesiense ; chagas disease, which is caused by t. cruzi ; and various forms of leishmaniasis caused by infection with different species of leishmania. those living in tropical and subtropical areas of the world are at risk of contracting these diseases. sleeping sickness, which is found predominantly across sub - saharan africa, is fatal if not treated, and all drugs used in the treatment of hat have issues relating to efficacy, administration, and side effects. additionally, increasing levels of drug resistance demonstrate the need for new, improved, and affordable drugs. we have previously identified the bifunctional enzyme fold as an interesting target for antibacterial drug discovery. this enzyme produces n - formyl - tetrahydrofolate (thf) in a two - step reaction. first, n, n - methylene - thf is converted to n, n - methenyl - thf by the nadp or nad - dependent n, n - methylene - thf dehydrogenase (dh) ; then, n - formyl - thf is produced by the action of n, n - methenyl - thf cyclohydrolase (ch, figure 1). proposed reaction mechanism of tbfold. fold crystal structures have been reported from different organisms, including leishmania major(8) but not from t. brucei. we initiated an inhibitor discovery program targeting the t. brucei enzyme (tbfold) and selected as a lead compound ly374571 (compound 1) developed by eli lilly and company because it inhibits bacterial and human fold. however, following the protocol reported for the synthesis of 1, we obtained a new compound whose structure has been unambiguously determined as 2 (figure 2). we judge it likely that the original report refers to compound 2 not 1. to confirm 2 as a suitable lead compound for the development of tbfold inhibitors, we were able to obtain the first x - ray structure of tbfold in the presence of nadp and the inhibitor. the x - ray crystal structure of the complex tbfold-2-nadp revealed molecular details that were relevant to the binding and inhibition of the enzyme, and allowed, with the help of molecular modeling, a rational design of several analogues (compounds 1620). multiple sequence alignment of fold from several different organisms (see figure s1) and superimposition of the corresponding three - dimensional structures provided insight into the major determinants of ligand binding and selectivity for the different fold forms (see figure s2). antiparasitic activity against the bloodstream form of t. brucei and cytotoxicity against human leukemia macrophages, differentiated from thp1 monocytes, were evaluated for all compounds under study. (a) structures of 1 and 2 ; (b) molecular formula of one symmetry - independent molecule of intermediate 3 and of the dmso unit showing the atom numbering scheme. compound 2 was prepared following a procedure originally reported to afford 1 (figure 2). however, in contrast to what was reported by schmidt. reaction of 4-hydroxy-2,5,6-triaminopyrimidine sulfate with ethyl 4-isocyanatobenzoate (scheme 1) afforded a derivative with the urea moiety linked at position 5 (compound 3) instead of position 6. our structural assignment is based on the x - ray analysis of 3, which was crystallized as a thin plate by slow evaporation from dimethyl sulfoxide (dmso). figure 2b shows the molecular formula of one symmetry - independent molecule of 3 with dmso and the atom numbering scheme (see supporting information for details on crystal determination : figure s3, s4, and s5). the latter can be obtained free of charge from the cambridge crystallographic data centre via www.ccdc.cam.ac.uk/data_request/cif. a series of new analogues of compound 2 were designed on the basis of the crystallographic data and molecular modeling studies, as discussed in detail later. as shown in scheme 1 intermediate 4 was reacted with a series of different -amino acids 69 or with -amino butyric acid 10, all as methyl esters, using n - methylmorpholine and 2-chloro-4,6-dimethoxy-1,3,5-triazine as coupling reagents, affording derivatives 1115, which were finally converted into the desired compounds 1620 by alkaline hydrolysis with 1 n naoh (scheme 2). the molecular structure of 3, as obtained from x - ray diffraction analysis, is shown in figure s3. the asymmetric unit consists of four independent molecules (labeled a, b, c, and d), each of them being strictly associated with a dmso host molecule (called e, f, g, and h) ; see figure s4. the four unique molecule 3 entities mainly differ by the torsion angles between the pyrimidine ring and the ureic moieties (table s1). in general, the pyrimidine ring is almost perpendicular to the phenyl mean plane, whereas the ureic group roughly lies in the same plane as the phenyl system. for example, the (o3-c10-n1-c7) angle is as low as 6(2), 0(2), 2(2) and 7(2) degrees in molecules a, b, c, and d, respectively. as for the crystal packing, a / b and c / d alternating pairs of closely associated independent molecules are arranged along the [110 ] axis (figure s5a). within each pair, a cyclic network of n3h3o4 hydrogen - bonded contacts is set up between almost coplanar facing pyrimidine rings (figure s5b). the other pyrimidinic nitrogen (n4) acts as an acceptor of an intermolecular hydrogen bond donated from the n6h2 group (figure s5c), determining the formation of two symmetry - independent infinite hydrogen - bonded zigzag ribbons in the (110) plane (figure s5a). moreover, the ureic system is involved in hydrogen - bonded contacts with the oxygen of the cocrystallized solvent, whereas the o3 oxygen is a hydrogen - bond acceptor interacting with a neighboring n5h2 group. table s2 summarizes all the relevant intermolecular hydrogen - bonded contacts in crystalline 3. the preparation of an efficient recombinant escherichia coli protein production system as well as the purification, crystallization, and structure determination of tbfold in a ternary complex with nadp and the inhibitor (s)-2-(4-(3-(2,4-diamino-6-oxo-1,6-dihydro pyrimidin-5-yl) ureido)benzamido) pentanedioic acid (2) is detailed in the supporting information. the inhibitor and adp component of the cofactor were clearly defined by the high - resolution electron density, but the density was less well ordered for the nicotinate moiety. the tbfold subunit, which is a polypeptide of 297 amino acids, displays a distinctive tertiary structure typical of this enzyme family. this subunit consists of 11 -helices and 11 -strands forming a two - domain structure.tbfold forms a dimer in solution, as shown by size exclusion chromatography, and a dimer constitutes the asymmetric unit of the crystal structure. approximately 10% of a subunit surface is occluded from the solvent by dimer formation. the c - terminal domain displays the rossmann fold and binds nadp in a deep cleft. the adenine occupies a hydrophobic pocket near the surface of the enzyme, and the nicotinate is then located between the c- and n - terminal domains. the amino acids that form the cofactor - binding site and interact with the cofactor are highly conserved. we previously noted in different crystal structures of bacterial fold that the conformation of a loop adjacent to the active site, the 8-10 loop, was variable. the loop occludes the active site in the structure of the pseudomonas aeruginosa fold (pafold). in the case of the tbfold, the loop adopts an open configuration, lining and helping to create the active site (figure s6). the folate - binding catalytic center is a deep, solvent - filled cavity on the n - terminal domain. here, 2 binds with an extended conformation (figure 3). the pyrimidine headgroup is wedged between k56 and q100 on one side and i174 (not shown) on the other. direct hydrogen bonding interactions between the inhibitor headgroup and the enzyme involve the side chain of d123, the main chain carbonyls of l101 and v99, and the main chain amide of l101. additionally, a number of solvent - mediated interactions link 2 o4 to the side chains of k56 and d123. the k56 side chain also binds to the carbonyl group of the linking amide. the 2 benzyl forms -stacking interactions on one side with y52 ; on the other side, there are van der waals interactions with g276, p277, and t279. the -carboxylate group of the ligand glutamate moiety participates in a direct charge - reinforced hydrogen bond with the g273 main chain nitrogen and a long - range ionic interaction with the side chain of r10. in similar fashion, the -carboxylate interacts with r54. in addition, there are several solvent - mediated interactions that link these acidic groups to g274, g276, and y250. the side chains of t51, l55, and l252 together with p272 form a hydrophobic clamp to position the tail of the inhibitor. the polypeptide is depicted as off - white ribbon, the interacting residues, and the nicotinate moiety of the cofactor, which was modeled, are represented with c atoms as orange sticks, the ligand as cyan sticks. all o, n, and h atom positions are red, blue, and white, respectively. compound 2 was tested for the inhibition of fold dehydrogenase activity using an established assay. to enlarge the sar study, we decided to include in the panel of compounds under evaluation also the synthetic intermediates 35. we selected compound ly354899 (21), a competitive inhibitor of the human (ki 29 nm), pseudomonas aeruginosa (30 nm) and l. major (105 nm) fold enzymes, as a reference compound, whose structure represents a rigid cyclized analogue of compound 2. superimposition of the available a. baumanii fold/21 structure (pdb code 4v4v) on the tbfold/2 complex, herein described, shows that the two ligands adopt the same binding conformation (figure s7 in supporting information). in accordance, compound 21 displayed tbfold inhibitory activity with a ki comparable to that of compound 2 (ki = 8.5 m and 1.1 m, respectively). in vitro growth inhibition expressed as ic50 (m) of all compounds against the t. brucei bloodstream form and human thp1 differentiated macrophages. compound structures, enzyme inhibition, antiparasitic activity, and human macrophage growth inhibition are reported. in stark contrast, the inactivity or very low level of inhibition observed with compounds 35 confirms that the aforementioned interactions displayed by the glutamate tail are essential for the inhibitory activity. indeed, both the elimination of the glutamate portion (compounds 34) and the esterification of the -carboxylate group (compound 5) led to a significant drop in the inhibitory properties of these compounds. on the basis of the tbfold/2/nadp ternary complex presented here, new compounds 1620 were designed, synthesized, and tested in an enzyme assay. specifically, compounds 18 and 19 were designed to fill the hydrophobic cleft formed by the side chains of y52, y250, l252, and t51. indeed, derivative 18 (ki = 0.54 m) displayed a 2-fold increase of the inhibitory potency, compared to that of compound 2. in contrast, the lower activity of 19 suggests that the hydroxyl group on the terminal phenyl ring is not well accepted, probably due to its electron - donating properties that unfavorably affect the interaction with y52 and y250. we considered that a direct interaction with r54 would be beneficial for inhibition, and compound 16 (ki = 0.48 m) was designed for that purpose. the 2-fold increase in the inhibitory potency of this compound, compared to that of compound 2, seems to confirm our hypothesis. however, compounds 17 and 20 were synthesized as proof - of - concept for the importance of the c and c carboxylate groups, respectively. both derivatives showed a decreased inhibitory potency, confirming, once again, that at least two strong interactions should be established by the amino acidic tail in order to achieve submicromolar inhibition, either one charge - reinforced h - bond and one ionic interaction, as for compounds 2 and 16, or one charge - reinforced h - bond and one hydrophobic interaction, as in the case of compound 18. potential binding poses of 16 and 18, the most potent inhibitors identified in this series, were calculated by means of glide 5.5 software in extra precision (xp) mode, using glidescore for ligand ranking (see below). as shown in figure 4, the elongation of the -carboxylate chain (16 vs 2) allows for a salt bridge interaction with the side chains of r54 or r10. specifically, in the tbfold-2-nadp x - ray structure the shortest distance between the 2 carboxylate oxygen and an r54 guanidine hydrogen was around 5, while in the docking - derived pose the same distance is reduced to 2.6. this direct and by implication stronger association is likely the reason for the 2-fold increase in inhibitory potency of 16 with respect to 2. (a) two possible binding modes of 16 within tbfold as resulted from docking studies. compound 18 was originally designed to fill the hydrophobic pocket shaped by the three residues y52, y250, and l252 residues. indeed, two favored binding poses have been found, one in which the phenyl ring occupies the above - mentioned hydrophobic cleft forming a t - shaping assembly with the two tyrosine residue side chains (see figure 4) and another in which the ligand phenyl group is directed toward r10 establishing a cation- interaction (data not shown). however, the lower activity of 19 would perhaps suggest that the latter pose may not be relevant but would support the interaction of the terminal phenyl ring with y52 and y25. these latter interactions could be, in turn, the reason for the increased inhibitory potency of 18 with respect to 2. superimposition of the three - dimensional x - ray structures of human and tbfolds demonstrates that the enzyme presents a similar structure in these two diverse species and that the amino acids lining the active sites are overall well conserved (figure s2, overall sequence identity of 40%). nevertheless, a number of residues differ between the two enzymes, and a close inspection of the different residues lining the active sites reveals structural differences that could be exploited for the discovery of new trypanocidal agents endowed with low activity against the human fold. specifically, in human fold, residues k10, l51, n54, v55, k175, c236, and i238 are replaced by r10, t51, r54, l55, d173, v236, and t238, respectively, in tbfold (figure 5). the lysine arginine asparagine arginine differences at positions 10 and 54 might be targeted to achieve selectivity in inhibitor both tbfold r10 and r54 are in close proximity to the glutamate tail of 2 and may be reached via appropriate modification of the ligand s glutamate residue. thus, the crystal structure of the tbfold-2-nadp ternary complex and comparison with the human fold structure may help address the challenging problem of how to achieve both potency and selectivity toward the parasite and not the human enzyme. superimposition of the human fold (pdb code 1dig) and tbfold/2 (pdb 4lrr) complexes, represented as green and orange ribbons and sticks, respectively. all compounds were screened against the bloodstream form of t. brucei and showed in vitro dose - dependent killing, as determined from the reduction of the resazurin marker for cell viability (table 1). despite the fact that several compounds within this series showed a micromolar or even submicromolar activity against the target enzyme tbfold, they did not display any remarkable antiparasitic activity. only compounds 25 showed a moderate antiparasitic activity, and 2 was the most active, with an ic50 value of 49 3.2 m. the reference compound, analogue 21, exhibited a similar activity. all compounds were less active than suramin, a standard drug used in hat therapy. a counter - screen testing all compounds with human macrophages differentiated from thp1 monocytes showed more effective toxic activity on the parasite than in mammalian cells. compounds 2, 5, and 21 were 4-, 2.2-, and 1.8-fold more effective, respectively, against the t. brucei bloodstream form than against that from human macrophages. we have reported the first crystal structure of tbfold, a potential therapeutic target, with the bonus of obtaining information on the molecular basis of inhibition by compound 2. this molecule displays a micromolar enzyme inhibitory activity against tbfold and modest antiparasitic properties. a key part of the analysis was the clear assignment of the molecular structure of 2 using single crystal x - ray analysis of a synthetic intermediate, 3. the crystal structure of the enzyme ligand complex provides an accurate template to support structure - based approaches in early stage drug discovery. in addition, we have suggested that, although fold is a highly conserved enzyme, as indicated by the primary sequence alignment and structural comparisons, some critical differences in the active sites could be exploited to reduce the activity on the human form of the enzyme, and this possibility will be pursued in future development of this class of compounds. moreover, because molecular recognition is a dynamic process, differences in structural flexibility between the human and t. brucei enzymes might provide additional information to support this effort. despite the fact that almost all compounds showed low micromolar or submicromolar inhibition of tbfold, the antiparasitic activity on the bloodstream form parasite was modest. considering the polar nature of these molecules, an explanation for the modest level of antiparasitic activity is that structural modification of the amino acidic tail, while facilitating a better interaction with the target enzyme, has compromised the ability to cross the parasite membrane, likely due to a reduced affinity for membrane transporters. indeed, very little modification of the glu chain, such as the one carbon homologation of compound 2 leading to 16, has produced a complete loss of antiparasitic activity. this suggests that, to obtain more efficacious analogues, also the interaction with membrane transporters should be considered at the onset of the design process. h nmr and c nmr spectra were recorded with a varian mercury 300 (300 mhz) spectrometer. chemical shifts () are expressed in ppm, and coupling constants (j) are expressed in hz. tlc analyses were performed on commercial silica gel 60 f254 aluminum sheets ; spots were further evidenced by spraying with a dilute alkaline potassium permanganate solution or ninhydrin. melting points were determined on a model b 540 bchi apparatus and are uncorrected. mass spectrometry was carried out on a triple quadrupole spectrometer type varian 320-ms coupled with esi source. elemental analyses were performed on a perkinelmer pe 2400 elemental analyzer, and the data for c, h, and n were within 0.4% of the theoretical values. all target compounds possessed a purity of 95% as verified by elemental analyses by comparison with the theoretical values. 4-hydroxy-2,5,6-triaminopyrimidine sulfate (0.91 g, 3.8 mmol) was suspended in water (2 ml) and mixed with a 1 n naoh solution (11.4 ml). a solution of ethyl 4-isocyanatobenzoate (0.73 mg, 3.8 mmol) in 6 ml of acetonitrile was added dropwise. after stirring at room temperature for 2.5 h, 1 n hcl (11.4 ml) was added, and the mixture was stirred for 5 min. the solid was filtered off and washed with water (50 ml), etoh (30 ml), and diethyl ether (30 ml). after drying, the product was obtained as a pale orange solid (0.95 g, 2.86 mmol, 75% yield). m.p. h nmr (300 mhz, dmso - d6) (ppm) : 10.0 (bs, 1h, -nhconh-), 8.98 (bs, 1h, -nhconh), 7.84 (d, 2h, j = 8.4, ar h), 6.76 (s, 1h, -nh), 6.16 (bs, 2h, -nh2), 5.96 (bs, 2h, -nh2), 4.26 (q, 2h, j = 7.0, ch2ch3), 1.31 (t, 3h, j = 7.0, ch2ch3). c nmr (75 mhz, dmso - d6) (ppm) : 166.22, 162.38, 160.97, 155.26, 154.02, 145.89, 130.89, 122.60, 117.61, 89.94, 60.85, 14.94. calcd for c14h16n6o4 : c 50.60, h 4.85, n 25.29 ; found, c 50.62, h 4.98, n 25.03. compound 3 (0.95 g, 2.86 mmol) was suspended in water (25 ml), and 1 n naoh (29 ml) was added. the mixture was stirred at room temperature for 3 h, and it slowly turned into a clear solution. 1 n hcl (29 ml) was added, and the precipitate was collected by centrifugation (5000 rpm for 3 min). the solid was sequentially washed and centrifuged (5000 rpm for 3 min) with water (20 ml), etoh (20 ml), and diethyl ether (20 ml). the product was collected as a pale orange solid (0.80 g, 2.63 mmol, 92% yield). m.p. : h nmr (300 mhz, dmso - d6) (ppm) : 12.50 (bs, 1h, cooh), 9.98 (bs, 1h, -nhconh-), 8.90 (bs, 1h, -nhconh), 7.80 (d, 2h, j = 7.6, ar h), 6.71 (s, 1h, -nh), 6.18 (bs, 2h, -nh2), 5.88 (bs, 2h, -nh2). c nmr (75 mhz, dmso - d6) (ppm) : 167.81, 162.20, 160.88, 155.26, 153.98, 145.58, 131.07, 123.50, 117.49, 90.05. calcd for c12h12n6o4 : c 47.37, h 3.98, n 27.62 ; found, c 47.41, h 4.06, n 27.40. a suspension of compound 4 (0.1 g, 0.33 mmol) in dry dmf (7 ml) was placed under a nitrogen atmosphere and sonicated for 5 min. n - methylmorpholine (0.146 ml, 1.32 mmol) was added to the mixture, followed by 2-chloro-4,6-dimethoxy-1,3,5-triazine (232 mg, 1.32 mmol), and the mixture was stirred at 35 c under nitrogen for 5 h. the suspension slowly turned into an orange - red solution. the desired amino acid (610) as methyl or ethyl ester hydrochloride (1.32 mmol) was added to the solution followed by n - methylmorpholine (0.146 ml, 1.32 mmol), and the mixture was stirred at 30 c overnight. the solvent was removed under vacuum, keeping the temperature below 45 c, and the crude mixture was resuspended in etoh (10 ml) and stirred for 5 min. the solid was recovered by vacuum filtration and washed with etoh (10 ml) and diethyl ether (10 ml) and finally dried under vacuum overnight. h nmr (300 mhz, dmso - d6) (ppm) : 9.95 (bs, 1h, -nhconh-), 8.82 (bs, 1h, -nhconh), 8.48 (d, 1h, j = 7.0, arco - nh-), 7.80 (d, 2h, j = 8.8, ar h), 6.70 (s, 1h, -nh), 6.18 (bs, 2h, -nh2), 5.88 (bs, 2h, -nh2), 4.404.37 (m, 1h, h), 4.07 (q, 2h, j = 7.3, ch2ch3), 4.00 (q, 2h, j = 7.3, ch2ch3), 2.452.38 (m, 2h, ch2ch2cooet), 2.171.95 (m, 2h, chch2ch2cooet), 1.17 (t, 3h, j = 7.3, ch2ch3), 1.13 (t, 3h, j = 7.3, ch2ch3). c nmr (75 mhz, dmso - d6) (ppm) : 172.92, 172.66, 166.98, 162.17, 160.88, 155.37, 153.95, 144.36, 129.06, 126.59, 117.37, 90.10, 61.17, 60.60, 52.65, 30.91, 26.46, 14.76 ; ms 490.2 [m + h ]. calcd for c21h27n7o7 : c 51.53, h 5.56, n 20.03 ; found, c 51.61, h 5.60, n 19.91. h nmr (300 mhz, dmso - d6) 9.96 (bs, 1h, conhar), 8.82 (bs, 1h, pyrim - nhco), 8.50 (d, 1h, j = 7.4, conhc), 7.77 (d, 2h, j = 8.8, ar h), 7.50 (d, 2h, j = 8.8, ar h), 6.69 (s, 1h, oh), 6.14 (bs, 2h, -nh2), 5.88 (bs, 2h, -nh2), 4.38 (dt, 1h, j = 6.9, 7.4, h), 3.62 (s, 3h, och3), 3.56 (s, 3h, och3), 2.32 (t, 2h, j = 7.3, ch2ch2ch2coome), 1.891.72 (m, 2h, ch2ch2ch2coome), 1.681.53 (m, 2h, ch2ch2ch2cooch3). c nmr (75 mhz, dmso - d6) 173.76, 173.45, 166.88, 162.29, 160.88, 155.37, 153.97, 144.37, 129.07, 126.53, 117.34, 90.05, 53.00, 52.51, 51.94, 33.46, 30.51, 22.00. calcd for c20h25n7o7 : c 50.52 ; h 5.30 ; n 20.62 ; found, c 50.62, h 5.58, n 20.43. h nmr (300 mhz, dmso - d6) 9.98 (bs, 1h, conhar), 8.82 (bs, 1h, pyrim - nhco), 8.55 (d, 1h, j = 6.9, conhc), 7.77 (d, 2h, j = 8.7, ar h), 7.50 (d, 2h, j = 8.7, ar h), 6.70 (s, 1h, oh), 6.17 (bs, 2h, -nh2), 5.89 (bs, 2h, -nh2), 4.43 (dq, 1h, j = 6.9, 7.1, h), 3.62 (s, 3h, och3), 1.37 (d, 3h, j = 7.1, chch3). c nmr (75 mhz, dmso - d6) 174.08, 166.52, 162.20, 160.88, 155.37, 153.95, 144.33, 129.03, 126.52, 117.31, 90.03, 52.50, 48.86, 17.49. calcd for c16h19n7o5 : c 49.35 ; h 4.92 ; n 25.18 ; found, c 49.38, h 4.95, n 24.98. dmso - d6) 9.98 (bs, 1h, conhar), 8.81 (bs, 1h, pyrim - nhco), 8.61 (d, 1h, j = 7.0, conhc), 7.70 (d, 2h, j = 8.8, ar h), 7.48 (d, 2h, j = 8.8, ar h), 7.307.21 (m, 4h, ch2c6h5), 7.207.13 (m, 1h, ch2c6h5), 6.70 (s, 1h, oh), 6.16 (bs, 2h, -nh2), 5.89 (bs, 2h, -nh2), 4.60 (ddd, 1h, j = 6.0, 8.0, 9.1, h), 3.61 (s, 3h, och3) 3.173.04 (m, 2h, j = 7.3, ch2-c6h5). c nmr (75 mhz, dmso - d6) 173.11, 166.74, 162.27, 160.87, 155.35, 153.96, 144.36, 138.49, 129.74, 128.99, 128.93, 127.15, 126.48, 117.34, 90.05, 54.71, 52.57, 36.97. calcd for c22h23n7o5 : c 56.77 ; h 4.98 ; n 21.06 ; found, c 56.96, h 5.18, n 20.82. h nmr (300 mhz, dmso - d6) 9.97 (bs, 1h, conhar), 9.19 (s, 1h, ch2c6h4oh), 8.83 (bs, 1h, pyrim - nhco), 8.55 (d, 1h, j = 7.8, conhc), 7.70 (d, 2h, j = 8.7, ar h), 7.48 (d, 2h, j = 8.7, ar h), 7.06 (d, 2h, j = 8.4, ch2c6h4oh), 6.71 (s, 1h, oh), 6.63 (d, 2h, j = 8.4, ch2c6h4oh), 6.15 (bs, 2h, -nh2), 5.89 (bs, 2h, -nh2), 4.564.45 (m, 1h, h), 3.60 (s, 3h, och3), 3.052.87 (m, 2h, ch2c6h4oh). c nmr (75 mhz, dmso - d6) 173.26, 166.73, 162.31, 160.89, 156.58, 155.36, 153.97, 144.34, 130.67, 129.00, 128.43, 126.53, 117.30, 115.71, 90.03, 55.37, 52.49, 36.26. calcd for c22h23n7o6 : c 54.88 ; h 4.82 ; n 20.36 ; found, c, 54.97 ; h, 4.97 ; n, 20.01., dec t > 210 c ; h nmr (300 mhz, dmso - d6) 9.98 (bs, 1h, conhar), 8.77 (bs, 1h, pyrim - nhco), 8.26 (t, 1h, j = 5.5, conhch2), 7.71 (d, 2h, j = 8.5, ar h), 7.47 (d, 2h, j = 8.5, ar h), 6.69 (s, 1h, oh), 6.16 (bs, 2h, -nh2), 5.88 (bs, 2h, -nh2), 3.56 (s, 3h, och3), 3.23 (dt, 2h, j = 5.5, 6.6, ch2ch2ch2coome), 2.34 (t, 2h, j = 7.1, ch2ch2ch2coome), 1.75 (tt, 2h, j = 6.6, 7.1, ch2ch2ch2coome). c nmr (75 mhz, dmso - d6) 173.88, 166.57, 162.28, 160.88, 155.39, 153.96, 143.94, 128.65, 127.49, 117.36, 90.11, 51.92, 39.09, 31.53, 25.28. calcd for c17h21n7o5 : c 50.62 ; h 5.25 ; n 24.31 ; found, c 50.52, h 5.38, n 24.33. compound 5 (or analogues 1115) (0.2 mmol) was dissolved in 1 n naoh (0.8 mmol), and the solution was stirred at room temperature for 5 h. the solution was neutralized with 1 n hcl, and the precipitate was recovered by vacuum filtration and washed with water (10 ml), etoh (20 ml), and diethyl ether (20 ml) and finally dried under vacuum overnight. h nmr (300 mhz, dmso - d6) (ppm) : 12.35 (bs, 1h, cooh), 9.98 (bs, 1h, -nhconh-), 8.82 (bs, 1h, -nhconh), 8.38 (d, 1h, j = 6.6, arco - nh-), 7.79 (d, 2h, j = 8.0, ar h), 6.68 (s, 1h, -nh), 6.18 (bs, 2h, -nh2), 5.90 (bs, 2h, -nh2), 4.404.30 (m, 1h, h), 2.382.30 (m, 2h, ch2ch2cooh), 2.102.00 (m, 1h, chch2ch2cooh), 2.001.90 (m, 1h, chch2ch2cooh). c nmr (75 mhz, dmso - d6) (ppm) : 174.58, 174.28, 166.85, 162.30, 160.90, 155.39, 153.98, 144.26, 129.01, 126.82, 117.36, 90.10, 52.55, 31.15, 26.68. ms : 434.1 [m + h ]. anal. calcd for c17h19n7o7 : c 47.11, h 4.42, n 22.62 ; found, c 47.20, h 4.50, n 22.46. h nmr (300 mhz, d6-dmso) 12.26 (bs, 2h, cooh), 9.97 (bs, 1h, conhar), 8.80 (bs, 1h, pyrim - nhco), 8.35 (d, 1h, j = 7.7, conhc), 7.78 (d, 2h, j = 8.7, ar h), 7.50 (d, 2h, j = 8.7, ar h), 6.69 (s, 1h, oh), 6.14 (bs, 2h, -nh2), 5.87 (bs, 2h, -nh2), 4.31 (dt, 1h, j = 5.1, 7.7, h), 2.22 (t, 2h, j = 7.4, ch2ch2ch2cooh), 1.891.68 (m, 2h, ch2ch2ch2cooh), 1.681.49 (m, 2h, ch2ch2ch2cooch3). c nmr (75 mhz, d6-dmso) 174.96, 174.54, 166.69, 162.27, 160.90, 155.38, 153.99, 144.21, 128.97, 126.95, 117.35, 90.14, 53.09, 33.97, 30.90, 22.15. ms 448.1 m / z [m + h ]. calcd for c18h21n7o7 : c 48.32 ; h 4.73 ; n 21.91 ; found, c 48.42, h 4.90, n 21.63. h nmr (300 mhz, dmso - d6) 12.44 (bs, 1h, cooh), 9.98 (bs, 1h, conhar), 8.82 (bs, 1h, pyrim - nhco), 8.40 (d, 1h, j = 7.1, conhc), 7.77 (d, 2h, j = 8.8, ar h), 7.49 (d, 2h, j = 8.8, ar h), 6.69 (s, 1h, oh), 6.13 (bs, 2h, -nh2), 5.87 (bs, 2h, -nh2), 5.74 (s, 1h, oh), 4.37 (dq, 1h, j = 7.1, 7.4, h), 1.36 (d, 3h, j = 7.4, chch3). c nmr (75 mhz, dmso - d6) 175.15, 166.33, 162.32, 160.90, 155.40, 153.99, 144.19, 128.94, 126.88, 117.33, 90.07, 48.85, 17.8. calcd for c15h17n7o5 : c 48.00 ; h 4.57 ; n 26.12 ; found, c 47.84, h 4.78, n 25.93. h nmr (300 mhz, dmso - d6) 12.76 (bs, 1h, cooh), 9.97 (bs, 1h, conhar), 8.79 (bs, 1h, pyrim - nhco), 8.46 (d, 1h, j = 8.2, conhc), 7.69 (d, 2h, j = 8.8, ar h), 7.48 (d, 2h, j = 8.8, ar h), 7.337.21 (m, 4h, ch2c6h5), 7.197.12 (m, 1h, ch2c6h5), 6.69 (s, 1h, oh), 6.14 (bs, 2h, -nh2), 5.87 (bs, 2h, -nh2), 4.56 (ddd, 1h, j = 4.6, 8.2, 10.5, h), 3.15 (dd, 1h, j = 4.6, 13.8, ch2-c6h5), 3.04 (dd, 1h, j = 10.5, 13.8, ch2-c6h5). c nmr (75 mhz, dmso - d6) 174.09, 166.64, 162.27, 160.88, 155.35, 153.97, 144.22, 138.99, 129.74, 128.91, 128.85, 126.99, 126.81, 117.33, 90.08, 54.88, 37.01. calcd for c21h21n7o5 : c 55.87 ; h 4.69 ; n 21.72 ; found, c 55.57, h 4.94, n 21.52. dec t > 198 c ; h nmr (300 mhz, dmso - d6) 12.61 (bs, 1h, cooh), 9.96 (bs, 1h, conhar), 9.16 (s, 1h, ch2c6h4oh), 8.81 (bs, 1h, pyrim - nhco), 8.39 (d, 1h, j = 8.2, conhc), 7.69 (d, 2h, j = 8.8, ar h), 7.47 (d, 2h, j = 8.8, ar h), 7.07 (d, 2h, j = 8.4, ch2c6h4oh), 6.69 (s, 1h, oh), 6.62 (d, 2h, j = 8.4, ch2c6h4oh), 6.15 (bs, 2h, -nh2), 5.89 (bs, 2h, -nh2), 4.534.41 (m, 1h, h), 3.02 (dd, 1h, j = 4.5, 13.8, ch2-c6h4oh), 2.91 (dd, 1h, j = 10.1, 13.8, ch2-c6h4oh) ; c nmr (75 mhz, dmso - d6) 174.24, 166.64, 162.27, 160.88, 156.48, 155.37, 153.96, 144.21, 130.66, 128.94, 126.86, 126.81, 117.32, 115.65, 90.03, 55.24, 36.25. ms 468.2 m / z [m + h ]. calcd for c21h21n7o6 : c 53.96 ; h 4.53 ; n 20.98 ; found, c 54.12, h 4.78, n 20.64. h nmr (300 mhz, dmso - d6) 12.02 (bs, 1h, cooh), 9.97 (bs, 1h, conhar), 8.78 (bs, 1h, pyrim - nhco), 8.25 (t, 1h, j = 5.8, conhch2), 7.72 (d, 2h, j = 8.8, ar h), 7.47 (d, 2h, j = 8.8, ar h), 6.68 (s, 1h, oh), 6.13 (bs, 2h, -nh2), 5.86 (bs, 2h, -nh2), 3.23 (dt, 2h, j = 5.8, 6.6, ch2ch2ch2cooh), 2.25 (t, 2h, j = 7.4, ch2ch2ch2cooh), 1.72 (tt, 2h, j = 5.8, 6.6, ch2ch2ch2cooh). c nmr (75 mhz, dmso - d6) 174.99, 166.56, 162.25, 160.88, 155.39, 153.96, 143.92, 128.64, 127.57, 117.37, 90.16, 39.24, 31.94, 25.36. calcd for c16h19n7o5 : c 49.35 ; h 4.92 ; n 25.18 ; found, c 49.32, h 4.78, n 25.02. with the aim to test the glide 5.5 program for its ability to reproduce the crystallized binding geometry of 2, the latter ligand was subjected to automated docking calculations using extra precision (xp) mode and glidescore for ligand ranking. before docking, the program was successful in reproducing the experimentally found binding mode of 2, as it corresponds to the best ranked solution with an rmsd of only 0.87. the herein reported x - ray structure of tbfold was prepared through the protein preparation wizard within the maestro 9.0.2112 package using the opls-2001 force field. compound 3 is poorly soluble in most organic solvents at room temperature, with the exception of dmso. we therefore tried to grow crystals from dmso (in different conditions) and from mixtures of dmso / ch3cn. eventually, crystallization by slow evaporation (16 days) of a solution of 3 in reagent - grade dmso (sigma - aldrich) at room temperature produced small plates suitable for the determination of the molecular connectivity. after testing several samples, a specimen (0.25 0.20 0.05 mm) grown at the liquor / air interface was selected for the crystallographic analysis. the specimen manifested a significant pleochroism (colorless to violet) under polarized light. it was mounted on the top of a glass capillary fiber in dense perfluorinated oil. the x - ray data collection was performed at room temperature on a 3-circle bruker smart apex goniometer equipped with a ccd apex - ii detector, using graphite - monochromated mo k radiation (= 0.71073). a total of 26180 reflections (8144 unique) were recorded up to (sin /)max = 0.5017. a high exposure time (120 s / frame) was employed due to the small dimensions of the sample and its very low scattering power. the compound was found to crystallize in the triclinic centrosymmetric p1 space group (no. 2), with unit cell parameters a = 8.0260(16), b = 10.054(2), c = 48.956(10), = 84.44(3), = 89.13(3), = 80.14(3), and v = 3873.8(14). careful visual inspection of the reciprocal lattice showed some weak off - lattice spots attributable to a minor non- merohedral epitaxial twin component of the same 3 phase. experimental structure factor amplitudes were extracted by integration of the diffraction frames through the saint+ program suite, taking into account the presence of the additional lattice. corrections for sample absorption and x - ray beam anisotropy were applied by the program twinabs. eventually, the molecular structure was solved and refined with the shelx program package. in the final least - squares refined model, the nonmerohedral twinning was explicitly accounted for, and the fractional contribution of the minority component was estimated to be 0.161(3). all of the hydrogen atoms in the asymmetric unit were idealized, and their coordinates were indirectly determined through a riding motion moreover, all the bonds involving non - h atoms were subjected to a rigid - bond restraint, i.e., the component of the atomic anisotropic displacement parameters along the bond direction were restrained to be equal within a tolerance of 0.01. this strategy was motivated by (i) the rather poor quality of the sample and, consequently, the relatively low maximum resolution available for the data ; (ii) the quite low data - to - parameter ratio (8), due to the high number of symmetry - unrelated molecules in a large unit cell. however, these drawbacks did not hamper us from reliably determining the molecular connectivity of 3, even though our estimates for bond distances and angles have, on average, a rather low precision. the latter can be obtained free of charge from the cambridge crystallographic data centre via www.ccdc.cam.ac.uk/data_request/cif. the gene encoding 5,10-methylene tetrahydrofolate dehydrogenase/5,10 methenyl tetrahydrofolate cyclohydrolase (dhch1) was identified in genedb (http://www.genedb.org, accession number tb927.7.1600). genomic dna from t. brucei (lister 927 strain) was used as template for pcr with the following primers designed to amplify the dhch1 open reading frame using ndei and xho1 restriction sites (bold), respectively : 5-cat - atg - cct - gag - gcg - gtt - g-3 and 5-ctc - gag - tca - caa - ggc - acg - aa-3. the pcr product was inserted into pcr - bluntii - topo vector using the zero blunt topo pcr cloning kit (invitrogen) prior to excision and ligation into a modified pet15b vector (novagen) containing a tobacco etch virus (tev) protease recognition sequence (pet15btev). this results in recombinant expression of a product carrying an n - terminal hexa - histidine tag (his - tag), which is cleavable with tev protease. the recombinant plasmid was amplified in xl-1 blue e. coli, and the gene sequence verified by dna sequencing services (dundee university), before being transformed into e. coli bl21 (de3) for protein production. protein was produced in cells cultured at 37 c in 1 l lb media containing 50 mg / ml ampicillin. when an od of 0.8 was attained, expression was induced with iptg (1 mm) for 16 h at 21 c. cells were collected by centrifugation at 4000 rpm for 30 min at 4 c and frozen at 80 c until required. after thawing, the cells were resuspended in 50 mm bicine and 50 mm nacl, ph 8.0 (using 2 ml / g pellet), with the addition of complete (roche edta - free protease inhibitor) then lysed by sonication (7 10 s bursts), with cooling to < 4 c between pulses. the lysate was clarified with centrifugation (40000 g, 30 min, 4 c). the supernatant was loaded, at 2 ml min, onto a histrap hp ml column (prepacked with ni sepharose high performance - ge healthcare) equilibrated with buffer 50 mm bicine and 50 mm nacl, ph 8.0. retained proteins were eluted with a linear gradient at 1 ml min of 0100% of buffer 50 mm bicine, 50 mm nacl, and 1 m imidazole, ph 8.0. the sample of tbfold was passed through a hitrap desalting (ge healthcare) column to exchange the buffer with 50 mm bicine, 250 mm nacl, 0.5 mm dtt, and 10% glycerol, ph 8.0. the addition of 10% glycerol in all buffers was required to prevent the loss of activity when the samples were flash - frozen. crystallization trials were carried out with a phoenix liquid handling system (art robbins instruments / rigaku) using commercially available screens (hampton research) with a 1:1 ratio of 100 nl of protein solution and an equivalent volume of reservoir equilibrated against a 70 l reservoir at 20 c. crystals, small orthorhombic blocks with approximate dimensions of 0.1 0.1 0.1 mm, were observed after 3 days in conditions with 20% peg 6000 and 0.1 m citric acid, ph 5.0. we were unable to increase the size of the crystals using hanging drop or sitting drop vapor diffusion methods. crystals were transferred into a cryo - solution containing the reservoir supplemented with 20% glycerol prior to flash freezing at 173 c. crystals were characterized in - house with a micromax-007 rotating anode generator and r - axis iv dual image plate detector (rigaku), prior to storage in liquid nitrogen. x - ray diffraction data were then collected on the microfocus beamline of id23 - 2 at the european synchrotron radiation facility (esrf). the crystals belonged to the orthorhombic space group p212121 with unit cell parameters a = 58.22, b = 77.33, c = 128.91, = = = 90. the molecular weight of a subunit is 31.9 kda, and the asymmetric unit consists of two subunits with a vm value of 2.79 da and solvent content of approximately 55%. the structure of tbfold was solved by molecular replacement and refined to a resolution of 2.05. the search model was a monomer of the l. major fold structure, which shares a sequence identity of 85% (pdb code a26), with side chains removed. the rotation and translation functions were determined with phaser with a log likelihood gain of 84. inspection of the solution in the graphics program coot showed that a dimer, consistent with gel filtration data, was indeed present. side chains were built into electron and difference density maps, followed by iterative rounds of restrained refinement, model manipulation, and addition of solvent molecules using coot and refmac5. geometric restrained parameters were manually adjusted and applied in addition to translation / libration / screw analysis (tls) during the latter stages of refinement. structure superpositions were calculated using lsqkab, and figures were prepared using pymol (schrdinger llc). the dehydrogenase activity of fold was measured spectrophotometrically, using a beckman du-640 spectrophotometer, following the formation of 5,10-ch = thf from 5,10-ch2-thf. 5,10-ch2-thf dehydrogenase assays were carried out in a 0.5 ml volume at 27 c and contained 25 mm mops, ph 7.3, 30 mm 2-mercaptoethanol, 35 m mthf, and 1 mm nadp. the km values of substrate and cofactor having been previously elucidated as approximately 35 and 70 m. the reaction was initiated by the addition of nadp and incubated for 5 min then stopped by the addition of an equal volume of 1 m hcl and the 5,10-ch = thf produced quantified at 350 nm, using an extinction coefficient of 24.9 mm cm. the enzyme activity was expressed as moles 5,10-ch = thf produced per minute. enzyme inhibition was measured using the dehydrogenase assay, with stocks of compounds dissolved in dmso (10 mm). data from inhibition assays were fitted to a competitive model by linear regression using origin (originlab corporation). the bloodstream - form t. brucei brucei strain 427 was grown in hmi-9 medium 17.66 mg / ml imdm (gibco), 3.020 mg / ml sodium bicarbonate (sigma - aldrich), 0.136 mg / ml hypoxanthine (sigma - aldrich), 0.039 mg / ml thymidine (sigma - aldrich), and 0.028 mg / ml bathocuproine sulfonic acid (sigma - aldrich) supplemented with 10% heat - inactivated fetal bovine serum (fbs) (biowhittaker), 1.5 mm l - cysteine hydrochloride monohydrate (merck), 0.2 mm -mercaptoethanol (sigma - aldrich), 100 u / ml penicillin, and 100 u / ml streptomycin (biowhittaker) at 37 c under a humidified 5% co2 atmosphere. cultures were grown in t25 or t75 vented cap culture flasks (sarstedt) and subcultured every 23 days by 100- to 1000-fold dilution, respectively. parasites were counted directly using a neubauer chamber (marienfeld) and diluted appropriately in complete hmi-9 medium. the compounds under study and 21 were tested in a serial drug dilution assay in order to determine the ic50 values (concentration of drug causing 50% growth inhibition) by using the alamar blue assay. serial drug dilutions were prepared in 96-well microtiter plates containing culture medium as described above, and wells were inoculated with approximately 2,000 bloodstream form t. b. brucei cells. cultures were incubated for 72 h at 37 c under a humidified 5% co2 atmosphere. after this time the plates were incubated for an additional 4 h, and then the fluorescence read in a microplate reader (sinergy 2, biotek) using an excitation wavelength of 528 nm and an emission wavelength of 590 nm. the cytotoxicity of the compounds under study was assessed by a colorimetric mtt assay. thp1 differentiated macrophages were seeded at a density of 10 cells / well in 96-well plates and allowed to adhere overnight. cells were incubated with the compound concentration range for 72 h at 37 c. at the end of the incubation period, 200 l of 0.5 mg / ml mtt reagent (thiazolyl blue tetrazolium bromide, sigma) solution was added to each well. the plates were further incubated for 4 h at 37 c in the dark. the culture medium was subsequently discarded, and 200 l of isopropanol was added to dissolve the dark - blue formazan crystals. cell viability was quantified spectrophotometrically by measuring the absorbance of the formazan product at wavelength 570 nm and the background at 660 nm with a microplate reader (synergy 2, biotek, usa). the data are expressed as the percentages of viable cells compared to the survival of a control group (untreated cells). the ic50 value, i.e., the concentration of compounds necessary to decrease cell viability to 50% of the untreated control was determined by linear regression analysis. differences between in vitro anti - trypanosoma and thp1 cell cytotoxicity were examined using student s t test. the data are presented as the means sd, and all experiments were independently repeated at least three times. p - values < 0.05 (two - sided) were considered to be statistically significant.	the bifunctional enzyme n5,n10-methylenetetrahydrofolate dehydrogenase / cyclo hydrolase (fold) is essential for growth in trypanosomatidae. we sought to develop inhibitors of trypanosoma brucei fold (tbfold) as potential antiparasitic agents. compound 2 was synthesized, and the molecular structure was unequivocally assigned through x - ray crystallography of the intermediate compound 3. compound 2 showed an ic50 of 2.2 m, against tbfold and displayed antiparasitic activity against t. brucei (ic50 49 m). using compound 2, we were able to obtain the first x - ray structure of tbfold in the presence of nadp+ and the inhibitor, which then guided the rational design of a new series of potent tbfold inhibitors.
obesity and central adiposity, in particular, are now a well - recognized risk factor for several malignancies including colon cancer in both men and women [13 ]. body mass index (bmi) is commonly used to assess obesity (bmi 30 kg / m), and a large number of epidemiological studies support a bmi - colon cancer risk association. this association is generally stronger for men than that for women [46 ], possibly due to sex differences in fat distribution and/or hormonal milieus. possible putative pathophysiologic mechanisms for the positive association of obesity with colon cancer include higher levels of circulating peptide hormones such as insulin and insulin - like growth factor-1 (igf-1), adipose and gut hormones, inflammatory cytokines, free radicals, fatty acid metabolites, and sex steroid hormones in the obese [7, 8 ]. both animal and human studies have demonstrated modulatory effects of reproductive hormones on tumor cell growth and proliferation [912 ] and estrogen, either endogenously produced in premenopausal females or administered exogenously in the form of hormone replacement therapy (hrt), has been consistently shown to exert a protective effect against colorectal cancer in women [4, 13, 14 ]. a number of epidemiological studies have shown that the obesity - colon cancer risk association is stronger in pre - menopausal women as compared to postmenopausal women [1, 15, 16 ], suggesting an interactive effect of obesity and estrogen in the development of colon cancer. we have previously demonstrated increased colon cancer risk for men and women with bmis over 30 kg / m at time of recruitment as well as for large bmi changes (10 kg / m) during the 30s or 20s decades in women. these results further strengthened the evidence for the role of obesity in increasing colon cancer risk, and suggest that adulthood bmi changes may be a sensible measure of obesity - related colon cancer risk in women. however, no studies have examined how changes in bmi over time might be affected by hrt use among postmenopausal women in colon cancer development. therefore, we evaluated potential associations between bmi and changes in adult bmi over time (since 20s, 30s, 40s, 50s, and 2 years before study recruitment (current)) and colon cancer risk in 1,457 postmenopausal women participating in a population - based case - control study. we hypothesize that hrt use may offset the risk effect of adult weight gain to the extent that the risk associated with large adult bmi change and colon cancer will be diminished in postmenopausal women taking hrt, as compared to those not taking hrt. briefly, patients with newly diagnosed colon cancer (rectal cancer excluded) were identified from the kentucky cancer registry, a participant in the national cancer institute 's surveillance, epidemiology, and end results (seer) program as well as the centers for disease control and prevention 's national program of cancer registries. the registry database was reviewed quarterly and all primary incident colon cancer cases were identified and invited to participate in the study via mailed letter. we excluded patients with known history of inflammatory bowel disease (ibd), family history of familial adenomatous polyposis (fap), and hereditary nonpolyposis colorectal cancer (hnpcc). area codes and exchanges of cases were utilized with random digit generation of the remaining four digits. among those reached, the controls were at least 40 years of age and could not have had any personal history of nonskin cancer. of those who answered the phone and allowed eligibility determination, 70.8% of cases and 66.7% of controls agreed to participate in the study. the institutional review boards of the university of kentucky, lexington and university hospitals case medical center approved the study and all participants provided written informed consent. for this analysis, we included postmenopausal women only (516 cases, and 941 controls), excluding 190 women who were premenopausal at the time of recruitment and all 1,125 male participants from the dataset. eligible cases and controls were sent a lifestyle risk factor questionnaire developed by the national cancer institute colon cancer familial cancer registry https://bioinformatics.dartmouth.edu/ccfrc/downloads/rfq.pdf. this survey includes questions on height and weight throughout the 20s, 30s, 40s, since the age of 50, and 2 years prior to diagnosis for cases and the previous 2 years for controls (current). bmi categories were calculated based on world health organization definition as follows : normal (bmi 18.5 to 6 mets) [19, 20 ]. we first examined all variables of interest univariately for association with colon cancer risk in our sample. continuous variables were evaluated using a standard student 's t - test ; categorical variables were evaluated using a chi - square test. we then used unconditional logistic regression modeling to evaluate the potential associations between overweight and obesity compared to normal body size and colon cancer risk in each decade (20s, 30s, 40s, 50s, and current). all analyses were adjusted for potential confounding by other known colon cancer risk factors including age, race, education, income, physical activity, family history of colon cancer, smoking, alcohol, nonsteroidal anti - inflammatory drug use, age at menarche, and parity. changes in bmi over time were calculated for each individual for different age decades through the time of recruitment. these were categorized into small, moderate, and large changes as described in the data collection section. small bmi changes were used as the referent group. in logistic regressions, we adjusted for all variables included in the logistic regression models for bmi, and additionally adjusted for current bmi. these logistic regressions were then stratified by hrt use to evaluate differential association of bmi changes by hrt use. we examined the interaction of hrt use and weight change by adding into the full regression model a cross - product term of hrt and weight change during various age decades. all p values are from two - sided tests and p values 0.05). table 2 summarizes the results of the logistic regression models to evaluate the association of bmi at different ages on colon cancer risk. in the overall sample, there were no statistically significant associations for bmi reported at different age decades or up to 2 years prior to recruitment. stratified analyses by hrt use only revealed a statistically significant increase of risk of colon cancer in obese postmenopausal women who used hrt (or = 1.74 ; 95% ci = 1.082.80). table 3 summarizes the association of changes in bmi over age decades and colon cancer risk with and without stratification by hrt use. among all women, weight gain or bmi changes taking place during the 20s and 30s decades moderately, but statistically significantly, increased risk for colon cancer (p for trend = 0.05 and = 0.02, resp.). stratified analyses revealed that the increase of risk for weight gain or bmi changes taking place during both age decades was limited to postmenopausal women not using hrt, with a greater than 2 fold increase of colon cancer risk for large bmi change (> 10 kg / m) in the 20s age decade (or = 2.21 ; 95% ci = 1.094.45 ; p for trend = 0.02). hrt use itself was statistically significantly associated with a decreased risk of colon cancer in both univariate and fully adjusted analyses (data not shown). we have previously shown that large adult weight gain or bmi changes taking place in the 20s or 30s age decade significantly increased the risk of colon cancer independent of current bmi (within 2 years prior to recruitment), particularly among women. our previous report was based on 438 cases (212 women) and 491 controls (380 women). in the current study, based on a much larger sample size of postmenopausal women (516 cases and 941 controls) taken together, these results suggest that weight gain occurring since early adulthood may be a rational indicator of visceral adiposity accumulation and an important phenotypic marker for assessing obesity - related colon cancer risk in women. bmi is commonly used in epidemiological studies to assess degree of obesity, and the bmi - colon cancer association has consistently been found to be stronger in men than in women [1, 2124 ]. in the current study, we found that overall there is no colon cancer risk association for bmi reported for most of the age decades or 2 years prior to recruitment. the statistically significant association for obesity (bmi 30 kg / m) reported for the 50s age decade among hrt users is somewhat unexpected. given that there is no consistent pattern of risk increase (or = 0.95 for overweight (bmi 25 to < 30 kg / m)) in this nor in any other age decade, we believe this is likely due to chance. it is speculated that central adiposity is the major driver for increased circulating levels of insulin, igfs, and inflammatory cytokines, as well as decreased levels of igf binding proteins all of which have been implicated as tumor - promoting [8, 2529 ]. as a result, some have advocated that alternative methods for body size characterization such as waist - hip ratio (whr) and waist circumference may more accurately reflect abdominal obesity. other studies have failed to demonstrate any increased association of whr or waist circumference, as compared to bmi, with colorectal cancer in women thereby arguing against body fat distribution as the sole determinant of gender - based differences in crc risk. consistent with our hypothesis of opposing effects of adult weight gain and hrt use, we have found that the increased risk of colon cancer associated with weight gain since early adulthood was largely limited to postmenopausal women reporting no prior use of hrt. although testing for multiplicative interaction between bmi change and hrt use was not statistically significant, this was likely due to limited power in our study. bmi changes during their 20s were more than twice as likely to develop colon cancer. similarly, this same group of women had a 77% increased risk of colon cancer if they experienced even moderate and large bmi change between the 30s decade and recruitment was associated with a higher risk of colon cancer, though this association was only statistically significant for those reporting moderate these findings confirm earlier studies showing that hrt use may modify the effects of obesity in postmenopausal women possibly by counteracting the known risks of weight gain during the early premenopausal years. modification of weight gain - associated crc risk by use of exogenous hrt may be partly explained by the opposing effects of hrt and adiposity on circulating levels of insulin and igf-1, and endogenous sex hormones such as estrogen. higher levels of insulin and bioactive igf-1 are both established risk factors for colon cancer and are believed to at least partially account for the obesity - associated increased risk of colon cancer. hrt use decreases hepatic synthesis of insulin - igf-1 axis factors [33, 34 ], thus offsetting the insulin - igf-1 mediated colon carcinogenic effect of adiposity. hrt use also increases the synthesis of sex hormone binding globulin, leading to reduced circulating levels of bioavailable endogenous estrogen. in postmenopausal women, adipose tissue is the main source of endogenous estrogen production. accumulating evidence suggests that in contrast to the well established protective effects of hrt use against colon carcinogenesis, increased lifetime exposure to and high circulating levels of endogenous estrogen promote the development of colon cancer [32, 3638 ]. it is thus plausible that hrt use may offset obesity - associated risk of colon cancer by reducing the circulating levels of bioavailable endogenous estrogen in postmenopausal women. study limitations include the possibility for information bias from our case - control study design as well as potential recall bias inherent in the use of self - reported height and weight to calculate bmi. however, correlations between self - reported and actual measured weight are generally quite high [39, 40 ]. additionally, in elderly patients, accurate recall of self - reported weight has been demonstrated for as long as 28 years prior. random digit dialing, although still prone to selection bias, because of preference toward individuals with landlines, was the most feasible method through which recruitment of a sample most representative of our case source population (state of kentucky) could be achieved. all analyses were adjusted, however, for well established risk factors including education and income using very finely defined categories. in summary, our results confirm earlier findings of the protective role of hrt in crc and suggest that, in postmenopausal women, hrt may ameliorate the negative effects of early pre - menopausal weight gain to some extent. this finding is of paramount clinical importance, especially in light of the fact that the use of hormone replacement has declined significantly since the women 's health initiative study findings were published in 2002 [42, 43 ]. further study is warranted to evaluate why earlier versus later weight gain is so strongly associated with increased crc risk. furthermore, a deeper understanding of the exact biological mechanisms via which hrt may modify crc risk due to obesity throughout adult life is necessary. prospective studies in both pre- and postmenopausal women of various body sizes, and with varying types of fat distribution patterns, employing serum biomarkers such as insulin, igf-1, estrogen, and how they change over time will be most instructive.	purpose. we recently reported an association of adult bmi change with colon cancer risk. here, we sought to further explore this association with respect to postmenopausal hrt use in a larger study population. methods. we included 1,457 postmenopausal women participating in an ongoing population - based case - control study of colon cancer. results. we confirmed a previously reported association of adulthood weight gain and increased risk of colon cancer : compared to those with 10 kg / m2) bmi changes since their 20s had or estimates of 1.54 (95% ci = 1.092.19) and 1.45 (95% ci = 0.902.33), respectively (p for trend = 0.05). stratified analyses showed that this association was limited to hrt nonusers : ors were 1.77 (95% ci = 1.023.05) and 2.21 (95% ci = 1.094.45), respectively (p for trend = 0.03), for bmi changes occurring between the 20s decade and time of recruitment among non - users. similar associations were observed for bmi changes since the 30s decade. there was no association among hrt users. conclusion. our results suggest early adulthood weight gain increases colon cancer risk in postmenopausal women who do not use hrt.
due to its generally malignant behavior, radical inguinal orchidectomy is the gold standard for the treatment of a testicular tumor. nevertheless, in some less frequent histological subtypes of tumors with low malignant potential organ - sparing approach : partial orchidectomy or even testicle - sparing excisions of tumors could be attempted. since there are no reliable non - invasive diagnostic tests available which would allow unequivocal differential diagnosis of the lesion in the preoperative period, it is recommended that the testicle should be first exposed and frozen section biopsy should be performed prior to main surgery. large cell calcifying sertoli cell tumor (lccsct) is a very uncommon sex cord stromal neoplasm. usually, the disease presents in adolescents and young adults with slowly enlarging painless testicular mass or gynecomastia. in up to 40% of cases, it coincidences with inherited genetic syndromes such as peutz. these autosomal dominant multiple neoplasia syndromes present with a variety of pigmented lesions of the skin and mucosae and a number of tumors affecting many organs. yet still, only a small fraction of syndromic patients develop lccsct in total, about 30 cases of testicular lesions have been previously reported in the english language literature in this subgroup of patients (reviewed by ulbright.). therefore, instead of standard radical orchidectomy, in case of lccsct suspicion partial orchidectomy is recommended. here, we report a case of bilateral lccsct in a 20-year - old man with skin lesions suggestive for pjs, which was successfully treated with bilateral testicle - sparing tumorectomies. a 20-year - old man was referred to the urological department due to bilateral testicular enlargement. on physical examination, two hard, non - tender tumors were detected. besides, patient presented with dispersed lentiginosis on the face, hands, penis and on the buccal mucosa (fig. 1). serum alpha - fetoprotein, beta - hcgh and testosterone levels were within normal range. lentigosis on the face (a), hands (b) and penis (c) characteristic skin lesions lentigosis on the face (a), hands (b) and penis (c) peculiar skin lesions and hyperechoic ultrasound image of the bilateral tumors suggested underlying constitutional genetic abnormality and hence plausibly benign character of the testicular lesions. frozen section intraoperative consultation was indicative of benign testicular neoplasm, confirming organ - sparing approach. 2microscopic features of the tumor a testicular tumor composed of trabecles, small tubules and cords of polygonal mildly pleomorphic cells with abundant eosinophilic cytoplasm embedded in myxoid stroma with scattered granulocytes. the tumor had also strong immunoreactivity for s-100 protein microscopic features of the tumor a testicular tumor composed of trabecles, small tubules and cords of polygonal mildly pleomorphic cells with abundant eosinophilic cytoplasm embedded in myxoid stroma with scattered granulocytes. the tumor had also strong immunoreactivity for s-100 protein in order to verify clinical suspicion of the peutz - jaghers syndrome, genetic testing was performed. dna was extracted from peripheral blood leukocytes in accordance with the standard phenol - chlorophorm protocol. mutational screening of the stk11 gene was performed by direct sequencing of all exons and adjacent intronic junctions of the gene. large genomic rearrangements were analyzed by multiplex ligation - dependent probe amplification method using salsa mlpa p101-a2 stk11 kit (mcr holland), as previously described. stk11 gene amplification confined to the promoter region and exon 1 was detected ; however, due to large size of the gene fragment in question and its high gc content, the exact breaking points of the rearrangement were not established. nevertheless, since amplification of the exon 1 results in frameshift and hence premature termination of translation the resulting aberrant mrna either will be subjected to nonsense - mediated decay or encode a truncated, malfunctioning protein comprising of mere 96 n - terminal amino acids instead of 433 residues present in the wild - type stk11 protein. skin lesions, histopathology type of the testicular tumors and results of genetic tests all confirmed diagnosis of peutz jeghers syndrome. peutz jeghers syndrome is a rare genetic disorder characterized by multiple gastrointestinal (gi) hamartomatous polyps, mucocutaneous pigmentation and increased predisposition to various neoplasms. typically, pjs diagnosis is made in young adults presenting with gastrointestinal problems such as obstruction, pain or bleeding caused by the presence of hamartomatous polyps in the digestive tract, who in addition to abdominal symptoms have peculiar melanin hyperpigmented spots on the face, especially around lips and nostrils, oral mucosa, genitals, anus and sometimes on hands or feet. even though 88% of pjs patients have gi polyps, the reported patient neither had ever manifested any symptoms of gastrointestinal disease, nor had polyps detected during eventual gi work - up. yet still, since the most common site of pjs polyps is the small intestine that is a difficult organ to examine by clinical or radiological means, it can not be excluded that in the future the patient becomes symptomatic. the second atypical feature of the presented case is the type of causative mutation. even though one - third of pjs patients is found to have rearrangements of the stk11 gene, on the whole these are large exonic deletions. in accordance with human gene mutation database (2011), our patient is the second case worldwide with an exonic amplification affecting stk11 gene, the other patient having exon 7 duplication. in view of the rarity of such mutation, no definite conclusions with respect to genotype phenotype correlations can be drawn. in addition to polyps and melanin spots that are the two constant features of the syndrome, pjs patients have increased risk of developing various tumors. the relative risk of cancer in these individuals is 15 times the general population risk and the cumulative cancer risk is 7681% by the age of 70 years [6, 7 ]. the most common sites are small intestine, colon, pancreas, stomach, lung, breast, uterus, and ovary. the occurrence of ovarian tumors far exceeds that of testicular tumors in this disorder, with the estimated cumulative risk of 621% in pjs women [6, 7 ]. conversely, lccscts have been observed in a few pjs patients only, nevertheless accounting for up to one - third of all lccsct reported in the available literature [3, 4 ]. in contrast to sporadic lccsct cases, pjs men develop cancer at earlier age (6.5 vs. 16 years) [3, 4 ]. in pjs, the first manifestation typically results from hormonal activity of the tumor and can be observed clinically as bilateral gynecomastia and precocious puberty, while sporadic cases usually present with non - tender testicular mass in adolescence. in general, pjs tumors are bilateral and may be multifocal, while only 20% of sporadic cases affect both testicles [3, 4 ]. by and malignant behavior can be observed in 17% of cases, mainly in lesions > 4 cm or in patients over the age of 20. preoperative work - up should include evaluation of retroperitoneal lymph nodes, bones, liver and lungs as these are the most common sites of cancer metastases. on ultrasound imaging lccsct are usually round, regular, and hyperechoic with acoustic shadow. distinctive ultrasound picture helps making preliminary diagnosis, but it must be remembered that intratumor hyperechoic elements may also be present in seminomas, carcinomas embrionale, mature teratomas as well as in benign lesions caused by post - traumatic or post - infectious inflammation. in comparison with traditional treatment of testicular tumors by means of radical inguinal orchidectomy, in case of lccsct partial orchidectomy / tumorectomy seems to be more beneficial surgical approach. nevertheless, testicle - sparing surgery is rational only if several conditions are fulfilled. to start with, benign character of the tumor secondly, the tumor has to be located far from the rete testis and be smaller than 2025 mm in order to have enough viable testicular tissue left. more to the point, after this kind of surgery, close follow - up must be implemented and it is usually done by testicular ultrasound imaging. in cases of lccscts that present with additional abnormalities suggestive of genetic syndromes such as heart myxoma in carney complex, or melanin spots and/or hamartomatous polyps in pjs detection of the testicular tumor may help to diagnose these entities and hence enable starting their adequate treatment and further lifelong oncological surveillance. he does not meet the generally acknowledged clinical criteria for pjs diagnosis since he had no signs of gi disease, or positive family history of pjs in a close relative(s). also clinical manifestation of lccsct was atypical, with no endocrinological disturbances and relatively late age at onset. nevertheless, stk1 mutational screening allowed for molecular confirmation of the disease pointing out the importance of molecular testing in lccsct patients due to a low malignant potential of lccsct, it may be very efficiently treated by testicle - sparing method, especially in cases of bilateral tumor. characteristic phenotypic features of the patients with genetic dysplastic syndromes that may coincide with lccsct and typical appearance of the tumor on ultrasound facilitate its diagnosing prior to surgery and hence reduce the invasiveness of the treatment. conversely, lccsct diagnosis enables identification of the underlying genetic syndrome that may predispose to increased risk of other neoplasia.	large cell calcifying sertoli cell tumor (lccsct) is an exceptionally rare neoplasm originating from sperm cord cells. the tumors have relatively low malignant potential and unlikely proceed to metastasis formation. the lesions may occur in an isolated form or in ca. 40% of cases may be associated with genetic abnormalities, by and large peutz jeghers syndrome and carney complex. at presentation, 20% of lccsct cases are bilateral and/or multifocal. owning to characteristic skin lesions and particular hyperechoic ultrasound image of the tumor, preliminary diagnosis of the syndromic lccsct is possible in the preoperative period. consequently, testicle organ sparing procedure can be attempted, which is especially justified in bilateral lesions. here, we report a case of a bilateral lccsct in a 20-year - old man with atypical peutz jeghers syndrome due to amplification of the exon 1 of stk11 gene who was successfully treated with bilateral testicle - sparing tumorectomies.
photoreceptors are the primary neurons in the vertebrate retina and their degeneration underlies many forms of visual impairment. studies using mouse models of photoreceptor degeneration have demonstrated successful rescue or restoration of visual function through transplantation of photoreceptor cells harvested from young retina or generated in vitro from mouse stem cells.1 - 3 this has generated great excitement in the vision community and in the general public. at the same time, it has heightened the scientific and societal importance of defining a reliable source of new photoreceptor cells. current research on deriving new photoreceptors for replacement, as regenerative medicine in general, centers on using embryonic stem cells and induced pluripotent stem (ips) cells to generate transplantable cells.4 - 10 great strikes have been made towards the ultimate goal of cell replacement using photoreceptor cells derived from the patient s own somatic cells through ips cell technology. it begins with awakening adult cells at the site of a wound to proliferate, followed by activating / reactivating cell differentiation programs to produce the desired cells. if an in vivo regeneration mechanism could be employed to produce new photoreceptor cells, then photoreceptor replacement could be attainable without cell transplantation and associated risks and complications. injuries induce photoreceptor regeneration from retinal stem cells residing at the ciliary margin in teleost fish eyes.11 however, this regeneration mechanism seems lacking in mammals.12,13 the previously reported presence of retinal stem cells in adult ciliary epithelium of the mammalian eye14,15 has been contested.16,17 a recent study reported the presence of multipotent stem cells in the retina of 4 - 8 weeks old mice that could be expanded in vitro for over 35 passages and produced different types of cells including functional photoreceptor cells.18 it would be interesting and important to demonstrate the presence of multipotent retinal stem cells in well matured and aged mice. muller glia in various species, including mammals, possess certain properties of progenitor cells, but their ability to efficiently give rise to photoreceptors needs to be demonstrated.19 - 28 the rather disappointing outcomes from exploring the neural retina for photoreceptor regeneration, along with the discoveries of multipotent stem cells from various tissues in adult mammals, have spurred interests in non - neural tissues of the eye for photoreceptor genesis. alternative sources being examined include the iris pigment epithelium,29 - 34 the ciliary body,14,15,32,35 the limbal epithelium,36 and the retinal pigment epithelium (rpe). excluding the rpe, none of the alternatives give rise to a significant number of, if any, photoreceptor - like cells. this may reflect the biological nature of these tissues ; it may also stem from the used approaches, as few of the studies used a regulatory gene with pro - photoreceptor activity to steer uncommitted cells toward the photoreceptor path or to initiate photoreceptor differentiation program in the otherwise non - neural cells. the anatomical location places the rpe at an ideal position for providing new photoreceptors to repopulate a damaged retina. the key question upfront is whether or not the rpe is biologically amenable to a reprogramming scheme to give rise to photoreceptor cells. developmentally, the rpe and the neural retina originate from the same structure, i.e. the optic vesicle. indeed, classic experiments have revealed an intriguing phenomenon, rpe becoming a neural retina, referred to as rpe transdifferentiation. in very young chick embryos, physically separating the rpe from the neural retina,37 or surgically removing most of the neural retina,38 results in the rpe developing into a neural retina. subsequent investigation led to the discovery of bfgf as a stimulus for this phenomenal transdifferentiation.39 rpe - to - neural retina transdifferentiation also occurs in amphibians.40 - 42 in culture, rodent rpe from young embryos has been shown to undergo rpe - to - retina transdifferentiation.43,44 mutations in regulatory factors involved in regulating rpe versus retinal fate or disruptions of bone morphogenetic protein (bmp) and wnt (int / wingless) signaling pathways can result in ventral rpe transdifferentiation into neural retina.45 - 52 notably, this rpe - to - retina transdifferentiation results in the rpe to be no longer present. the rpe plays important roles in the well - being and function of photoreceptor cells and the retina as a whole. dysfunctional rpe is believed to be an underlying pathological condition in age - related macular degeneration, a leading cause of blindness in the elderly in developed countries. adult mammalian rpe is well - known for two biological properties : proliferation and plasticity. in the mature eye, most cells in the rpe remain quiescent, except a small population in the periphery where cell proliferation has been observed.53 however, rpe cells can re - enter the cell cycle to proliferate upon retinal detachment,54 - 56 when stimulated physically,57 or under disease conditions.60 - 62 this proliferative response may result in rpe regeneration / wound healing.61 - 65 it may also lead to proliferative retinopathy when progeny cells from rpe proliferation differentiate into cells with tractional force causing retinal detachment.66 on the other hand, these very traits of proliferation and plasticity raise an intriguing possibility of exploring the rpe as a source of new photoreceptor cells. with mounting knowledge on the regulatory guidance of photoreceptor production during retinal development, an alternative approach emerged to produce new photoreceptor cells, i.e. reprogramming the rpe by genes with pro - photoreceptor activities, to channel rpe proliferation and plasticity towards photoreceptor production. the feasibility of channelling rpe proliferation and plasticity to photoreceptor production using genes that steer unspecified cells toward the path of differentiating into photoreceptors was first tested with the chick system, taking advantage of abundant rpe tissue from chick embryos and readily achievable wide - spread gene transduction from replication - competent retrovirus rcas. dissociated rpe cell culture was established from chick embryos at day 6 (e6) and thereafter, stages at which the rpe has already lost its competence to undergo the classic rpe - to - retina transdifferentiation. cells in the primary rpe cell culture were then infected with rcas expressing a gene with pro - photoreceptor activities. using this system, over 20 regulatory genes shown or implicated to be important for the development of the eye, the retina, and/or photoreceptor cells have been screened for activities to guide cultured rpe cells to the path of differentiating towards photoreceptors.67 the screening identified neurogenin1 (ngn1) and ngn3 as the two top - performers for eliciting rpe - to - photoreceptor reprogramming.67 both genes induced the production of neural clusters from otherwise monolayer rpe cell cultures (figures 1a and b)67 and de novo generation of large numbers (as high as 80% of the cells present in the culture) of cells (referred to as reprogrammed cells) positive for photoreceptor protein visinin (figure 1c).67 molecularly, the reprogrammed cells expressed transcription factors crx, nr2e3, raxl, rxr, and neurod, which participate in initiating the photoreceptor differentiation program. reprogrammed cells also expressed components of phototransduction, including red opsin, the -subunit of cng channels, and cone -transducin. red opsin+ cells displayed dot - like immunostaining at the apices of the cells (figures 1f and g),67 reminiscent of that in the retina, indicating proper localization of red opsin in the reprogrammed cells. morphologically, in contrast to the hexagonal rpe cells, visinin+ cells resembled young photoreceptor cells, with an elongated cell body, an axon - like process, an inner segment - like compartment, and a lipid droplet - like structural feature (figure 1d and e).67 electron microscopy showed that reprogrammed cells developed a cellular compartment rich in mitochondria, resembling the inner segment of photoreceptor cells.67 on the apex of the inner segment - like structure, reprogrammed cells displayed ciliary expansions, reminiscent of the developing outer segments of retinal photoreceptors in e17 eye or in culture. fluorescent calcium imaging showed that reprogrammed cells exhibited both hallmarks : they responded to light by decreasing their ca2 + levels (figure 2) and responded to 9-cis - retinal by increasing their ca2 + levels (figure 3).67 these results indicate that reprogrammed cells were able to develop advanced photoreceptor traits at molecular, structural, and physiological levels. to be a convenient source of new photoreceptor cells, rpe in the eye needs to be responsive to the reprogram scheme. in e7.5 chick eyes, either the wild type or those infected with control retrovirus rcas expressing gfp (rcas - gfp), developing photoreceptor cells positive for visinin were confined within the neural retina at the prospective location of the outer nuclear layer (onl), and the rpe layer lacked visinin+ cells (figures 4a and b).68 in embryonic chick eyes infected with retrovirus rcas - ngn3, the rpe layer contained visinin+ cells. this was unequivocally observed in regions where the rpe and the retina were separated (figures 4c and d).68 the visinin+ cells accounted for ~37.24.3% of cells in the rpe layer. a thin process could be seen on some of the visinin+ cells (arrows in figures 4e and f), indicating they were morphologically more similar to neurons than to rpe cells, while other visinin+ cells were more rpe - like. some of the visinin+ cells in the rpe layer retained dark pigmentation typical of rpe cells (arrowheads in figures 4e and f). in addition, visinin+ cells in the rpe predated their presence in the retina.68 thus visinin+ cells in the pre layer likely originated from the rpe and possibly were in transitional stages in rpe - to - photoreceptor switching process. the above results provide compelling evidence for the feasibility of reprogramming embryonic chick rpe to produce photoreceptors. nonetheless, the study used the chick, a non - mammalian vertebrate that is evolutionally more ancient and may manifest phenotypical changes that are lacking in mammals after comparable experimental manipulations. to move forward towards the ultimate therapeutic goal of inducing in situ photoreceptor generation, transgenic mice were created with a dna construct that would express ngn1 under the control of rpe bestrophin1 (vmd2) promoter69 or ngn3 under the control of rpe65 promoter.70 the logic was to use ngn1/ngn3 to induce in mouse rpe cells the expression of genes that initiate photoreceptor differentiation and suppress the expression of rpe genes, including the transgene itself from the rpe promoter (figure 5). this would emulate transient ngn1/ngn3 expression in the developing neural retina.71,72 continuation of the process would lead to the production of mature photoreceptor cells. a pronounced phenotypical change was the presence of photoreceptor - like (pr - l) cells in the subretinal space (figure 6).73 immunohistologically and morphologically these cells appeared similar to those in the onl. they displayed outer segments discernible with bright - field microscopy, decorated by anti - red opsin and anti - rhodopsin immunostaining (figure 7), and containing stacks of electron - dense disc membrane that constitutes the outer segments of photoreceptors.73 the very approach of reprogramming the rpe to give rise to photoreceptor cells inherently raises a concern of whether new photoreceptor cells would be produced at the expense of the pre, an undesirable outcome as the rpe plays essential roles in maintaining the health of the retina, particularly of photoreceptors. this concern was eased by the presence of the monolayer rpe in eyes with pr - l cells (figure 7)73 and at the place with cells seemingly en route rpe - to - photoreceptor transition.73 it appears that the rpe might have regenerated itself, after some of its cells had taken on the route to becoming pr - l cells. after experimental rpe debridement in the pig eye, the rpe heals.62 rpe wound healing has also been reported in aged - related macular degeneration patients after debridement of defective rpe monolayer.65 rpe repair / wound healing initially involves cell sliding migration and subsequently cell proliferation61 and may require the presence of neural retina for the new rpe to structurally and functionally mature.63 a question highly relevant to the potential application of the reprogramming scheme to photoreceptor generation is whether the rpe in an aging mouse eye would be responsive to the gene - directed reprogramming. cells seemingly en route rpe - to - photoreceptor transition were present in the subretinal space in a 9-month - old pvmd2-ngn1 animal.73 dark pigment granules were present in these cells, as in cells of the adjacent, monolayer rpe. yet, these transitional cells were positive for recoverin, a photoreceptor protein involved in phototransduction, and displayed an elongated cell body typical of young photoreceptor cells. distal to the domain of the transitional cells was a well - defined layer of pr - l cells, which no longer displayed conspicuous rpe marks.73 these observations suggest that the aging rpe in the 9-month - old animal was responsive to the reprograming scheme. additionally, there were recoverin+/brdu+ cells in eyecup explants derived from a 6-month - old transgenic mouse, indicating that the eyecup from the animal that was well into adulthood was able to give birth to pr - l cells in vitro.73 in conclusion, results from experiments using chick and mouse systems provided support to the biological feasibility of using gene - directed reprogramming of the rpe to produce new photoreceptor cells in situ in the eye. if applicable to the human eye, gene - directed reprogramming may offer a tantalizing prospect of using the rpe as a convenient source of new photoreceptor cells for retinal repair in situ, without involving cell transplantation and its associated risks and complications. however, enticing as it may seem, it is imperative to carry out rigorous studies to address many issues important to both basic science and potential clinical applications.	recent success in restoring visual function through photoreceptor replacement in mouse models of photoreceptor degeneration intensifies the need to generate or regenerate photoreceptor cells for the ultimate goal of using cell replacement therapy for blindness caused by photoreceptor degeneration. current research on deriving new photoreceptors for replacement, as regenerative medicine in general, focuses on the use of embryonic stem cells and induced pluripotent stem (ips) cells to generate transplantable cells. nonetheless, naturally occurring regeneration, such as wound healing, involves awakening cells at or near a wound site to produce new cells needed to heal the wound. here we discuss the possibility of tweaking an ocular tissue, the retinal pigment epithelium (rpe), to produce photoreceptor cells in situ in the eye. unlike the neural retina, the rpe in adult mammals maintains cell proliferation capability. furthermore, progeny cells from rpe proliferation may differentiate into cells other than rpe. the combination of proliferation and plasticity opens a question of whether they could be channeled by a regulatory gene with pro - photoreceptor activity towards photoreceptor production. studies using embryonic chick and transgenic mouse showed that indeed photoreceptor - like cells were produced in culture and in vivo in the eye using gene - directed reprogramming of rpe cells, supporting the feasibility of using the rpe as a convenient source of new photoreceptor cells for in situ retinal repair without involving cell transplantation.
pharmacological agents known to inhibit a membrane enzyme involved in the oxidation of various primary amines, and hitherto named semicarbazide - sensitive amine oxidase (ssao, e.c. most of these observations of such slimming effect with the tested agents were not expected since they were evidenced in studies initially focused on vascular pharmacology. the first reported observation was made by yu and coworkers in the obese and diabetic kkay mice. whilst the authors aimed to demonstrate an antiatherogenic effect of (e)-2-(4-fluorophenethyl)-3-fluoroallylamine (fpfa), they observed that this compound reduced weight gain in obese kkay mice fed an atherogenic diet. in the same report, yu and colleagues demonstrated that fpfa was able to inhibit both ssao and monoamine oxidases (mao). more recently, by performing pharmacological research on arterial thickness alteration, mercier and colleagues repeatedly administrated the reference inhibitor of ssao, namely, semicarbazide, at 100 mg / kg bw / d to sprague - dawley, and brown - norway rats. in both models, the authors observed a dramatically reduced body weight gain in response to the ssao inhibitor, which also induced a decrease in the pressure resistance of arteries, as initially expected. we have also reported that aminoguanidine, which inhibits ssao together with nitric oxide synthases (nos) and diamine oxidase (dao), was able to limit wat extension without notably altering calorie intake and body weight gain in obese zucker rats. then, we observed that the combined inhibition of ssao and mao, obtained by repeated injections of semicarbazide plus pargyline, or by daily i.p. administration of phenelzine (an antidepressant which inhibits both mao and ssao), produced concomitant limitation of body weight gain in the obese zucker rat [7, 8 ]. therefore, at least four distinct pharmacological agents tested in vivo to inhibit ssao were able to alter energy balance and to lower body weight gain in rodents. ssao is historically known for its presence in vessels : in endothelial cells, where it is known as ssao / vap-1 owing to its vascular adhesion properties and in smooth muscle cells, where it is involved together with another copper - containing amine oxidase, the lysyl oxidase, in extracellular matrix maturation [2, 11 ]. in fact, ssao is also highly expressed in white adipose tissue (wat). tissue - distribution studies have recently evidenced that the ssao amount in adipocytes is extremely elevated, regarding gene expression, protein abundance, or activity level, including in man. with an approach aiming at unravelling the role for such ssao abundance at the surface of fat cells, we observed in vitro that exogenous amines exert insulin mimicry when added to adipocyte preparations. actually, at submillimolar concentrations, benzylamine elicits, in a ssao - dependent manner, an activation of glucose transport and an inhibition of lipolysis in isolated fat cells, from human or rodent origin. in addition, benzylamine, methylamine, or other ssao substrates, activate adipocyte differentiation in several preadipocyte lineages and therefore partly reproduce the adipogenic action of insulin. lastly, in vitro experiments showed that the hydrazine derivative phenelzine (which inhibits ssao) alters the adipocyte differentiation of cultured human and mouse preadipocytes. we therefore hypothesized that endogenous or dietary amines may reproduce in vivo such anabolic insulin - like effects, and if the amines can reach wat, any sustained pharmacological inhibition of their oxidation could hamper fat deposition. in this context, it appeared essential to verify whether inhibition of fat mass extension was an important issue occurring during the body weight gain reduction induced by the above - mentioned agents, all of them sharing the property to inhibit ssao. this prompted us to further test whether the per os administration of the prototypical ssao inhibitor, semicarbazide, was limiting fat deposition. the following results show that, when given in the drinking water, semicarbazide not only inhibited the ssao activity in wat, but also limited food consumption, and to a larger extent, hampered fat accretion in both visceral and subcutaneous fat depots. moreover, the prevention of body weight gain observed during oral administration of semicarbazide was not accompanied by any worsening of the plasma levels of metabolites or oxidative stress markers. finally, since semicarbazide oral toxicity has been recently suspected [2123 ], the need for further investigations of the putative antiobesity effects of other ssao inhibitors is discussed. fvb / n male mice (charles river, l'arbresle, france) were separated in two groups of 8 mice with equivalent body weight at the age of 5 weeks. they were housed at 2 animals per cage with unlimited access to standard rodent chow (global rodent diet, harlan, france) and water (control), or to a semicarbazide solution (semicarbazide - drinking). semicarbazide hydrochloride (sigma - aldrich, saint quentin fallavier, france) was dissolved in drinking water and given as a 0.125% solution that was changed weekly. body mass, food, and water consumption were checked weekly, and at the end of an 8-week treatment period, the mice were sacrificed after overnight fasting. ten other 10-wk - old fvb / n mice grown under standard conditions were used for tissue distribution study of ssao activity and preliminary lipolytic studies. housing conditions and experimental procedures followed in the ifr 150 animal unit were in accordance with the european union regulations on the use of animals for scientific research. once obtained, plasma was immediately frozen at 80c, and circulating metabolites (glucose, insulin, triglycerides, fatty acids, etc.) were determined using a cobas - mira + multi - analyser, according to the manufacturer ' instructions (roche, neuilly, france). once weighed, the perirenal, retroperitoneal, and epididymal white adipose tissues were pooled (and named as visceral wat), immediately digested to obtain adipocyte preparations for glucose uptake assays and lipolysis measurements, as previously described, or for cell size determination under microscope using lucia g software (nikon). portion of the tissues were also frozen for further determinations of dna and protein contents and for amine oxidase assays. dna content was assessed in wat after proteinase k digestion, chloroform / ethanol extraction, and 260/280 nm spectrophotometric readings. protein content was determined using dc protein assay kit (biorad, hercules, ca). freshly isolated adipocytes were diluted in around 10-fold their volume of krebs - ringer containing 15 mm sodium bicarbonate, 10 mm hepes, 2 mm pyruvate, and 3.5% serum bovine albumin. then 400 l of cell suspension was distributed into plastic incubation vials and incubated 45 min at 37c with the tested agents, just before 10 min exposure to 0.1 mm [h]-2-deoxyglucose (2-dg). separation between extracellular and internalized hexose was performed on 200 l aliquots by centrifugation through dinonyl - phthalate layer which allowed to separate buoyant intact fat cells from medium, as previously described. 2-dg uptake was expressed as fold increase over basal uptake or even as percentage of maximal response to insulin, with basal uptake set at 1 or at 0%, respectively. oxidative deamination was measured by extracting the oxidation products of 0.1 mm [c]-benzylamine (from amersham biosciences, buckinghamshire, uk) or 0.5 mm [c]-tyramine (from sigma - aldrich, st quentin fallavier, france) after incubation for 30 min in 200 l of 200 mm phosphate buffer, ph 7.5, in the presence of protease inhibitors and approx. 100 g protein of homogenates prepared just before assays from thawed tissues, as previously described. 15-min preincubation with 1 mm semicarbazide or 0.5 mm pargyline was used to selectively inhibit ssao or mao activity, respectively, as previously reported. real - time quantitative rt - pcr was performed using oligonucleotide primers specific for the indicated genes, designed with primer express software (perkin - elmer life sciences, courtaboeuf, f), and the sequences of which are reported in, for mao - a, mao - b, aoc2, and aoc3, or in for adipokines, or either given as supplemental data to. total rnas were extracted from mature adipocytes, using rneasy minikit, then reverse - transcribed using random hexamers and superscript ii reverse transcriptase (invitrogen, cergy - pontoise, f). reactions without reverse transcriptase (rt-) were performed in parallel to estimate dna contamination. real - time rt - pcr was performed using the primers in the presence of 6.25 ng cdna and sybr green universal pcr mix (eurogentec, angers, f). fluorescence was analysed in an abi prism 7500 sequence detection device (taqman, applied biosystems, foster city, ca). analysis of 18 s rna was performed in parallel using the ribosomal rna control taqman assay kit (applied biosystems) to normalize gene expression as already reported [25, 26 ]. for each gene, results were expressed as arbitrary units : 2 (1 1/(2)) 10, where ct corresponds to the number of cycles reaching fluorescence threshold. comparisons between semicarbazide - treated and control groups were determined using an unpaired student 's t - test. table 1 shows that the ssao activity was higher in adipose depots than in any other studied organ. as previously reported, the oxidation of the prototypic ssao substrate benzylamine was inhibited by semicarbazide in all the homogenates. however, the visceral wat of fvb / n mouse was richer in ssao activity than the subcutaneous wat and than the interscapular brown fat. aorta, duodenum, and kidney exhibited a lower capacity to oxidize benzylamine than the adipose depots. n mice exhibits a rapid growth after weaning, which accounts for a week increase of about 10% of their body mass. we have therefore tested whether semicarbazide oral treatment could hamper adipose tissue development during such period of intense anabolism and substantial fattening. to this aim, semicarbazide was administered at 125 mg/100 ml in the drinking water of 5-week - old mice. since changes in body mass and in water consumption occurred between the beginning and the end of the 8-week treatment, the ingestion of such dose of semicarbazide was calculated to reach a mean daily intake comprised between 123 and 150 mg / kg / day (equivalent to 11001350 mol / kg / d). this range is similar to the dose used in toxicology studies showing no signs of any distress and is lower than the oral ld50 stated in mouse (225 mg / kg, available at http://www.carl-roth.de/jsp/en-de/sdpdf/4681e.pdf). an immediate decline was detected in the growth curve after the first week of treatment, while retardation persisted throughout the treatment (figure 1). at the end of the treatment, the semicarbazide - drinking mice weighed 15% less than their control, since body weights were 24.7 0.6 and 29.1 0.6 g, respectively (the subsequent lowered body weight gain found after 8 weeks of semicarbazide ingestion (2.4 0.5 g) represented only one third of that observed in the control group (6.9 0.7 g, n = 8, p <.001). it was concomitant with a significant reduction in the cumulative water and food consumption, observed during the same period (equivalent to a 29% and 12% reduction, resp.. a clear reduction of adiposity was found in the semicarbazide - drinking mice, at least when comparing the mass of the epididymal, perirenal, retroperitoneal, and inguinal fat pads (figure 2). since the body weight was lower in the semicarbazide - treated mice, it was more convenient to compare the adiposomatic index between the two groups, that is, the percentage of the sum of dissected fat depots relative to body weight. this index fell from 5.8 0.3% to 4.0 0.3% (n = 8, p <.001), corresponding to a 31% reduction. the mass of the interscapular brown fat pad was also reduced by approximately 20% in semicarbazide - drinking mice : 82 4 versus 103 6 mg in control (p <.01). first, the determination of adipocyte diameter distribution clearly showed a fall in the proportion of the larger adipocytes, together with an increase in the proportion of small fat cells (figure 3(a)). this resulted in a significant decrease of the mean fat cell size (figure 3(b)), reflecting an impaired lipid accumulation inside the fat cells. second, the dna content per 100 mg tissue was greater in wat from semicarbazide - drinking mice than in control, indicating that more numerous and smaller cells were present per unit of tissue mass in the treated than in the control animals, the fat depots of which contained larger, heavier, lipid - laden adipocytes (figure 3(c)). these two approaches, together with the lack of increased adipogenic markers (see below), indicated that the wat of semicarbazide - drinking mice partially escaped to the age - dependent lipid accumulation and contained adipocytes having a reduced cell size / mass when compared with age - matched control mice. moreover, the maximal lipolytic activity of the adipocytes was elevated in the semicarbazide - treated group : in fat cells isolated from epididymal plus retroperitoneal and perirenal fat pads (pooled as visceral wat, to obtain sufficient amount of biological material for the various determinations), the glycerol released in response to 10 m isoprenaline was 3.14 0.72 versus 1.90 0.20 mol/100 mg lipids/90 min in control (n = 4, p <.05). such observation supported that these small adipocytes were more metabolically active than larger fat cells. therefore, chronic semicarbazide ingestion clearly appeared to prevent adipocyte hypertrophy and to facilitate lipid mobilization. of worth was to investigate whether the insulin - like effects of amines were abolished in fat cells after ssao blockade. the submaximal stimulation of deoxyglucose uptake by 10 nm insulin reached 2.9 0.3-fold increase over basal in control adipocytes, while it was equivalent to a 4.5 0.5-fold increase in semicarbazide - drinking mice (n = 8, p <.01). again, the smaller fat cell size of semicarbazide - treated mice likely accounted for such enhanced insulin responsiveness. on the opposite, the insulin - like effect of benzylamine was dramatically impaired in the semicarbazide - treated mice, especially when the ssao substrate was tested at 0.1 mm in the presence of 0.1 mm vanadium (figure 4). the combination of vanadium and benzylamine produced more than 80% of the maximal insulin effect in control cells, as previously reported, whereas this effect did not exceed 25% after semicarbazide treatment. similarly, the weak effect of benzylamine alone on glucose uptake also exhibited a tendency to be reduced in semicarbazide - treated mice (not shown). it was then verified whether ssao activity was really blocked in wat after semicarbazide treatment. as shown in figure 5(a), the oxidation of benzylamine was totally abolished in the subcutaneous wat of semicarbazide - drinking mice. this was in agreement with the fact that, in control wat, benzylamine oxidation was exclusively ssao - dependent. the remaining tyramine oxidation found after semicarbazide treatment was explained by an unaltered mao - dependent oxidation together with an almost total disappearance of the ssao component (figure 5(b)). a similar obliteration of ssao activity was found in visceral wat after semicarbazide treatment : the oxidation of 0.1 mm benzylamine fell from 2.74 0.28 to 0.06 0.02 nmol / mg protein / min (n = 6, p <.001). the total blockade of ssao activity obtained after semicarbazide treatment was not accompanied by any alteration of mao activity. this was supported by the lack of change in the expression of genes encoding for the monoamine oxidases a and b in adipocytes (table 2). similarly, there was no change for aoc2 gene, which is poorly expressed in wat. more surprising was the decline in aoc3 expression, the gene encoding for ssao, traducing that prolonged semicarbazide treatment did not only inhibit the ssao enzymatic activity but also reduced its expression in wat. to further analyse the changes occurring in adipose tissue after semicarbazide treatment, the expression of several genes was quantified in adipocytes from visceral wat. as expected, the leptin expression was strongly inhibited in adipocytes from the smaller fat pads found in the semicarbazide - treated group (table 2). however, no noticeable change in the expression of other adipokines was found, irrespective of their detrimental (tnf, il-6, resistin) or beneficial (adiponectin) effect on insulin resistance, with the exception of apelin, the mrna abundance of which exhibited a 50% reduction that failed to reach statistical significance (p <.1). regarding the genes involved in energy supply to adipocytes, there was no clear change in lpl or fas expression (table 2). the expression of the ubiquitous glucose transporter glut1 was reduced, but not that of the insulin - sensitive glut4. lastly, the expression of the adipogenic markers ap2 and ppar2 genes was unchanged in the semicarbazide - treated group (they were equivalent to 77% and 93% of the levels found in control, resp.) while a tendency to decrease was found for the macrophage marker f4/80 (not shown). taken together, these last observations argued that the increased dna content per mass unit of wat was not due to an increased adipogenesis or to an infiltration of immune cells but was rather the consequence of the reduced size of the adipocytes : being of smaller mass, they were in higher number per 100 mg tissue than in control, the wat of which was constituted with larger fat cells. regarding the adverse effects that might be provoked by semicarbazide prolonged ingestion, no change was found at the plasma level for the circulating values of glucose, insulin, triglycerides, free fatty acids, or cholesterol (not shown). similarly, the plasma markers of lipid peroxidation did not differ between control and semicarbazide - drinking mice (malonyldialdehyde equivalents were 23.9 2.0 and 22.5 3.2, resp. the present study highlights decrease in both body weight gain and fattening as marked consequences of semicarbazide subchronic ingestion. the total ssao blockade found in wat, together with the decreased lipogenic effect of benzylamine, which can be considered as one representative of the dietary amines reaching visceral fat during the digestion process, led us to propose that pharmacological inhibition of ssao can be considered as a way to mitigate obesity. since ssao is largely expressed in fat depots, a peripheral mode of action can be envisaged, but other central mechanisms, participating to the observed 12% reduction of calorie intake can not be excluded. the clear - cut blockade of benzylamine oxidation found in the wat of treated mice agrees with the fact that benzylamine oxidation is almost exclusively catalyzed by ssao activity in this tissue. as a consequence, the insulin - like action of the ssao substrate benzylamine on glucose transport was abolished in adipocytes from semicarbazide - treated mice. this was concomitant with substantial reductions in the fat mass and in the mean fat cell size. whether the decrease in ssao expression was a consequence of fat cell size reduction appears unlikely since the shrinking of lipid stores observed during prolonged fasting is not accompanied by a reduction of ssao activity in adipose depots. on the opposite, on the basis of these previous observations, it can be proposed that inhibition of lipogenic effects of endogenous / dietary ssao substrates participates to the limitation of fat deposition. the reduced number of large adipocytes together with the increased dna content per wat mass and the unchanged adipocyte differentiation markers (ap2, ppar2), indicated that the differentiated adipocytes already present at the beginning of treatment, at the age of 5 weeks, were less capable to accumulate lipids in semicarbazide - drinking mice than in the control group. the resulting fat cells of moderate size were present in a larger number per mass unit and consequently gave higher dna richness per mass unit of wat, higher lipolytic capacity, and higher insulin responsiveness. in this view, the inhibition of peripheral ssao, which is abundant in fat cells [15, 25 ], but which is not expressed in neurones, may represent a fascinating novel approach to modulate wat development. such paradigm is relevant only whether endogenous or dietary amines spontaneously exert insulin - like effects, which is far from being demonstrated. indeed, it is widely accepted that numerous amines are spontaneously present in foods [30, 31 ] or may be generated during digestion process by the intestinal flora : for instance, tyramine is present at around 45 mg / kg in rodent chow and benzylamine reaches around 310 mg / kg in edible vegetables. however, it has never been demonstrated (or ruled out) whether such dietary amines might exert in vivo the lipogenic effects they exhibit in vitro, and so facilitate fat accretion once ingested. moreover, ssao is not only expressed in fat cells but also in vessels, in endothelial cells, where it is involved in leukocyte extravasation, and in smooth muscle cells, where it is involved in glucose transport and in collagen - elastin maturation [2, 3 ]. the tendency to decrease the f4/80 macrophage marker level found in mice after semicarbazide exposure is probably related to the anti - inflammatory properties of ssao / vap-1 blocking agents. whether the improved in vitro responsiveness to insulin in the adipocytes from semicarbazide - drinking mice is a consequence of such reduced low - grade inflammation or is related to the smaller size of fat cells remains to be determined. in the treated adipocytes, there was no change in the expression of tnf and il-6, two cytokines involved in insulin resistance, together with a decrease of the proinflammatory adipokine leptin and a tendency to lower the expression of apelin, another adipokine recently shown to facilitate glucose utilization. such absence of enhanced wat inflammation was associated with an absence of oxidative stress (evidenced by unchanged lipid peroxidation markers or catalase content). this context may explain the increased ex vivo insulin responsiveness of adipocytes, which was not sufficient to fill the fat cells of semicarbazide - drinking mice via sustained lipogenic pathways, since it was occurring together with a reduced calorie intake. of interest is to test whether the insulin - sensitizing action of ssao blockade also occurs acutely in obese insulin - resistant models, and improve the insulin - dependent and independent antilipolytic responses, since adipose tissue lipolysis regulation has been proposed to be instrumental in the treatment of obesity and metabolic syndrome. the major concern is that semicarbazide itself can not currently be proposed as antiobesity drug since this derivative has been described to exert deleterious toxic effects. in 2003, the european food safety authority has banned the use of azodicarbonamide as a blowing agent for plastics used in food processing or packaging, or as a flour treatment, since it can be transformed into semicarbazide, which is suspected to be a food contaminant exerting toxicological effects on consumers. however, this hydrazine derivative is not only a food contaminant, the origin of which is limited to the transformation of known chemical substances used (or even prohibited) in food processing, but can also naturally occur in certain foods [38, 39 ]. as a consequence, the dna damaging effects of ingested semicarbazide have been intensively studied to bring health risk assessments. but these recent toxicological studies inconsistently demonstrated the genotoxic properties of semicarbazide, since they raised variations according to the model and the protocol used. briefly, semicarbazide has very poor genotoxic effects on human lymphocytes when tested in vitro and is not genotoxic in mice when administered in vivo at 120 mg / kg b.w.. however semicarbazide exhibits carcinogenic potential with marginal statistical relevance when orally given to rats at 150 mg / kg. of note, other aspects of semicarbazide toxicity have also been stated very recently and have produced more consensual results. indeed, a toxicological study showed a dramatic decrease in body weight with orally given semicarbazide as mixed in the food at doses up to 1000 ppm, giving a daily intake of 65 to 70 mg / kg in male and female rats. however, the relative weights of the brain, heart, and kidneys were unchanged or even increased, depending on the sex, while body weights were clearly lower in the semicarbazide - treated rats. this may indicate that while semicarbazide ingestion limits wat expansion, as assessed in our conditions, other organs are preserved during the body weight loss. unfortunately, exposure to semicarbazide was accompanied by serious adverse effects such as deformation of bones and lesions of articular cartilages. in the aorta, alteration of connective tissues was suspected to increase fragility of arterial walls. nonetheless, no inflammatory disease was found. in view of these toxicological effects of chronic exposure to semicarbazide, the no observed adverse effect level (noael) was estimated by the authors to be lower than 1821 mg / kg / day in rat. accordingly, the noael in juvenile rats on another strain has recently been proposed to be lower than 40 mg / kg for semicarbazide oral administration. in this study describing the toxicity of semicarbazide on biochemical and behavioural parameters, the authors observed a clear - cut inhibition of body weight gain and defects in cartilage mineralization. during the completion of the present work, the same italian group reported that semicarbazide behaves as an endocrine disrupter, being able to reduce estrogen levels or to alter testosterone catabolism. anyhow, semicarbazide inhibited body weight gain and food consumption in postweaning male and female rats when orally given from the dose of 40 to 140 mg / kg / d period. thus, semicarbazide administration is univocally recognized to reduce body weight gain in diverse obese or nonobese models [2, 3, 7, 8, 21 ] and regarding this aspect our findings are confirmatory. our detailed analysis of adiposity of juvenile mice, together with the high expression of ssao in adipocytes, brings novel insight, focusing the attention on wat as a major target of semicarbazide. however, this hydrazine derivative can not be safely used as a slimming agent for toxic issues in bones, joints, and vessels. although other ssao inhibitors have also been reported to limit body weight gain, several semicarbazide effects other than inhibiting ssao will be discussed here in an attempt to detect whether ssao blockade is necessary and/or sufficient to limit fat deposition. another well - recognized effect of semicarbazide is to inhibit lysyl oxidase, a copper - containing amine oxidase involved in the stabilization of extracellular matrix by cross - linking of proteins such as collagen and elastin [2, 3 ], therefore explaining part of the semicarbazide toxic effects on bones and joints. however, the lysyl oxidase inhibitor -aminopropionitrile, reported to decrease insoluble elastin and collagen content and to impair collagen cross - linking does not decrease body weight gain, excepted when combined with semicarbazide. semicarbazide has been reported to inhibit glutamic acid decarboxylase, a -amino butyric acid - synthesizing enzyme [41, 42 ], and its administration is therefore believed to impair -amino butyric acid (gaba) formation and to lower gaba brain levels. this has been proven to induce freezing behaviour in rodents after central administration. however, it is striking to note that relatively high doses of gamma - vinyl gaba, an irreversible inhibitor of the enzyme gaba - transaminase, which is involved gaba catabolic pathway, have been recently reported to produce significant weight loss in rats. likewise, another inhibitor of the gaba degradation exhibits anorectic potency in obese rats, while it has been proposed that a rat strain resistant to high - fat diet feeding reduces its food intake via an elevation of brain gaba levels. in fact, it is difficult to understand how the chronic in vivo administration of semicarbazide, supposed to inhibit gaba synthesis, alters the gaba - ergic systems in a manner that results in reduced food intake, as it is the case with inhibitors of gaba degradation. to pour more complexity, it has been recently reported that several semicarbazide derivatives elevate gaba levels in the midbrain and exert anticonvulsant activity. it could be therefore of interest to determine the relative proportion of the central gaba - ergic alterations and of the peripheral ssao blockade in the slimming action of semicarbazide before establishing whether more selective ssao blockers, devoid of interaction with the gaba - ergic system, also elicit antiobesity effects. otherwise, the current number of ssao - interacting agents that have been also reported to reduce weight gain (fpfa, aminoguanidine, phenelzine) suggests a nonnegligible influence of the component related to ssao inhibition in the observed effect of semicarbazide. semicarbazide has also been described to inhibit sphingosine-1-phosphate (s1p) lyase, but it is not clear whether such nonspecific inhibition may have some bearing on our observations. as in the case of gaba - ergic system, it must be quoted that novel semicarbazide derivatives act differently from semicarbazide itself, since instead of inhibiting a s1p - degrading enzyme, they are selective inhibitors of a subset of the g protein - coupled s1p receptors (s1p2). this emphasizes that semicarbazide is a molecule with multiple aspects, each one being selectively developed by the association of a functional and selective moiety to the semicarbazide turntable structure. such drug design is out of the scope of the present study, while ssao inhibition, obtained by the various above - mentioned pharmacological agents was always found to be concomitant with a decreased body weight gain. the need for searching novel, selective ssao inhibitors is not only a concern for our proposed antiobesity pharmacology, but also relies with the general biology of the enzyme, which will soon change of denomination, from ssao to primary amine oxidase, a fact indicating that its definition based on its sensitivity to semicarbazide will become historical once the many other inhibitors under development will be fully characterized. taken together, our results demonstrate that oral administration of the prototypical ssao inhibitor semicarbazide limits weight gain and fat deposition in mouse. in spite of the toxicity of the high doses of this agent, the use of other specific ssao inhibitors might reveal more beneficial for the pharmacologic treatment of obesity.	an enzyme hitherto named semicarbazide - sensitive amine oxidase (ssao), involved in the oxidation of primary amines, is abundantly expressed in adipocytes. although ssao physiological functions remain unclear, several molecules inhibiting its activity have been described to limit fat accumulation in preadipocyte cultures or to reduce body weight gain in obese rodents. here, we studied whether oral administration of semicarbazide, a prototypical ssao inhibitor, limits fat deposition in mice. prolonged treatment with semicarbazide at 0.125% in drinking water limited food and water consumption, hampered weight gain, and deeply impaired fat deposition. the adiposomatic index was reduced by 31%, while body mass was reduced by 15%. such treatment completely inhibited ssao, but did not alter mao activity in white adipose tissue. consequently, the insulin - like action of the ssao substrate benzylamine on glucose transport was abolished in adipocytes from semicarbazide - drinking mice, while their insulin sensitivity was not altered. although semicarbazide is currently considered as a food contaminant with deleterious effects, the ssao inhibition it induces appears as a novel concept to modulate adipose tissue development, which is promising for antiobesity drug discovery.
bioprostheses do not require a lifelong anticoagulation as opposed to mechanical prostheses and offer a satisfactory haemodynamic profile. this is not only due to an increasingly older patient cohort, where biological prostheses are favoured [1, 2 ]. the higher durability enabled by improved anticalcification treatment and other optimizations of valve design led to a higher implantation rate in a younger patient population. the two most commonly implanted biological prostheses are either stent - mounted native porcine aortic heart valves or bovine pericardial valves. they have undergone modifications in design during the past decades to optimize haemodynamic performance and prolong durability. pericardial prostheses are credited to have a favourable haemodynamic profile compared with porcine valves [6, 7 ]. however, the impact of improved postoperative transvalvular gradients on outcome is still a matter of debate. furthermore, high transvalvular gradients in porcine heart valves seem to be at least partly caused by an echocardiographic phenomenon. the aim of the current study was to compare two commonly implanted bioprostheses at a single centre in a real - world setting. the medtronic mosaic porcine biological aortic heart valve and the carpentier - edwards magna pericardial aortic bioprosthesis were analysed regarding survival, reoperation rate, valve - related complications and echocardiographic data. data of all consecutive patients undergoing isolated aortic valve replacement with either a carpentier - edwards magna pericardial prosthesis (edwards lifesciences, irvine, ca, usa) or a medtronic mosaic porcine prosthesis (medtronic, st paul, mn, usa) during the same time period between 2002 and 2008 at a university hospital were prospectively collected and analysed. the surgeon made the decision regarding the type of valve prosthesis according to his or her preference independent of this analysis. there were no strict rules established to guide the decision to one or the other prosthesis. all patients with concomitant surgical procedures except of root and/or annular enlargement were excluded from this analysis. patients ' characteristics and risk factors were documented prospectively in the electronic documentation system of our institution. risk scores [additive and logistic european system for cardiac operative risk evaluation (euroscore) ] were calculated and stored. the follow - up was performed in accordance with the current guidelines for reporting mortality and morbidity after cardiac valve interventions. all postoperative hospitalizations and outpatient visits in public hospitals of the same city were assessed. the databank 's closing interval was from mid - february 2013 to end of march 2013 (6 weeks). furthermore, a second databank search to update survival and adverse event information for final revision was performed in september 2014. in addition to our follow - up, overall mortality was yearly crosschecked with the countrywide database maintained by the national statistical institute (statistics austria, vienna, austria). all deaths in austria are registered in the database with the full name, date of birth and date of death. every austrian citizen who was operated at our department and died thereafter in austria could be identified. therefore, the follow - up for survival is considered as complete except for foreign patients or patients who left the country. the follow - up time ranged from 0 to 11 years in the mosaic group and from 0 to 12 years in the magna group, with a mean time of survival follow - up of 6 3 years for both valves (p = 0.61). reoperations including interventional valve - in - valve procedures were recorded and categorized into reoperations for structural valve disease, non - structural valve disease, valve thrombosis and endocarditis. furthermore, valve - related adverse events including stroke, transient ischaemic attack, peripheral emboli, endocarditis, valve thrombosis, bleeding and myocardial infarction were assessed during the follow - up. ten percent of patients were lost to the follow - up for valve - related complications after the early postoperative period with no significant difference between valve types (p = 0.90). the follow - up time ranged from 0 to 11 years in the mosaic group and from 0 to 12 years in the magna group. the mean time (total amount) of morbidity follow - up was 4 3 (1158) years in the magna group and 4 3 (626) years in the mosaic group. at least one echocardiographic follow - up with a mean time from surgery to echo of 9 12 months could be performed in 42% of this population. we calculated the projected effective orifice area index (eoai) according to the implanted valve size using previously published effective orifice area measures and the actual body surface area. the test was performed to analyse the frequencies of binary outcomes between treatment groups. continuous variables were presented as mean and standard deviations and compared by the independent samples t - test between valve types. the kaplan meier method with a log - rank test was performed to compare survival. further, we calculated the average linearized event rates per patient - year by dividing the observed number of events by the number of follow - up years. cumulative survival of an age- and sex - matched austrian standard population was computed by the life table method, based on age sex - specific mortality data of the year 2005 published online by the austrian federal statistical agency. cumulative survival and 95% confidence intervals (cis) for the study population were computed using the product - limit (kaplan meier) method and compared with the standard population according to the methods outlined in finkelstein.. in particular, a standardized mortality ratio with 95% confidence limits was calculated, which expresses the ratio of observed number of deaths in the study population and expected number of deaths in an age- and sex - matched reference population with an equal follow - up. this was done for the complete follow - up time as well as separately for the first and the subsequent years. for further survival analysis, a multivariable cox regression analysis was performed including variables valve type, year of surgery, log2 of logistic euroscore and demographic variables with a p - value below 0.2 between groups (body size in cm). non - linear effects of year of surgery, body size and logarithm of logistic euroscore were accounted for by b - splines with three degrees of freedom and tested using likelihood ratio tests against a model assuming only linear effects of these variables. interactions of the valve type with these four covariables were tested using likelihood ratio tests as well. finally, the proportional hazards assumption was assessed for all four variables in the model by computing the correlation of scaled schoenfeld residuals with time. the two - sided significance level was set to 5%. the r statistical computing software (version 3.1.1 ; r foundation for statistical computing, vienna, austria) and ibm spss statistics 20 (ibm, armonk, ny, usa) were used for statistical analysis. data of all consecutive patients undergoing isolated aortic valve replacement with either a carpentier - edwards magna pericardial prosthesis (edwards lifesciences, irvine, ca, usa) or a medtronic mosaic porcine prosthesis (medtronic, st paul, mn, usa) during the same time period between 2002 and 2008 at a university hospital were prospectively collected and analysed. the surgeon made the decision regarding the type of valve prosthesis according to his or her preference independent of this analysis. there were no strict rules established to guide the decision to one or the other prosthesis. all patients with concomitant surgical procedures except of root and/or annular enlargement were excluded from this analysis. patients ' characteristics and risk factors were documented prospectively in the electronic documentation system of our institution. risk scores [additive and logistic european system for cardiac operative risk evaluation (euroscore) ] were calculated and stored. the follow - up was performed in accordance with the current guidelines for reporting mortality and morbidity after cardiac valve interventions. all postoperative hospitalizations and outpatient visits in public hospitals of the same city were assessed. the databank 's closing interval was from mid - february 2013 to end of march 2013 (6 weeks). furthermore, a second databank search to update survival and adverse event information for final revision was performed in september 2014. in addition to our follow - up, overall mortality was yearly crosschecked with the countrywide database maintained by the national statistical institute (statistics austria, vienna, austria). all deaths in austria are registered in the database with the full name, date of birth and date of death. every austrian citizen who was operated at our department and died thereafter in austria could be identified. therefore, the follow - up for survival is considered as complete except for foreign patients or patients who left the country. the follow - up time ranged from 0 to 11 years in the mosaic group and from 0 to 12 years in the magna group, with a mean time of survival follow - up of 6 3 years for both valves (p = 0.61). reoperations including interventional valve - in - valve procedures were recorded and categorized into reoperations for structural valve disease, non - structural valve disease, valve thrombosis and endocarditis. furthermore, valve - related adverse events including stroke, transient ischaemic attack, peripheral emboli, endocarditis, valve thrombosis, bleeding and myocardial infarction were assessed during the follow - up. ten percent of patients were lost to the follow - up for valve - related complications after the early postoperative period with no significant difference between valve types (p = 0.90). the follow - up time ranged from 0 to 11 years in the mosaic group and from 0 to 12 years in the magna group. the mean time (total amount) of morbidity follow - up was 4 3 (1158) years in the magna group and 4 3 (626) years in the mosaic group. at least one echocardiographic follow - up with a mean time from surgery to echo of 9 12 months could be performed in 42% of this population. we calculated the projected effective orifice area index (eoai) according to the implanted valve size using previously published effective orifice area measures and the actual body surface area. in addition to our follow - up, overall mortality was yearly crosschecked with the countrywide database maintained by the national statistical institute (statistics austria, vienna, austria). all deaths in austria are registered in the database with the full name, date of birth and date of death. every austrian citizen who was operated at our department and died thereafter in austria could be identified. therefore, the follow - up for survival is considered as complete except for foreign patients or patients who left the country. the follow - up time ranged from 0 to 11 years in the mosaic group and from 0 to 12 years in the magna group, with a mean time of survival follow - up of 6 3 years for both valves (p = 0.61). reoperations including interventional valve - in - valve procedures were recorded and categorized into reoperations for structural valve disease, non - structural valve disease, valve thrombosis and endocarditis. furthermore, valve - related adverse events including stroke, transient ischaemic attack, peripheral emboli, endocarditis, valve thrombosis, bleeding and myocardial infarction were assessed during the follow - up. ten percent of patients were lost to the follow - up for valve - related complications after the early postoperative period with no significant difference between valve types (p = 0.90). the follow - up time ranged from 0 to 11 years in the mosaic group and from 0 to 12 years in the magna group. the mean time (total amount) of morbidity follow - up was 4 3 (1158) years in the magna group and 4 3 (626) years in the mosaic group. at least one echocardiographic follow - up with a mean time from surgery to echo of 9 12 months could be performed in 42% of this population. we calculated the projected effective orifice area index (eoai) according to the implanted valve size using previously published effective orifice area measures and the actual body surface area. descriptive statistical methods were applied to depict the study population regarding preoperative risk factors. the test was performed to analyse the frequencies of binary outcomes between treatment groups. continuous variables were presented as mean and standard deviations and compared by the independent samples t - test between valve types. the kaplan meier method with a log - rank test was performed to compare survival. further, we calculated the average linearized event rates per patient - year by dividing the observed number of events by the number of follow - up years. cumulative survival of an age- and sex - matched austrian standard population was computed by the life table method, based on age sex - specific mortality data of the year 2005 published online by the austrian federal statistical agency. cumulative survival and 95% confidence intervals (cis) for the study population were computed using the product - limit (kaplan meier) method and compared with the standard population according to the methods outlined in finkelstein.. in particular, a standardized mortality ratio with 95% confidence limits was calculated, which expresses the ratio of observed number of deaths in the study population and expected number of deaths in an age- and sex - matched reference population with an equal follow - up. this was done for the complete follow - up time as well as separately for the first and the subsequent years. for further survival analysis, a multivariable cox regression analysis was performed including variables valve type, year of surgery, log2 of logistic euroscore and demographic variables with a p - value below 0.2 between groups (body size in cm). non - linear effects of year of surgery, body size and logarithm of logistic euroscore were accounted for by b - splines with three degrees of freedom and tested using likelihood ratio tests against a model assuming only linear effects of these variables. interactions of the valve type with these four covariables were tested using likelihood ratio tests as well. finally, the proportional hazards assumption was assessed for all four variables in the model by computing the correlation of scaled schoenfeld residuals with time. the two - sided significance level was set to 5%. the r statistical computing software (version 3.1.1 ; r foundation for statistical computing, vienna, austria) and ibm spss statistics 20 (ibm, armonk, ny, usa) were used for statistical analysis. the medtronic mosaic was implanted in 163 patients (35.6%) and the carpentier - edwards magna in 295 patients (64.4%). the mean implanted valve size was 22.4 1.5 mm for the medtronic mosaic, which was significantly larger than the carpentier - edwards magna size (21.8 1.8 mm ; procedural characteristics including cross - clamp time and intraoperative risk factors were comparable (table 2). table 1:preoperative patient characteristicsfactorporcinepericardialp - valueage (years)74 873 90.70sex (f / m)93 (57%)/70 (43%)160 (54%)/135 (46%)0.56height (cm)167 9166 120.095weight (kg)77 1478 180.75body surface area (m)1.92 0.211.91 0.230.69nyha iii and iv80 (62%)133 (63%)0.82additive euroscore8 37 30.15logistic euroscore12 1210 100.21ejection fraction > 50% (%) 104 (72%)202 (75%)0.64heart rate (bpm)70 1572 130.35systolic blood pressure (mmhg)131 24131 220.99diastolic blood pressure (mmhg)69 1371 160.34fvc (l)2.7 0.92.7 0.80.76fev1 (%) 86.1 23.887.7 23.60.64haemoglobin (g / dl)12.8 1.513.0 1.80.24platelets (g / l)228 73227 690.98creatinine (mg / dl)1.2 0.81.2 0.70.62mean preoperative gradient (mmhg)61 2259 240.42porcine : medtronic mosaic ; pericardial : carpentier - edwards magna ; continuous data are presented as the mean standard deviation ; categorical data as total number and percentage ; bpm : beats per minute ; fvc : forced vital capacity ; fev1 : forced expiratory volume in 1 second ; euroscore : european system for cardiac operative risk evaluation. table 2:procedural specifications and early follow - upfactorporcinepericardialp - valueduration of anaesthesia (min)274 57275 640.84cross - clamp time (min)60 1957 140.12red blood cell units (packs)2.5 1.52.8 2.20.28valve size (mm)22.4 1.521.8 1.80.001revision for bleeding7 (4.3%)21 (7.1%)0.23early mortality5 (3.1%)8 (2.7%)0.83porcine : medtronic mosaic ; pericardial : carpentier - edwards magna ; continuous data are presented as the mean standard deviation ; categorical data as total number and percentage. preoperative patient characteristics porcine : medtronic mosaic ; pericardial : carpentier - edwards magna ; continuous data are presented as the mean standard deviation ; categorical data as total number and percentage ; bpm : beats per minute ; fvc : forced vital capacity ; fev1 : forced expiratory volume in 1 second ; euroscore : european system for cardiac operative risk evaluation. procedural specifications and early follow - up porcine : medtronic mosaic ; pericardial : carpentier - edwards magna ; continuous data are presented as the mean standard deviation ; categorical data as total number and percentage. early mortality was 2.8% and comparable between groups (table 2). during the follow - up, 54 (33%) of porcine valve patients and 100 (34%) of pericardial valve patients died (p = 0.87). the long - term survival rate was 76 and 56% after 5 and 10 years for the medtronic mosaic, which was comparable with the carpentier - edwards magna (77 and 57% ; p = 0.92). multivariable cox regression analysis revealed significant association with survival of the logistic euroscore [hazard ratio (hr) = 1.67 per doubling, 95% ci : 1.451.91, p 0.65 cm / m) and severe prosthesis patient mismatch (eoai 0.65 cm / m) was significantly worse for the medtronic mosaic prosthesis (moderate : 57 vs 19% ; severe : 2 vs 0% ; p < 0.001). blue : porcine valve ; green : pericardial valve ; a smoothing line for each group was obtained by a local regression function. blue : porcine valve ; green : pericardial valve ; a smoothing line for each group was obtained by a local regression function. herein, we present a single - centre, direct, non - randomized comparison of two bioprostheses currently implanted in the majority of surgical aortic valve replacements in an elderly patient cohort. in contrast to other recent publications, we evaluated a very distinct patient group, which was limited to isolated aortic valve replacements [1315 ]. thereby, we avoided possible confounding factors such as concomitant coronary artery bypass grafting or other surgical procedures. this resulted in a higher survival rate compared with the publication by said.. overall long - term survival was equal in both groups of our study. a multivariate cox regression analysis corrected for the potential effect of euroscore and demographic parameters did not show any difference regarding the valve type or year of surgery. moreover, the long - term survival was also comparable with the age- and sex - matched austrian population (fig. 2). our data are inline with previous publications reporting favourable survival with these valves in an elderly population [4, 5, 15, 16 ]. we previously demonstrated that older patient cohorts are more likely to achieve the predicted survival after aortic valve replacement compared with younger patients. the age- and sex - matched standard population represents the expected overall austrian survival for the year 2005. the age- and sex - matched standard population represents the expected overall austrian survival for the year 2005. the observed operative mortality led to a decreased survival compared with the matched population in the first year after surgery for the magna valve, but this effect could be overcome thereafter. a similar number of patients in the mosaic group would have probably also resulted in a significantly decreased survival in the first year. the valve - related complication rate was low and comparable with other reports (table 3). this may reduce the threshold for reinterventions in the near future and avoid death due to structural valve deterioration, which was observed in one of our patients. a recent meta - analysis by yap. described a lower rate of valve - related adverse events for pericardial valves. it may require a higher sample size to find similar differences of adverse events in our population. on the other hand, our report describes only recent and currently available types of pericardial and porcine prostheses, which may have improved results regarding adverse events due to new tissue preservation protocols. the central question, which is always addressed in the literature regarding porcine heart valves, is the presence, cause and effect of high transvalvular gradients [7, 14 ]. as expected, transvalvular gradients were also increased in our postoperative echocardiographic follow - up of the medtronic mosaic valve. a potential explanation may be that the observed differences in gradients as such do not affect long - term outcome in this elderly patient cohort. otherwise, the observed higher gradients measured in the mosaic valve could also be caused by an echocardiographic phenomenon. the concept of this phenomenon, called pressure recovery, was previously published and advocates that the majority of the observed gradient in the medtronic mosaic group is again transferred to aortic pressure after the aortic prosthesis due to the laminar flow pattern. previous publications highlighted the variable labelling of valve sizes and also reported a difference in the inner diameter of size - matched prostheses up to 2 mm [19, 20 ]. therefore, studies comparing different aortic valve prostheses according to the labelled implanted valve size have to be interpreted with caution. our department introduced the medtronic mosaic early and previously published a randomized analysis comparing implanted valve sizes in relation to the real annular diameter measured with a hegar dilator. the implanted carpentier - edwards magna labelled sizes were smaller compared with the medtronic mosaic valve for a standardized annular measurement. not one 19 mm medtronic mosaic valve was implanted in this patient population and the average diameter according to the labelled size was 0.6 mm higher in the medtronic mosaic group. the size of the implanted prosthesis is a major determinant for prosthesis - patient mismatch. a severe mismatch below 0.65 cm / m has been identified as a potential risk factor for long - term mortality. severe prosthesis -patient mismatch may induce turbulent flow in the ascending aorta, which would theoretically diminish the pressure recovery effect. the analyses regarding survival were not included in this paper due to a low number of patients with severe prosthesis patient mismatch. although the follow - up for survival was complete due to the crosscheck with the statistical institute, the follow - up for valve - related adverse events was based solely on a database research and telephone follow - up. restoration of normal life expectancy in elderly patients should not be extrapolated to younger age groups, as death from competing causes obscure valve - related mortality. furthermore, elderly patients accepted for cardiac surgery at a given age may be in a better general health condition compared with the general population. only routine echocardiographic studies without a distinct time schedule therefore, the projected rather than the measured eoai was used for prosthesis patient mismatch grading. although the follow - up for survival was complete due to the crosscheck with the statistical institute, the follow - up for valve - related adverse events was based solely on a database research and telephone follow - up. restoration of normal life expectancy in elderly patients should not be extrapolated to younger age groups, as death from competing causes obscure valve - related mortality. furthermore, elderly patients accepted for cardiac surgery at a given age may be in a better general health condition compared with the general population. therefore, the projected rather than the measured eoai was used for prosthesis patient mismatch grading. in conclusion, both types of aortic bioprostheses offer excellent long - term results as documented by comparison with the austrian standard population. funding to pay the open access publication charges for this article was provided by the department of cardiac surgery, medical university of vienna.	objectivesoutcome of aortic valve replacement may be influenced by the choice of bioprosthesis. pericardial heart valves are described to have a favourable haemodynamic profile compared with porcine valves, although the clinical notability of this finding is still controversially debated. herein, we compared the long - term results of two commonly implanted bioprosthesis at a single centre.methodsall consecutive patients undergoing isolated aortic valve replacement with either a carpentier - edwards magna pericardial prosthesis or a medtronic mosaic porcine prosthesis between 2002 and 2008 were analysed regarding preoperative characteristics, short- and long - term survival, valve - related complications and echocardiographic findings.resultsthe medtronic mosaic was implanted in 163 patients and the carpentier - edwards magna in 295 patients. the sizes of implanted valves were 22.4 1.5 mm for the mosaic and 21.8 1.8 mm for the magna (p = 0.001). the long - term survival rate was 76 and 56% after 5 and 10 years for the medtronic mosaic, which was comparable with the carpentier - edwards magna (77 and 57% ; p = 0.92). overall long - term survival was comparable with an age- and sex - matched austrian general population for both groups. valve - related adverse events were similar between groups. the postoperative mean transvalvular gradient was significantly increased in the mosaic group (24 9 mmhg vs 17 7 mmhg ; p < 0.001).conclusionsboth types of aortic bioprostheses offer excellent results after isolated aortic valve replacement. despite relevant differences in gradients, long - term survival was comparable with the expected normal survival for both bioprostheses. patients with a porcine heart valve had a higher postoperative transvalvular gradient.
invasive pulmonary aspergillosis is extremely difficult to diagnose, and a combination of culture tests, clinical attributes, imaging tests, and serological diagnosis must be used. in approximately 40% of all cases, invasive pulmonary aspergillosis is not clinically diagnosed while the patient is still alive ; it is only discovered during post - mortem examination. the diagnostic criteria for chronic progressive necrotizing pulmonary aspergillosis is defined as follows : (1) it progresses > 1 month with lower respiratory tract symptoms ; (2) the clinical manifestations are exacerbated on new imaging findings ; (3) the exacerbated imaging findings confirm an aspergillus infection serologically or pathologically ; and (4) the condition of the patient on antifungal treatment is inexplicably necessary in terms of general bacterial infection or another disorder ; and (5) elevated inflammatory reactions or advanced lesions attributable to aspergillosis must meet criteria 14 even in the absence of an elevated inflammatory response. invasive pulmonary aspergillosis is normally diagnosed on the basis of the european organization for research and treatment of cancer / invasive fungal infections cooperative group and the national institute of allergy and infectious diseases mycoses study group (eortc / msg) diagnostic criteria for invasive fungal infections. the presence of calcium oxalate in sputum culture samples is regarded as potentially useful in the diagnosis of aspergillus niger and aspergillus fumigatus. a study comparing 65 patients who tested positive for aspergillus and 60 control patients (with or without other types of infection) showed that calcium oxalate crystals are specific markers of an aspergillus infection. however, the value of calcium oxalate in a sputum culture for the diagnosis of pulmonary aspergillosis has very rarely been examined. we treated two patients in whom calcium oxalate crystals in the sputum proved to be a useful clue in the diagnosis of aspergillus. since calcium oxalate in sputum samples aids in diagnosing a. niger and a. fumigatus, which account for most cases of pulmonary aspergillosis, this suggests that it should be added to the diagnostic criteria for all cases of aspergillosis, including a. niger. a 66-year - old man presented to an outlying hospital with complaints of coughing, hemoptysis, and back pain. he had type 2 diabetes managed with kinesitherapy and dietotherapy but without medication. he had no particular contact with any disease agents, no history of owning pets or of traveling north america (los angeles), south america (cancun and mexico). he smoked 40 cigarettes / day and had no history of alcohol use or allergies. he developed chest and back pain around november 8, 20xx (day 7). on november 12 (day 3), he visited a local ear - nose - throat clinic ; leukocytosis and an inflammatory response were detected. on november 15 (day 0), he was referred to our hospital 's emergency department for detailed testing and treatment. after imaging tests were performed, he was admitted for further testing and treatment (table 1). upon arrival, the patient was hyperthermic (38.7 c) with a fast pulse (99 beats / min). he had a ground - glass shadow in the left upper lung field on thoracic plain radiograph (fig. 1), and there was a reverse halo shadow in the left s3 with ground - glass attenuation surrounding the consolidation in s1 and s2 on the thoracic computed tomography (ct) scan (fig. 2). on the basis of the clinical course and imaging findings, the differential diagnosis in case 1 included community - acquired pneumonia, pulmonary mycobacteriosis (tuberculosis or nontuberculous mycobacteriosis), pulmonary mycosis (chronic pulmonary aspergillosis, cryptococcosis, or histoplasmosis), pulmonary nocardiosis, actinomycosis, alveolar cell carcinoma, pulmonary malignant lymphoma, and cryptogenic organizing pneumonia. the samples were tested for the human immunodeficiency virus antibody, -d - glucan, aspergillus and cryptococcus antigens, and sputum cytology. sputum gram stain test was negative ; however, considering the frequency of the community - acquired pneumonia, ceftriaxone (2 g / day) was started on day 1. in addition to the aforementioned tests, that for the aspergillus galactomannan antigen yielded positive results ; however, the sputum smear test (three consecutive expectorations) for mycobacterium yielded negative results, and the sputum culture was negative. no significant findings were detected in the sputum culture or the cytology acquired by bronchoscopy. given the presence of calcium oxalate crystals (fig. 3) as well as the fact that the diagnosis of chronic pulmonary aspergillosis was consistent with the imaging findings, treatment with voriconazole (loading dose : 300 mg intravenously every 12 h on the first day ; subsequent doses : 200 mg intravenously every 12 h) was started on day 8. since the patient 's symptoms improved, as did the blood test results, the medication was switched to oral voriconazole (600 mg / day) from day 16. the patient was discharged on day 23, and oral voriconazole was continued. a 66-year - old man presented to an outlying hospital with complaints of fever and a productive cough. the patient had emphysema and stomach cancer (total gastrectomy). he smoked 20 cigarettes / day for 40 years (until 6 years previously) and drank 350 ml of beer / day. the patient developed a fever, coughing, and expectoration around september 12, 20xx (day 12). emphysematous changes in the upper lobe of the right lung and niveau formation in the bulla were evident, and consolidation was also present in the left upper lung. the patient was monitored as an outpatient but was eventually admitted to our hospital 's department of respiratory medicine on september 26 (day 0) after hemoptysis was observed (table 2). he had ground - glass shadow in the left upper lung field on the thoracic plain radiograph (fig. 4) and consolidation associated with air bronchogram in the left s3 on thoracic computed tomography scan (fig. 5). klebsiella pneumoniae was identified from an initial sputum sample, which was collected at day 12, and treatment with ampicillin / sulbactam (3 g intravenously every 6 h) was started on day 1. however, his symptoms failed to improve, and since the tests for the aspergillus antigen and antibody were both positive on day 10, our department was consulted. the calcium oxalate crystals were also evident in the sputum culture (figs. 6 and 7) ; thus, chronic necrotizing pulmonary aspergillosis was suspected and treatment with voriconazole was started (loading dose : 300 mg intravenously every 12 h on the first day ; subsequent doses : 200 mg intravenously every 12 h). however, the patient 's general condition on admission was poor, and he died on day 14. the study was approved by the ethics committee of kanto rosai hospital, and all the patients provided written informed consent to publish the manuscript. a 66-year - old man presented to an outlying hospital with complaints of coughing, hemoptysis, and back pain. he had type 2 diabetes managed with kinesitherapy and dietotherapy but without medication. he had no particular contact with any disease agents, no history of owning pets or of traveling north america (los angeles), south america (cancun and mexico). he smoked 40 cigarettes / day and had no history of alcohol use or allergies. he developed chest and back pain around november 8, 20xx (day 7). on november 12 (day 3), he visited a local ear - nose - throat clinic ; leukocytosis and an inflammatory response were detected. on november 15 (day 0), he was referred to our hospital 's emergency department for detailed testing and treatment. after imaging tests were performed, he was admitted for further testing and treatment (table 1). upon arrival, the patient was hyperthermic (38.7 c) with a fast pulse (99 beats / min). he had a ground - glass shadow in the left upper lung field on thoracic plain radiograph (fig. 1), and there was a reverse halo shadow in the left s3 with ground - glass attenuation surrounding the consolidation in s1 and s2 on the thoracic computed tomography (ct) scan (fig. 2). on the basis of the clinical course and imaging findings, the differential diagnosis in case 1 included community - acquired pneumonia, pulmonary mycobacteriosis (tuberculosis or nontuberculous mycobacteriosis), pulmonary mycosis (chronic pulmonary aspergillosis, cryptococcosis, or histoplasmosis), pulmonary nocardiosis, actinomycosis, alveolar cell carcinoma, pulmonary malignant lymphoma, and cryptogenic organizing pneumonia. the samples were tested for the human immunodeficiency virus antibody, -d - glucan, aspergillus and cryptococcus antigens, and sputum cytology. sputum gram stain test was negative ; however, considering the frequency of the community - acquired pneumonia, ceftriaxone (2 g / day) was started on day 1. in addition to the aforementioned tests, that for the aspergillus galactomannan antigen yielded positive results ; however, the sputum smear test (three consecutive expectorations) for mycobacterium yielded negative results, and the sputum culture was negative. no significant findings were detected in the sputum culture or the cytology acquired by bronchoscopy. given the presence of calcium oxalate crystals (fig. 3) as well as the fact that the diagnosis of chronic pulmonary aspergillosis was consistent with the imaging findings, treatment with voriconazole (loading dose : 300 mg intravenously every 12 h on the first day ; subsequent doses : 200 mg intravenously every 12 h) was started on day 8. since the patient 's symptoms improved, as did the blood test results, the medication was switched to oral voriconazole (600 mg / day) from day 16. a 66-year - old man presented to an outlying hospital with complaints of fever and a productive cough. he smoked 20 cigarettes / day for 40 years (until 6 years previously) and drank 350 ml of beer / day. the patient developed a fever, coughing, and expectoration around september 12, 20xx (day 12). emphysematous changes in the upper lobe of the right lung and niveau formation in the bulla were evident, and consolidation was also present in the left upper lung. the patient was monitored as an outpatient but was eventually admitted to our hospital 's department of respiratory medicine on september 26 (day 0) after hemoptysis was observed (table 2). he had ground - glass shadow in the left upper lung field on the thoracic plain radiograph (fig. 4) and consolidation associated with air bronchogram in the left s3 on thoracic computed tomography scan (fig. 5). klebsiella pneumoniae was identified from an initial sputum sample, which was collected at day 12, and treatment with ampicillin / sulbactam (3 g intravenously every 6 h) was started on day 1. however, his symptoms failed to improve, and since the tests for the aspergillus antigen and antibody were both positive on day 10, our department was consulted. the calcium oxalate crystals were also evident in the sputum culture (figs. 6 and 7) ; thus, chronic necrotizing pulmonary aspergillosis was suspected and treatment with voriconazole was started (loading dose : 300 mg intravenously every 12 h on the first day ; subsequent doses : 200 mg intravenously every 12 h). a. fumigatus was identified in a sputum culture test on day 12. however, the patient 's general condition on admission was poor, and he died on day 14. the study was approved by the ethics committee of kanto rosai hospital, and all the patients provided written informed consent to publish the manuscript. the main causative species of pulmonary aspergillosis are a. fumigatus, a. niger, aspergillus nidulans, and aspergillus flavus, of which a. fumigatus accounts for most cases. calcium oxalate is formed when oxalic acid is released into tissue as a metabolite when aspergillus binds with tissue calcium. studies have suggested that calcium oxalate may be useful in the diagnosis of a. niger [46 ], and some have suggested that it may also be useful in the diagnosis of a. fumigatus, as it is present in the pathological samples. however, no previous study has investigated the value of calcium oxalate for diagnosing pulmonary aspergillosis caused by a. fumigatus in living patients. the aspergillus galactomannan antigen is one of the parameters used for diagnosing chronic necrotizing pulmonary aspergillosis and is among the microbial parameters of the eortc / msg diagnostic criteria for invasive fungal infections. however, it has been reported that its sensitivity for a. fumigatus may be lower than that for non - fumigatus infections, and it is possible that the presence of calcium oxalate in particular may be a sufficient cause for suspecting a. fumigatus pulmonary aspergillosis in chronic lower respiratory lesions. in conclusion, calcium oxalate may aid in the diagnosis of pulmonary aspergillosis caused by a. fumigatus. the exact use of calcium oxalate as a diagnostic parameter of chronic necrotizing pulmonary aspergillosis as well as a microbial parameter of the eortc / msg diagnostic criteria for invasive fungal infections should be confirmed through the examination of more similar cases.	we present two cases of pulmonary aspergillosis in which calcium oxalate crystals in the sputum proved to be a useful diagnostic clue. in case 1, aspergillus hyphae was not identified ; however, calcium oxalate crystals were present, and chronic necrotizing pulmonary aspergillosis was diagnosed. in case 2, calcium oxalate was detected and aspergillus fumigatus was identified later. thus, the presence of calcium oxalate in the sputum may be an important indicator for an a. fumigatus infection.
, tamoxifen has been the antiestrogen of first choice. however, about half of the recurrences in er - positive breast cancer do not respond to tamoxifen, which is due to either acquired resistance or to intrinsic insensitivity to tamoxifen [1, 2 ]. from experimental studies, many different mechanisms have been suggested to explain resistance, including activation of kinase pathways or inactivation of prb, that render the tumor cell independent of the er pathway for its proliferation. however, with exception of cerbb2 (neu) overexpression, which mostly, but not exclusively occurs in er-negative breast cancer [3, 4 ], currently none of the resistance mechanisms identified have been translated into clinical implementation. therefore, they should be examined together as an integrated set of predictive markers for diagnosis of individual patients. not only the number of clinically relevant indicators for tamoxifen resistance is unknown, but also the proportion in which a particular marker contributes to resistance in patients is unclear. the relative contribution of these factors should be defined and potentially integrated into a combined set of predictive markers for tamoxifen responses of individual patients. these two opposing effects of tamoxifen on cell growth can be explained by the fact that tamoxifen is a partial antagonist, acting as an agonist under particular conditions. tamoxifen resistance is usually due to a direct effect on er ; tamoxifen may acquire agonistic properties for transactivation of er. therefore, a molecular understanding of the underlying mechanism of tamoxifen resistance could result in markers that specify how patients will respond to endocrine therapy. the potential translation of these markers into clinical evaluation has to be examined with historical material and ultimately in a prospective study. identification of markers predicting the antibreast cancer response to tamoxifen would have major clinical implications. currently, the clinical benefit of tamoxifen is similar to that of aromatase inhibitors, although the side effects of the drugs markedly differ. ultimately, by finding predictive markers, responsiveness to tamoxifen can be defined before treatment and patients will only receive tamoxifen if they are likely to benefit from it. and in case of resistance, patients may still respond to another treatment modality, such as aromatase inhibitors or the full antiestrogen antagonist, like fulvestrant, which may still be beneficial. the estrogen receptor superfamily consists of two homologous nuclear receptors : er and er. er is encoded by a different gene, and the two receptors exhibit different transcriptional activities and functions in breast cancer. because the phosphorylation of er and a potential role in tamoxifen resistance have not been well characterised, we will not discuss this estrogen receptor subtype. in this chapter, we focus on phosphomodifications of er in tumor cells that, by themselves, do not affect the female hormone estradiol (e2) dependency of the tumor cells for proliferation, but could affect the response to tamoxifen. which molecular pathways upstream or downstream of the phosphorylated er are involved in this form of tamoxifen resistance ? this complicates characterisation of structural changes upon ligand binding or posttranslational modifications, such as phosphorylation. furthermore, a conformational change due to phosphorylation could have consequences for the action of estrogens and antiestrogens. x - ray crystallography studies have thus far been performed on the ligand binding domain (lbd) of er. estradiol binds to amino acids glu353 from helix 3(h3), arg394 from h5, and to his524 from h11 in the lbd of er, whereas d351 in the lbd is critical for the interaction with the antiestrogen. specific mutation of d351 into d351y resulted in a receptor that shows an estrogenic, instead of an antiestrogenic, response to tamoxifen. coactivators have a common signature motif, lxxll, with which they can interact with er in a hormone - dependent manner. whereas in a nonligand - bound state helix 12 is highly mobile, upon binding of an agonist it takes a more fixed position, stabilising the conformation of er. helix 12 forms a charge clamp with helix 3, creating a hydrophobic groove to which a coactivator can bind. in contrast, crystallography shows that, when an antagonist, such as tamoxifen, binds to the lbd, helix 12 itself occupies the coactivator binding site, rendering er inactive [1113 ]. structural changes of er can influence coregulator binding and hence potentially the response to ligands. besides binding to the lbd in the af-2 domain, coactivators also bind to the af-1 domain of er, in a ligand - independent manner. phosphorylation of sites within or outside the af-1 region may affect the af-1-dependent binding of cofactors as well. phosphorylation of particular sites, especially of s118 and s305, affects the binding of coactivators in the presence of tamoxifen. in case of s305, this is due to an altered conformation of er, which can be measured by fluorescence resonance energy transfer (fret). in the presence of tamoxifen, an altered conformation of er due to phosphorylation of s305 resulted in a tamoxifen - resistant phenotype of er, not only measured by fret, but also by biological assays [6, 16 ]. not only tamoxifen but also arzoxifene is converted from an antagonist into an agonist after the s305 phosphorylation - induced conformational arrest of er. these findings strongly suggest that subtle changes in the conformation of er upon binding to antiestrogens are at the basis of resistance to antiestrogens. this provides the framework to consider a role for phosphorylation of er in resistance to tamoxifen. several kinase pathways have been associated with tamoxifen resistance, including activation of the protein kinase a (pka), mitogen - activated protein kinase (mapk) and p21-activated kinase-1 (pak-1) signaling pathways. this paper focuses on the phosphorylation sites on er that could contribute to an altered response to tamoxifen and on which kinase pathways and upstream activators are involved. a summary of the putative phosphorylation sites in er is presented in figure 1 and table 1. serine residues s102, s104, and s106 at the n - terminal af-1 region of er are phosphorylated by glycogen synthase kinase-3 (gsk-3) and by extracellular signal - regulated kinases 1 and 2 (erk1/2) and mitogen - activated protein kinase (mapk) (= mek1/2) pathways. these modifications lead to ligand - independent transcription of er and to an agonistic activity of tamoxifen [22, 23 ]. s102, a phosphorylation site discovered by mass spectrometry, requires concurrent phosphorylation of s104. er phosphorylation by gsk-3, which also targets s118, stabilizes er without ligand and modulates er transcriptional activity upon ligand binding. cyclin a has been reported as a predictive marker for tamoxifen resistance in breast cancer patients. serine 118 is one of the most reported phosphorylation sites of er. it is targeted by a number of kinase pathways : mapk, gsk-3, ikk, cdk7, and mtor / p70s6k. s118 phosphorylation by mapk increases binding of coactivator src3 and renders er hypersensitive to estradiol. phosphorylated s118 decreases er affinity for tamoxifen and reduces binding to dna, when er is tamoxifen bound. in a tamoxifen - resistant cell line obtained by selection after prolonged exposure to tamoxifen, mapk activity was found to be elevated and s118-p was increased. upstream, the ras / mapk pathway can be activated by igf stimulation inducing phosphorylation of er s118 and resulting in er activation and enhanced response to estradiol. estradiol and egf can induce the erk1/2 mapk pathway, which also leads to s118 phosphorylation of er. estrogen - dependent phosphorylation of s118-p can occur not only through the erk1/2 mapk pathway, but also by ikk and cdk7, a subunit of transcription factor ii h. in mcf7 cells, the receptor tyrosine kinase ret mediates er phosphorylation at s118 and s167 via the mtor / p70s6k pathway. activation of ret leads to estrogen - independent transcriptional activation of er - dependent genes and resistance to tamoxifen, strongly suggesting that ret activity acts through the estrogen receptor. this hypothesis is supported by a chromatin immunoprecipitation (chip) study on er and s118 mutants. phosphorylation of s118 influences the recruitment of coregulators to er-regulated genes ps2, c - myc, and cyclin d1 and affects e2-induced gene expression. the nonphosphorylatable s118a mutant has a greater impact on genes regulated through nonclassical mechanisms, such as er binding to fos / jun on an ap-1 promoter, than on estrogen responsive elements (ere). the clinical relevance of s118 phosphorylation in tamoxifen resistance is still unresolved. on the one hand, s118-p has been associated with a more differentiated phenotype, good prognosis, and better response to tamoxifen, which is supported by other studies (see, wigerup. most importantly, these studies reported that the s118 phosphorylation had no effect on progression of disease or survival without tamoxifen treatment, thereby emphasizing that s118 phosphorylation is a clear predictive marker for response to tamoxifen in these studies. on the other hand, s118 phosphorylation was negatively correlated with response to endocrine therapy in patients in other studies [3234 ]. nontreated patients have a better prognosis when they are positive for s118-p in these studies [32, 33 ]. these results are not easily reconciled with the previously mentioned studies (see [28, 31 ], wigerup. besides differences in patient series and tumor types, it is not clear which kinase activities in the tumors are resulting in s118 phosphorylation. in patients, both mapk and ret expressions activation of the erk1/2 mapk pathway apparently results in s118 phosphorylation, but it also induces a bypassing of the er pathway, thereby rendering tumors hormone - independent. cdk7-mediated phosphorylation is indicative of an active er. whereas the mapk mechanism may well be responsible for a worse outcome of disease, irrespective of tamoxifen treatment, the cdk7 mechanism would indicate a proper functioning of er, being an adequate target for tamoxifen treatment. egfr overexpression induces s167 phosphorylation, increases binding of er to dna, enhances the binding of coactivator src3 to er in the presence of e2, and consequently enhances transcription. moreover, in vitro, s167-p reduces sensitivity to tamoxifen [25, 32 ]. other kinases that target s167 include erk1/2 mapk [32, 37 ] and, upon e2 binding of er, casein kinase ii (ck2). in er-positive, tamoxifen - treated patients, activated akt (pakt) is associated with high risk for relapse and decreased overall survival, which would imply that s167-p is associated with a worse disease outcome. however, it is important to realise that akt, like erk1/2 mapk, has many other targets, which could well bypass the estrogen - receptor - dependent signaling. notwithstanding, in a set of 75 primary breast carcinomas of patients with metastatic breast cancer who received first - line endocrine treatment after relapse, those staining high for s167-p relapsed later. the metastases responded well to endocrine treatment and s167-p correlated with longer survival after relapse. this implies that s167-p is a predictive marker for a good response to endocrine therapy [34, 37 ]. serine 282 resides in the hinge region and, like s167, can be phosphorylated by ck2. estradiol increases phosphorylation of s282, stabilizes er, and induces transcriptional activity. in patients, low levels of s282 phosphorylation are associated with reduced overall survival in er - positive breast tumors from tamoxifen - treated patients, suggesting that s282 phosphorylation can be predictive for response to tamoxifen. serine 305 resides at the c - terminus of the hinge region that provides a centre of rotation to the total er. the region around ser305 is a multifunctional domain that binds to many coregulatory proteins and is involved in the regulation of activity and stability of er. phosphorylation of ser 305 occurs by protein kinase a and is associated with resistance to tamoxifen in patients (see [4345 ], wigerup. this domain also controls er ubiquitination and subsequent proteosomal degradation of er, that is influenced by ligands. different ligands can induce different conformations of er and hence affect accessibility to the hinge region for modifying proteins, such as ubiquitin ligases. this implies that ligands can be selective for specific posttranslational modifications. within the hinge region, lysines k302/303 are involved in proteasomal degradation of er by fulvestrant and are the targets of polyubiquitination. k302 and k303 are both required for monoubiquitination by the brca1/bard1 e3 ligase of e2- or tamoxifen - bound er. k303 is also target for acetylation (inhibiting er activity) and for methylation (stabilizing er and increasing activity), whereas s305 phosphorylation prevents acetylation of k303, thereby stimulating er activity. the reverse is also true : a k303r mutation is frequently found in breast cancer, which prevents acetylation and increases phosphorylation of ser305 by pka. these findings indicate that the hinge region is affected by various posttranslational modifications that affect structure and functioning of er. some of these modifications and their cross - talk are shown in figure 2. besides pka, p21-associated kinase 1 (pak1) has been suggested as an upstream kinase involved in the phosphorylation of ser305. pak1 phosphorylation of er s305 can lead to a secondary event on s118, presumably due to a conformational change of the estrogen receptor [15, 19 ]. pak1 overexpression by itself is associated with resistance to tamoxifen in vitro as well as in patients [32, 44, 45, 47 ]. notably, in an experimental tamoxifen - resistant setting, tamoxifen induces pak1, maintaining er in the tamoxifen - insensitive state. the evidence that pak1 phosphorylates s305 was indirect and was not confirmed by a direct inspection of the phosphorylation of er ser305 using specific antibodies or by the introduction of a dominant - active pak1 into breast cancer cells. moreover, overexpression of pak1 was not correlated with s305 phosphorylation in two different studies on breast cancer, indicating that these two events are independent (see, wigerup. pka phosphorylates s305, keeping er in an active conformation when tamoxifen is bound, which means that it mimics an estrogen - bound er. clinical studies show that tamoxifen resistance occurred in endocrine - treated patients with detectable s305-p in the primary human breast tumor. since s305 phosphorylation has no effect on patients that were not endocrine treated, this ser305p markers appears to be a predictive marker for treatment outcome and not for general disease progression. a combination of pak-1, phosho - pka, a marker of activated pka, and the phosphorylated s305 marker identified approximately 6070% of all tamoxifen resistant cases in breast cancer. this occurred in series of breast cancer from premenopausal and postmenopausal patients, in early to advanced stages of disease, indicating that the marker is independent of clinical stage of disease and of the hormonal status of the patient (see [31, 43, 45 ], wigerup. phosphorylation of this tyrosine inhibits er dimerisation and estrogen binding and reduces transcriptional activity of er. tyr 537 is located at the n - terminus of helix 12, and mutation of this tyr into a nonphosphorylatable alanine facilitates the rotation of helix 12 into an active conformation of er in the absence of any ligand. there is no apparent clinical evidence that y537 phosphorylation influences tamoxifen response in patients. of note, a naturally, but rarely occurring, y537 mutation to asparagine (y537n) in breast cancer metastasis constitutively activates the estrogen receptor by a conformational change of helix 12, which may contribute to breast cancer progression and resistance to endocrine treatment. several other phosphorylation sites of er have been found, which have not been associated with tamoxifen, either since tamoxifen was not included in the studies or because the phosphosite has not been included in clinical studies on tamoxifen resistance. ser-46/47 phosphorylation plays a role in ligand - dependent activation of er. mutation of ser-46/47 or ser-294 to alanine markedly reduced estradiol - dependent reporter activation. s47 phosphorylation may influence other posttranslational modifications of er. s46 is a putative recognition site for protein kinase c and seems to hold a predominant effect on transcriptional activity, rendering s47 phosphorylation a bystander effect. tyrosine 52 and 219 are phosphorylated by c - abl, a src - like nonreceptor tyrosine kinase. this results in enhanced er transcriptional activity, both in absence and presence of estradiol. stabilisation of er through c - abl ultimately leads to proliferation and invasion of breast tumor cells. serine 154, 212, 294, and 554 are putative er phosphorylation sites discovered by mass spectrometry on phosphopeptides. in vitro, an alanine mutation of s294 reduces estradiol - dependent transcription, suggesting that s294 phosphorylation is needed for a functional er. furthermore, s294 phosphorylation has been detected by immunohistochemistry (ihc) in human breast carcinoma but no significant effect of s294-p on tamoxifen response in terms of recurrence or overall survival has been observed. it is phosphorylated by pka, upon which er dimerisation and dna binding in the absence of ligands are lost, rendering er transcriptionally inactive, but both estradiol and tamoxifen can overcome this inhibition. threonine 311 is the only known threonine phosphorylation site on er. an active ras / mapk pathway stimulates er phosphorylation at thr-311. phosphorylation of t311 can be detected by immunohistochemistry, but thus far has not been significantly associated with altered tamoxifen sensitivity in breast cancer patients. serine 559, like y537, resides in the f domain of the estrogen receptor, in helix 12. this is of particular interest, because the position of helix 12 determines interaction with coactivators and corepressors and regulates response to (ant)agonists. therefore, s559 phosphorylation can probably influence er binding to coregulators, such as src-1, by changing the position of helix 12 and as a consequence the response to er ligands. phosphorylation inhibits ligand - independent activation of er. er is phosphorylated at s559 in human breast carcinoma biopsies. we presented, thus far, the effects of phosphorylation of relevant sites in er as single events. of course, reality is more complex and modifications not only occur on er itself, but also take place on the associated cofactors and on targets outside the er signaling pathways that could have an effect on er-mediated signaling. phosphorylation of carm1 by pka enhances its interaction with s448 in the lbd of er and creates a novel, more firm platform for binding of other cofactors. the net result is tamoxifen resistance by the buildup of a pka - specific coactivator complex. because the arginine methyltransferase carm1 is involved in methylation of histones h3 and h4 that is crucial for transcription to occur, the er-phosphocarm1 complex provides a specific regulatory unit for transcription. still, additional events are needed for tamoxifen resistance, among which possibly the phosphorylation of er s305 by pka. pak1 phosphorylates an alternate, but in breast cancer frequently present, isoform of the src3 steroid - receptor cofactor (src3 - 34), allowing it to bridge between egf - r and fak1 (focal adhesion kinase 1) and thereby activating erk1/2 mapk. these phosphorylated sites determine the optimal interaction with other transcription factors and are required for different physiological functions. these three examples demonstrate that there is a complex interrelated network of regulatory circuits influencing er transcriptional activity and that, by modification of one circuit, other circuits are affected. most of the studies have addressed only one significant mode of action, but it is evident that many factors can play a role in the resistance to endocrine treatment. how does phosphorylation of era affect resistance to tamoxifen ? the estrogen receptor is a nuclear receptor, which binds to specific sequences in the dna and regulates the expression of er - dependent genes. phosphorylation of er can affect dna binding, for example, by inhibiting dimerization of the receptor, and can influence er activity by changing the binding to coactivators or the orientation of components of the transcription factor complex. which genes are then affected ? in the classical way, an estradiol - bound estrogen receptor dimerizes, binds to an estrogen responsive element (ere), and transcribes the gene that lies within its proximity. the estrogen receptor can also regulate transcription of genes in an indirect manner, by binding to other transcription factors : ap-1, sp-1, or activated nfkb. when these interactions occur, transcription of the ap-1-, sp-1-, or nfkb - dependent genes becomes also dependent on er. when tamoxifen is bound to er, the classical estrogen responsive genes are not expressed but tamoxifen - bound er has its own, different transcriptome, most likely generated through the nonclassical pathway [62, 63 ]. different kinase pathways can be activated chemically in cells by adding growth factors (egf or igf) or camp, which induces pka. kinases were activated, and gene expression profiles were compared in the presence or absence of tamoxifen. tamoxifen treatment resulted in differential gene expression with either growth factor stimulation or pka activation. which of these genes is essential for tamoxifen resistance remains a crucial question. a more complete, but also more complex, picture arises from microarray analyses performed in tumors of tamoxifen - treated er-positive breast cancer patients. frasor. described a set of genes associated with disease recurrence, a subset of which is associated with treatment with tamoxifen. they developed a gene classifier to predict clinical outcome in tamoxifen - treated er-positive breast cancer patients. this classifier contains genes involved in invasion (slit2 and reck), anti - inflammatory response (tgfbr4, ptger4, c3, and gng2), and cell cycle regulation. in later studies, this group validated a number of hits by qpcr and hence demonstrated that ezh2 downregulation is associated with a favourable outcome and that downregulation of siah2, an e3-ubiquitin ligase, would imply tamoxifen resistance. they also showed that an extracellular matrix cluster of genes (timp3, fn1, lox, and sparc) is associated with tamoxifen resistance. in any of these studies, it is unclear whether phosphorylation of the estrogen receptor plays a role in tamoxifen resistance in these patients. looking at multiple genes, instead of only one, could be more informative for treatment outcome. therefore, kok. compared three gene classifiers [6971 ] for tamoxifen. this comparison indicates that a multigene approach would improve the prediction of response to tamoxifen. there is as of yet only one microarray study on tamoxifen - treated er-positive human breast cancers that addresses a specific phosphorylation site, s305p, and the effect on gene expression. a pathway analysis highlighted several pathways being affected, including pka, erk1/2 mapk, egf signaling, cdk regulation, and interferon alpha signaling. in total, 19 phosphorylation sites have been identified in er thus far, as summarized in figure 1. phosphorylation of s167, s118, s282, and y537 is beneficial for tamoxifen response according to experimental and, for s167, s118 and s282, because of reported clinical data. the contribution of phosphorylation of other target sites to tamoxifen resistance remains to be determined. some of these phosphorylation sites have been shown with fret technology to induce a conformational change of er, when exposed to other antiestrogens, such as fulvestrant and raloxifene. thereby, they may affect the antagonistic behavior of these compounds but the molecular mechanisms remain to be elucidated. dependent on the pathway and the phosphorylation sites involved, tamoxifen response can be affected either directly through er modification or by activation of other signaling pathways. phosphorylation of s118 is described as an example of this : an activated erk1/2 mapk pathway phosphorylates s118 but possibly induces tamoxifen resistance through the erk1/2 mapk pathway itself, rather than er signaling. s118 phosphorylation by the er - associated cdk7 indicates an activated er which would imply a beneficial effect on tamoxifen treatment in patients. tamoxifen response may be restored by blocking egfr with gefitinib [3, 72 ]. in a clinical study, blocking cerbb2 with trastuzumab restores er signaling in er-positive tumors and improves response to the aromatase inhibitor letrozole. this would suggest a better response to tamoxifen as well. it is challenging to extrapolate experimental data from er activation to the clinic and vice versa. in in vitro studies, it is feasible to examine differential gene expression after treatment and compare the profile before and after treatment, or in absence or presence of phosphorylation. translation of this information to the clinic can, however, be troublesome, since adjuvant tamoxifen treatment is started after surgical removal of the primary tumor. the in vitro experiments measure gene expression changes associated with acquired resistance, whereas investigation of primary tumors that respond better to treatment highlights genes which play a role in intrinsic resistance to tamoxifen. because primary tumors have not been exposed to tamoxifen they may occur at random during normal tumor development or may well coincide with other tumor progression markers. for example, overexpression of cerbb2 or of egfr in breast cancer marks worse course of disease not only in er-negative, but also in er-positive tumors and is a marker for tamoxifen resistance as well. it is also a marker for the choice of treatment, since a combination of s305-p, s118-p, and overexpression of src-1 or cyclin d1 coactivators dictates resistance to different antiestrogens [14, 15 ]. since outgrowth of micrometastases into tamoxifen resistant tumors occurs over longer periods of time (up to 15 years), extra alterations in the micrometastases outgrowths, in addition to the s305 phosphorylation status, potentially influence tamoxifen resistance. phosphorylation of ser305, however, was still maintained in the few metastases samples that could be examined. alternatively, one could study acquired and intrinsic resistance during neoadjuvant treatment with antiestrogens, where patients are treated up to three months prior to the surgical removal of the primary tumor. hence, samples can be obtained before and after treatment for comparison. in another study by this group, activation of the erk1/2/mapk pathway was a major factor associated with acquired resistance to tamoxifen. phosphorylation of s305 has experimentally been linked to resistance to tamoxifen, because of an altered conformation of er, where tamoxifen behaves as an agonist in fret and expression reporter assays. in patients, s305p was associated with alterations in the pka pathway that result in stimulation of pka activity [15, 44 ]. also experimental enhancement of pka activity in breast tumor cells led to proliferation of t47d breast tumor cells in the presence of tamoxifen. it is, however, still possible that s305p is a marker for tamoxifen resistance without any direct involvement. it could merely mark pka related events that bypass the estrogen receptor and hence induce tamoxifen resistance. the altered orientation of components of the transcription factor complex and the conformational changes in er strongly suggest, but are no proof of, a direct involvement. s305-p is, however, one of the few selective markers that predict resistance to tamoxifen in breast cancer patients. definition of the activation of relevant signaling pathways in the er-positive breast tumors (that constitute the bulk of human breast cancers) prior to endocrine treatment is essential for treatment success and will ultimately lead to personalised treatment of breast cancer patients.	about two thirds of all human breast cancer cases are estrogen receptor positive. the drug of first choice for these patients is tamoxifen. however, about half of the recurrences after removal of the primary tumor are or become resistant to this drug. while many mechanisms have been identified for tamoxifen resistance in the lab, at present only a few have been translated to the clinic. this paper highlights the role in tamoxifen resistance of phosphorylation by different kinases on different sites of the estrogen receptor. we will discuss the molecular pathways and kinases that are involved in phosphorylation of er and how these affect tamoxifen resistance. finally, we will elaborate on the clinical translation of these observations and the possibility to predict tamoxifen responses in patient tumor samples before treatment onset. the findings made originally on the bench may translate into a better and personalized treatment of breast cancer patients using an old and safe anticancer drug : tamoxifen.
deficits in social cognitive skills in schizophrenia patients (sz) have been demonstrated in numerous studies. facial affect recognition (far) is particularly relevant for effective social interaction (johnston., 2010 ; sachs., 2012 ; wlwer., 2012). as impaired social cognitive skills are linked to functional impairment (e.g., poole., 2000 ; sachs., 2004 ; hofer., 2009), remediation programs have targeted social - cognitive skills including far (e.g., wlwer., 2005 ; habel., 2010 ; mazza., 2010 ; wlwer and frommann, 2011 ; kurtz and richardson, 2012). in their meta - analysis of 19 studies including 692 sz, kurtz and richardson affirmed moderate - to - large effects on social cognitive measures (including facial affect identification tasks) and on observer - rated social function, with training effects varying with age, duration of illness, and extent of training. understanding brain processes contributing to social cognition deficits should facilitate the development and evaluation of tailored remediation strategies. numerous studies have studied cortical and subcortical correlates of emotion processing in sz including far (for hemodynamic imaging evidence, see pinkham., 2007 ; seiferth., 2010 ; for event - related brain potential evidence, turetsky., 2007 ; wlwer., 2012 ; wynn., 2013 ; for oscillatory activity, singh., 2011 ; these studies provide substantial evidence of deviant brain activity related to social cognition, including far. yet few studies have evaluated the effects of social - cognition training on brain activity (habel., 2010 ; wlwer., 2012 ; luckhaus., popov. (2013) proposed a neural mechanism for such disrupted facial affect processing and its remediation. the study demonstrated group differences in 1015 hz (alpha) neuromagnetic oscillatory power modulation in bilateral sensorimotor regions while sz and healthy controls (hc) viewed 5 s videos of dynamic facial stimuli that changed from neutral to fear or to happy expressions. during the period prior to correct affect recognition, hc exhibited a significant alpha power increase relative to baseline, whereas the significantly smaller increase in sz varied with poorer discrimination accuracy. because sensorimotor alpha activity has been linked to social information processing including far (e.g., singh., 2011), the present study employed a targeted intervention to test the hypothesis that this recruitment of neural processes facilitates the recognition of unfolding facial affect in hc and is apparently impaired in sz. support for the hypothesis would mean that appropriate training can address deficits in recognition skills. the present study evaluated a new facial affect recognition training (fat) protocol, designed specifically to address mechanisms facilitating far, and assessed alpha - power modulation as a possible mechanism of the training effect. because beneficial effects of specific cognitive and/or social cognitive training protocols as add - ons to general sz remediation programs have been reported (e.g., keefe and harvey, 2012 ; sacks., 2013), the present study compared fat with a well established cognitive training protocol, cognitive exercises (ces ; positscience, sf, usa) already shown to be effective in sz (fisher., 2014 ; see also popov., 2011, 2012). ce focuses on perceptual and cognitive skills and does not include facial or emotional judgments. thus, it served as an active control for fat 's use of a training regimen. in between - group analyses, fat and ce were compared with the inpatient unit 's treatment as usual (tau), which provided a nonspecific control for the passage of time and general treatment efforts. analyses addressed a series of questions : does training affect brain dynamics, and does specific training (fat) affect specific far - related oscillatory dynamics ? does training normalize brain dynamics (does fat reduce pre - training differences in far - related alpha dynamics between sz and hc to nonsignificance) ? to what extent are changes dependent on an active intervention in general (does fat do so better than tau) or on specific far - focused training (does fat do better than ce) ? inpatients with an icd diagnosis of paranoid - hallucinatory schizophrenia (code number 20.0) were recruited at the regional center for psychiatry. inclusion criteria were normal intellectual function and no history of any neurological condition or disorder such as epilepsy or head trauma with loss of consciousness. according to the standard treatment regimen of the center, all patients were stably medicated at the time of the study. from the pool of eligible sz (n = 114 1), n = 80 were randomly assigned to three intervention groups, of which n = 62 completed the interventions and all pre- and post - intervention assessments (symptom ratings, neuropsychological assessment, magnetoencephalographic (meg) recording). after providing written informed consent, sz were randomly assigned (with some adjustment to ensure balanced group sizes) to one of three groups : the two computer - based training methods, fat or ce, or tau. recruitment continued until at least 20 sz were enrolled in each intervention. at post - intervention assessment, data from one subject in each group were discarded because of meg artifact or missing meg data. thus, results are reported for n = 19 patients in each group. symptom severity pre- and post - intervention was assessed via the positive and negative symptom scale (panss, kay., 1987) and the global assessment of functioning (gaf) scale of dsm - iv. upon treatment assignment, groups did not differ in gender, age, educational level, iq, symptom severity (panss), global function (gaf), medication (evaluated by cpz equivalent), or neuropsychological test performance (below and table 2). in each group 2 patients were left - handed, and 24 were ambidextrous, as assessed by a modified version of the edinburgh handedness questionnaire (oldfield, 1971). sz participants completed a protocol consisting of (1) assessment of clinical and demographic data, neuropsychological test performance (matrics consortium cognitive battery, mccb, nuechterlein., 2008), and a far criterion task during meg, all prior to intervention, (2) 4-week intervention (fat, ce, or tau), and (3) post - intervention assessment of clinical data, mccb, and the far criterion task during meg. the study was approved by the ethics committee of the university of konstanz and registered as a clinical trial (http://clinicaltrials.gov registration nct01781000). pre - intervention meg data from 14 of the present sz were included in popov. in addition to primary analyses comparing these three patient groups, data from two groups of healthy comparison participants (hc), each tested once, were included in targeted analyses. following a standard protocol, hc were recruited from the community by oral advertisement and flyers and were included if they did not meet the criteria for a lifetime diagnosis of mental illness (screened with mini international neuropsychiatric interview ackenheil., 1999), did not report any history of head trauma with loss of consciousness, and were free of psychoactive medication. the hc19 group (n = 19) was recruited when sz were provided a pre-/post - intervention comparison of oscillatory activity in the far outcome criterion task. the hc24 group (n = 24) was recruited after the meg study ended, in order to provide a normative performance standard for the newly developed fat training tasks. none of the dependent measures overlapped for the two hc groups (no fat performance data available for hc19, and no meg data for hc24). the hc19 sample included 15 subjects from the hc group reported in popov. gender distribution of the hc19 (12 m, 7 f) did not differ from that of the 57 sz, though the hc19 were younger (27.0 3.7 years vs. 37.2 9.1 years, f(1,74) = 22.18, p.1), reflecting some general improvement over time in the pooled sample, and an intervention time interaction, f(1,36) = 4.78, p =.04 (p =.05), reflecting improvement in the fat group (fig. thus, changes in alpha power modulation were specific to training. here and below, no main effects of intervention emerged. that was as anticipated, given random assignment to group, with group variance therefore expected only in intervention time effects. the final a priori anova assessed whether this improvement in alpha response with active training was specific to fat. an intervention (fat vs. ce) emotion time analysis produced an effect of time, f(1,36) = 8.54, p =.006 (p =.008), reflecting improvement with training, but no intervention time interaction, indicating similar improvement in the two active treatment groups (fig. 4). an omnibus intervention (fat, ce, tau) emotion time analysis is of some value as a control for experiment - wise error. because the primary relevant hypothesis such an analysis addresses, that intervention improves brain function, is directional, a one - tailed test of the time effect would be appropriate, or equivalently a p =.10 criterion for the anova effect. the time effect exceeded that criterion, f(1,54) = 4.81, p =.03 (p =.06), with alpha power increase from prestimulus baseline during the pre - recognition period of the far criterion task larger after training than before training. a secondary hypothesis that an omnibus test could address is that interventions differed in their impact, thus a nondirectional intervention time effect, which was marginal, f(2,54) = 2.44, p.1). change in alpha power increase from baseline between pre- and post - intervention was not related to changes in neuropsychological test performance (mccb) or in clinical status (symptom severity, gaf). popov. (2013) identified induced alpha power modulation as a measure that varied with performance during a dynamic facial affect recognition task. schizophrenia patients exhibited less induced alpha power modulation than healthy subjects, corresponding to poorer behavioral performance (popov. the present study addressed the power and specificity of a computer - aided intervention targeting facial affect discrimination to ameliorate this deficit in schizophrenia patients. present results confirmed that deficient alpha modulation can be modified by psychological intervention : poor pre - intervention alpha power modulation during the far criterion task improved after training in facial affect discrimination. although neither performance on the training tasks nor alpha dynamics on the far criterion task reached normal levels, findings indicate that relevant brain dynamics in sz have considerable plasticity. similar training - induced neuroplasticity has been shown in other studies involving cognitive training (adcock., 2009 ; edwards., 2010 ; subramaniam., kelly and garavan (2005) proposed principles of redistribution and reorganization as potential mechanisms of neuroplasticity. redistribution refers to a change in activation within neural networks as a function of practice, without altering neural network structure. in contrast, reorganization is thought to reflect remodeling of structure and of functional activity related to task performance in the course of practice. normalization via reorganization would be expected if training adequately addresses the neurobiological mechanisms of cognitive deficiency. present results support the conclusion that the observed changes in alpha power as a function of training result from redistribution of neuronal processing resources that support facial affect discrimination. of course, additional changes may have resulted that the present meg measures did not detect. changes in brain dynamics were larger after the two dense, computer - aided training procedures than after a treatment - as - usual program. in a meta - analysis genevsky and colleagues (2010) concluded that, rather than fostering broad skills, efficient remediation should involve targeted, computer - aided with high - dose schedules, supplemented by psychosocial intervention. the present fat and ce training protocols provided the first two elements, i.e., targeted, high - dose computer training, relative to tau. moreover, both specific training protocols targeted functions that are dysfunctional in schizophrenia such as working memory, attention, or discrimination accuracy. this is in line with the success of the training of affect recognition (tar) protocol developed by wlwer and colleagues (wlwer., 2005 ; wlwer and frommann, 2011 ; sachs., 2012 ; luckhaus., 2013), which includes a specific facial affect discrimination task within a broader spectrum of tasks. tar improved social cognition indices and increased activity in various brain regions related to attention and cognitive processing. (2013) combined ce with specific social cognition training and found improvements on social cognitive outcome measures in sz. training protocols targeting functions known to be dysfunctional in sz may be beneficial whether applied alone or within a broader spectrum of tasks. in the present study, the fat and ce training protocols were similar in various ways : both targeted neuroplasticity by intensity (20 consecutive 1-hour sessions), shaping (performance - based adjustment of task demands), and motivational context (tasks embedded in computer game, frequent motivating feedback) ; both addressed working memory and visual learning. it is conceivable that these training protocols, though designed to target specific processes, modify more basic, general processes such as neural information sampling, readiness for information intake, and excitation inhibition and segregation integration balance, thus general computation mechanisms supporting diverse functions (buzsaki, 2010 ; buzsaki and watson, 2012 ; buzsaki.. fat may have improved item discrimination and memory in general, not only facial affect recognition. within present data, nonspecific enhancement of visual learning did not extend to (unchanged) performance on the visual - learning domain of the mccb. in order to distinguish specific and nonspecific effects, fat effects on other tasks an effect of the specific facial affect training on the social cognition mccb domain, examined with the msceit (mayer., 2003), might have been expected. similarly, the training of affect recognition protocol (wlwer and frommann, 2011) provides various strategies for affect management and regulation. improvement in such skills may be better reflected in msceit than the effects of the present training that specifically addressed facial affect discrimination and working memory. the msceit managing emotions subtest, used in the measures is a multifaceted construct and may not be suitable to test effects of the more specific functions (dawson., 2012) targeted by, i.e., facial affect discrimination accuracy and working memory. the extent to which modulation of brain dynamics can be directly related to complex constructs such as social cognition as measured by neuropsychological tests is a matter of debate (miller, 2010). (2014) anticipated an impact of training - enhanced basic processes on higher - order cognitive processes. additional dependent measures (both specific and general) would be needed to clarify the impact of fat on social cognitive functions in sz. it is also possible that the present 20-hour training was not intense enough to achieve more extended effects. for example, effects of intense cognitive training on social cognitive measures and global function were prominent after 50 training sessions (fisher. relevant to present hypotheses, training - induced change in performance on fat varied with improvement in alpha response. the parallel change in and the correlation between fat task performance and alpha power modulation suggest a functional relationship, thus alpha power increase as a mechanism facilitating facial affect recognition. the effect of fat on alpha power modulation in the far criterion task (a) supports the hypothesis (popov., 2013) that alpha power increase contributes to facial affect recognition and (b) demonstrates that a potential impairment of this mechanism can be modified by targeted training. first, results indicated that targeted training can affect neuronal processes believed to contribute to facial affect recognition and shown to be compromised in sz. however, conclusions about specificity of training effects would require further evaluation of effects of different types of training on diverse tasks, for example identifying double dissociations. moreover, specific vs. more general effects of fat on social cognition were not comprehensively examined : performance on the fat tasks was not assessed in the ce and tau groups, and as noted above the mccb social cognition domain may not appropriately interrogate the effects of specific, targeted training (fat) on higher - order social cognitive functions. a broader range of tests of social cognitive function would be necessary to fully evaluate the specificity of training effects. second, the present study focused on alpha - power modulation based on the previous finding of specific modulation in the 1015 hz range during the process of affect recognition. whereas sz differed from hc in this measure, no difference was observed in other measures such as the event - related potential to face presentation, specifically the m170 component (popov., 2014). this result, together with the lack of training - related changes in lower or higher frequency bands in the evaluated range (020 hz, see fig. 4), supports the proposal of alpha activity as a mechanism supporting facial recognition (popov., nevertheless, the contribution of other neuronal processes to facial affect recognition and an impact of the present training protocols on other neuronal phenomena, potentially evident in evoked high - frequency activity or event - related magnetic field variation, can not be ruled out. third, conclusions are always limited by the statistical power provided by the sample size, a common challenge in clinical settings (keefe., 2013). hc19 served to confirm dysfunctional alpha power modulation for the present sz samples, as had been shown in larger, well matched samples in popov. although a matched hc group for the entire group of 57 trained sz would have been of some interest, the present hc19 sufficed for evaluating this. taken together, present findings for an intervention targeting abnormal brain dynamics associated with deficient facial affect recognition in schizophrenia indicate considerable neuroplastic potential in schizophrenia. the intervention effects support the hypothesis of a neural mechanism facilitating facial affect recognition and its disruption in schizophrenia.	deficits in social cognition including facial affect recognition and their detrimental effects on functional outcome are well established in schizophrenia. structured training can have substantial effects on social cognitive measures including facial affect recognition. elucidating training effects on cortical mechanisms involved in facial affect recognition may identify causes of dysfunctional facial affect recognition in schizophrenia and foster remediation strategies. in the present study, 57 schizophrenia patients were randomly assigned to (a) computer - based facial affect training that focused on affect discrimination and working memory in 20 daily 1-hour sessions, (b) similarly intense, targeted cognitive training on auditory - verbal discrimination and working memory, or (c) treatment as usual. neuromagnetic activity was measured before and after training during a dynamic facial affect recognition task (5 s videos showing human faces gradually changing from neutral to fear or to happy expressions). effects on 1013 hz (alpha) power during the transition from neutral to emotional expressions were assessed via meg based on previous findings that alpha power increase is related to facial affect recognition and is smaller in schizophrenia than in healthy subjects. targeted affect training improved overt performance on the training tasks. moreover, alpha power increase during the dynamic facial affect recognition task was larger after affect training than after treatment - as - usual, though similar to that after targeted perceptual cognitive training, indicating somewhat nonspecific benefits. alpha power modulation was unrelated to general neuropsychological test performance, which improved in all groups. results suggest that specific neural processes supporting facial affect recognition, evident in oscillatory phenomena, are modifiable. this should be considered when developing remediation strategies targeting social cognition in schizophrenia.
the u4 snrnp core domain was reconstituted from the seven sm proteins and a variant of human u4 snrna16,22. crystals in space group p31 with 12 complexes per asymmetric unit were grown by vapour diffusion and diffracted x - rays anisotropically to 3.45 resolution. initial phases were determined by the mad method using semet substitution within sm sub - complexes. the structure containing 8101 protein and rna residues has been refined under 12-fold ncs restraints at 66.2 - 3.6 resolution to rfree of 32.1% with excellent geometry (table s1).	the spliceosome is a dynamic macromolecular machine that assembles on pre - mrna substrates and catalyses the excision of non - coding intervening sequences (introns)1 - 3. four of the five major components of the spliceosome, u1, u2, u4 and u5 snrnps, contain seven sm proteins (smb / b, smd1, smd2, smd3, sme, smf and smg) in common4,5. following export of the u1, u2, u4 and u5 snrnas to the cytoplasm6,7, the seven sm proteins chaperoned by the survival of motor neurons (smn) complex assemble around a single - stranded, u - rich sequence called the sm site in each snrna, to form the core domain of the respective snrnp particle8,9. core domain formation is a prerequisite for re - import into the nucleus10, where these snrnps mature via addition of their particle - specific proteins. here we present a crystal structure of the u4 snrnp core domain at 3.6 resolution, detailing how the sm site heptad (auuuuug) binds inside the central hole of the heptameric ring of sm proteins, interacting one - to - one with sme - smg - smd3-smb - smd1-smd2-smf. an irregular backbone conformation of the sm site sequence combined with the asymmetric structure of the heteromeric protein ring allows each base to interact in a distinct manner with four key residues at equivalent positions in the l3 and l5 loops of the sm fold. a comparison of this structure with the u1 snrnp at 5.5 resolution11,12 reveals snrna - dependent structural changes outside the sm fold, which may facilitate the binding of particle - specific proteins that is crucial to biogenesis of spliceosomal snrnps.
the incidence of degenerative arthritis of the knee has been increasing due to the longer life expectancy and growing senior population. accordingly, the numbers of patients who undergo total knee arthroplasty (tka) and develop postoperative periprosthetic fractures have been on the rise as well. recent reports estimated that periprosthetic fractures occurred in 0.6%-2.5% of the total tka patients1 - 3). most of the fractures around the prosthesis occur in the supracondylar region of the femur during daily living activities, although those in the femur, tibia, and patella have also been reported4 - 6). however, the conservative approach has been associated a variety of problems, such as increased pain and limited ambulation7). thus, efforts have been made to develop new treatment methods for supracondylar fractures of the femur following tka. minimally invasive plate osteosynthesis for distal femoral fractures has recently been introduced as a promising technique that is effective for preserving periosteal blood supply and bone perfusion and minimizing soft tissue dissection while providing satisfactory clinical and radiological outcomes9). in this study, we evaluated the clinical and radiological outcomes of minimally invasive plate osteosynthesis for the treatment of distal femoral fractures following tka and investigated the efficacy of the surgical technique. a total of 16 patients who had been treated for periprosthetic fractures following tka at our institution between july 2008 and october 2011 were retrospectively reviewed. patients who had been transferred from other clinics after tka were excluded in this study. of the 16 patients, 2 patients who had rorabeck.10) classification type 3 fractures were excluded from the study because a revision tka was determined necessary in them. the remaining 14 patients were available for a mean follow - up of 19.2 months (range, 10 to 37 months) until bony union was achieved. there were 2 males and 14 females with a mean age of 69.7 years (range, 52 to 78 years) (table 1). the cause of tka was degenerative osteoarthritis in 11, rheumatoid arthritis in 2, and posttraumatic osteoarthritis in 1 patient. the fractures were type 1 in 2 patients and type 2 in 12 patients when categorized according to the rorabeck.10) classification, whereas type 33-a1 in 10 patients and type 33-a2 in 4 patients according to the orthopaedic trauma association (ota) classification. the cause of fracture was slip and fall in 12 patients and a car accident in 2 patients. the affected side was the right side in 7 patients and left side in 7 patients. clinical results were obtained by a questionnaire designed to assess the knee range of motion (rom), functional score, and knee society score during outpatient clinical visits or telephone interviews. radiological results were assessed using anteroposterior and lateral radiographs obtained every month until the 6th postoperative month and every three months thereafter : the time to bony union and femorotibial angle were assessed and the distance between the anterior flange and the fracture line was measured to identify the cause of failure according to the presence of notching. bony union was defined as the absence of fracture site tenderness and false motion, absence of pain on full weight - bearing, and the presence of bridging of three of the four cortices on the anteroposterior and lateral radiographs. all the operations were performed by a single surgeon under general or spinal anesthesia using zplp (zimmer periarticular locking plate ; zimmer inc., warsaw, in, usa) in 8 patients and lcp - df (locking compressing plate - distal femur ; synthes, solothurn, switzerland) in 6 patents. the patient was placed in the supine position on the operating table and the unaffected side was lowered to facilitate identification of the trans - lateral view during surgery., direct reduction and fixation were performed through a lateral parapatellar arthrotomy. in the proximal area, a long incision was made for screw fixation of the lcp - df, whereas several small incisions were made using a jig system for fixation of each screw in cases of zplp. after reduction under traction, c - arm fluoroscopy was used to verify the reduction state. the knee was flexed on the towel bump placed beneath the femoral supracondyle and the distal femur was hyperextended. when anatomical reduction was considered obtained by adjusting the axis, rotation, and length, a metal plate was placed to be over the fracture site to the metaphysis. if necessary, the metal was contoured to fit the bone shape using a plate bender. to maintain the length, the first screw was fixated to be in parallel with the joint surface, and more than 4 screws were used in the region distal to the fracture line and more than 3 screws in the proximal region. continuous passive motion exercises of the hip and knee were initiated within 5 - 7 days after surgery when pain was subdued. partial weight - bearing with crutches was permitted at 6 weeks after surgery depending on the type of fracture and was gradually increased according to the degree of osseous bridging on radiographs. for statistical analysis, the pre- and postoperative clinical results and the immediate postoperative and last follow - up radiological results were compared using a paired t - test. the distance between the fracture line and the anterior femoral flange according to the presence of notching was compared using the mann - whitney test. the mean rom was decreased from 108.414.6 (range, 80 to 130) preoperatively to 107.320.3 (range, 70 to 130) at the last follow - up, but the change was not statistically significant (p>0.05). the functional score and knee society score decreased between the preoperative and last follow - up examinations from 82.37.3 points to 77.18.6 points and from 82.69.8 points to 78.911.6 points, respectively, but no statistical significance was observed in the changes (p>0.05). radiographic bony union was observed at 3.90.9 months (range, 2 to 5 months) after surgery. the femorotibial angle was decreased from 6.10.9 valgus preoperatively to 4.62.8 valgus postoperatively, but the change was not statistically significant (p>0.05). 1) was measured as 58.418.9 mm in patients without notching (n=9) and 1.84.6 mm in patients with notching (n=5), showing statistically significant difference according to the presence of notching (p=0.011). when the patients were subdivided according to the presence of notching, there was no significant difference in radiological results between groups (table 2). according to the presence of notching, there are radiographs after tka state, preoperatively, and immediately postoperatively in fig. 2. at the last follow - up, complications, such as infection, nerve or vascular damage, fixation failure, and component loosening, were not noted. periprosthetic fractures after tka are complicated by delayed union, nonunion, or metal failure in 25% to 75% of the cases4,8,11). most of the fractures occur due to combined action of rotational and axial force in the event of a slip and fall, an accident, or a fall12,13). the treatment can be challenging due to following reasons : 1) most of the fractures occur in the elderly patients ; 2) osteoporosis, periprosthetic osteolysis, and limited capacity for bony union are present in most cases ; and 3) instrument insertion is difficult due to the implanted joint prosthesis8,14 - 18). a variety of treatment methods have been introduced to overcome these difficulties and the common principles of those methods are to secure stable fixation and facilitate early joint movement. thus, a treatment method should be determined by the prospect of maintaining proper rom after surgery, preserving mechanical axis of the femur, and achieving internal fixation for bony union. however, it has been associated with a high incidence of nonunion, does not allow for early joint exercises, and requires a prolonged period of bed rest. accordingly, bed sore and cardiorespiratory dysfunctions have become major problems in the elderly patients treated conservatively. culp.12) reported that rom was reduced in 15 of the 30 patients after conservative treatment and harlow and hofmann19) reported that surgical intervention was necessitated in 29 out of 142 patients after a conservative treatment ; thus, they recommended surgical approaches for periprosthetic fractures. different surgical measures may be employed according to the stability of tka. in general, rorabeck.10) classification type 3 fractures are treated with revision tka due to the presence of implant instability, whereas type 1 and 2 fractures have been treated with open reduction and metal fixation, increasing the chances of nonunion, breakage of an internal fixation device, and infection due to excessive soft tissue dissection. on the other hand, retrograde intramedullary nailing may not be feasible if the intercondylar region of the femoral component is narrow, a fracture line is extended to the lateral cortex due to the difficulty of securing strong fixation with locking screws, or the knee prosthesis has a box20 - 22). recently, locking compression metal plates have been introduced as an alternative to these metal plates and screws. surgical techniques using the new metal plates that exhibit high biomechanical strength facilitates firm fixation through indirect reduction of major bone fragments without anatomical fracture reduction and bony union through preservation of blood supply to bone fragments. in the meantime, there has been advancement in minimally invasive plate osteosynthesis technique that minimizes soft tissue damage. this technique enables strong fixation of distal fragments, allows for multiple screw fixation for insufficient bone fragments, and provides strong resistance to varus force. nayak.9) reported that bony union was obtained in all cases and satisfying knee function score was achieved in 93.5% after minimally invasive plate osteosynthesis for distal femoral fractures, and phillips and christie23) showed satisfying results. in our study, there was no significant change in the rom of the knee, functional score, and knee society score after minimally invasive plate osteosynthesis for periprosthetic fractures following tka. kregor.24) reported that minimally invasive plate osteosynthesis resulted in a mean of 90 of rom and bony union in all cases (n=13), and bone grafting was required in 8%. in the study by kolb.25) the mean postoperative rom was 102, bony union was obtained in the total 23 cases, and varus malalignment was noted in 4%. in our study, bony union was obtained in all patients as was in the above - mentioned studies and the postoperative complication rate was lower. in our study, we paid attention to the presence of notching as a risk factor for fracture because it is responsible for most of the fractures after tka. the distance between the anterior femoral flange and the fracture line was shorter in patients with notching than those without. it is our understanding notching blocks load transfer from the femoral metaphysis to diaphysis, resulting in concentration of the load on the femoral epicondyle. thus, care should be taken during tka to prevent a fracture caused by notching. although minimally invasive osteosynthesis using locking compression plates yielded satisfying results in all of the 14 patients, we think the results should be confirmed in further studies involving larger study populations. in addition, the influence of metal plates from different manufacturers on the study results should also be taken into consideration. the distance from the fracture line to the anterior femoral flange was shorter when notching was present in a periprosthetic fracture after tka. minimally invasive plate osteosynthesis can be considered as a promising surgical treatment technique that provides good results without any complications.	purposeto evaluate the outcomes of the treatment of distal femoral fractures using minimally invasive plate osteosynthesis following total knee arthroplasty (tka).materials and methodsfrom july 2008 to october 2011, 14 patients were treated with minimally invasive plate osteosynthesis for periprosthetic fractures following tka. the mean duration of follow - up was 19.2 months and the mean age was 69.7 years. lewis and rorabeck classification was used to categorize the type of fracture. pre- and postoperative range of motion, femorotibial angle, and knee society score, time to bony union, and complications were evaluated.resultsthe mean range of motion was 108.4 degrees preoperatively and 107.3 degrees postoperatively. no significant difference was observed in the pre- and postoperative mean range of motion. the average time to bony union was 3.9 months. the knee society score was 82.6 points preoperatively and 78.9 points postoperatively. the mean femorotibial angle was changed from 6.1 degrees valgus postoperatively to 4.6 degrees valgus postoperatively. there was no complication during the follow-up.conclusionsminimally invasive plate fixation for distal femur fractures after tka showed good results. minimally invasive plate osteosynthesis is a recommendable treatment method for periprosthetic fractures.
the homeotic / hox genes encode a network of evolutionarily conserved homeodomain transcription factors that are involved in the specification of segmental identity along the anterior - posterior body axis of animals as diverse as insects and vertebrates [1, 2, 3, 4, 5, 6 ]. in drosophila, these genes are arranged on the chromosome in two gene clusters known as the antennapedia and bithorax complexes. there is a correlation between the relative position of the hox genes within the cluster and their spatial and temporal expression pattern in the body in that genes located towards the 3 ' end are expressed more anterior and earlier than genes located towards the 5 ' end (spatial and temporal colinearity) [7, 8, 9, 10, 11 ]. given their central role in developmental processes, it has been proposed that the homeoproteins do not act directly to specify morphological differences but rather control a battery of subordinate genes encoding cellular functions directly required in differentiation [12, 13 ]. in search of these subordinate genes, various strategies such as enhancer trapping, immunoprecipitation of chromatin fragments, subtractive hybridization, selection for binding sites in yeast, and heat - shock - induced overexpression have been used [9, 14, 15, 16, 17, 18, 19, 20, 21].only a small number of target genes of homeoproteins have been identified to date, however ; most of these encode either transcription factors or cell - signaling molecules. in contrast to these results, recent studies suggest that homeoproteins may bind at significant levels to the majority of genes in the drosophila embryo and regulate a large number of downstream genes [22, 23 ]. here it is the most proximal gene within the drosophila antennapedia complex ; it encodes an antennapedia - like q50 homeodomain transcription factor and is one of the most anteriorly expressed homeotic genes along the anterior - posterior body axis [24, 25, 26, 27 ]. genetic studies have demonstrated that lab is required for proper head formation and for the specification of cellular identity in the midgut as well as in the embryonic brain. the lab gene and its vertebrate hox1 orthologs are among the best - characterized examples of evolutionary conservation of structure, expression and function of hox genes in animal development [31, 32, 33, 34, 35 ]. to address the question of which and how many downstream genes are under control of lab, we used a combination of in vivo overexpression techniques and quantitative transcript imaging with oligonucleotide arrays. by using transgenic flies carrying the lab gene under the control of a heat - inducible promoter, we ubiquitously overexpressed lab following heat - shock treatment in drosophila embryos. we then used high - density oligonucleotide arrays representing 1,513 identified drosophila genes for large - scale detection and quantification of induced gene expression [36, 37, 38, 39 ]. quantitative reverse - transcriptase pcr on a selection of these genes verified the differential expression levels in response to heat - shock - induced overexpression of lab. our findings identify a number of novel candidate downstream genes for lab and thus show that oligonucleotide arrays are powerful tools for analyzing, at a genome - wide level, the number, identity and quantitative expression level of genes in the drosophila embryo. in this study, transgenic fly strains carrying the lab coding sequence under control of the heat - inducible hsp70 promotor were used. stage 10 - 17 embryos were given a 25 minute heat pulse to overexpress lab, and allowed to recover for 25 minutes (see materials and methods for heat - shock protocol). ubiquitous overexpression of lab was verified by whole mount in situ hybridization with a lab - specific antisense rna probe. ubiquitous overexpression of labial protein (lab) was verified by immunocytochemistry with an anti - lab antibody. these experiments demonstrated that both lab rna and lab protein were strongly overexpressed 50 minutes after the onset of heat shock in these strains (figure 1). wild - type control flies were subjected to an identical heat - shock regime. following ubiquitous overexpression of lab, transcript profiles were analyzed using a high - density oligonucleotide array and compared to the transcript profiles of heat - shocked wild - type control embryos. for each of the two experimental conditions (' hs - wt'and ' hs - lab '), four replicate experiments were performed and the data set was analyzed with an unpaired t - test (see and materials and methods). the genes represented on the oligonucleotide array correspond to probe sets that are complementary to 1,513 identified and sequenced drosophila genes. most of these genes can be grouped into 14 functional categories according to the nature of the encoded protein. at a significance level of p 0.01, a total of 96 genes were found to be differentially regulated following lab overexpression compared with heat - shocked wild - type control embryos. this corresponds to 6.3% of the genes represented on the array. at a significance level of p 0.05, 205 genes were found to be differentially regulated following lab overexpression compared with heat - shocked wild - type control embryos (data not shown). the relative distribution of lab - regulated genes in particular functional classes, as well as the percentage of genes regulated within a given functional class, were comparable between the p 0.01 group and the p 0.05 group. only genes that were differentially expressed at a significance level of p 0.01 are considered further. we propose these genes to be potential direct or indirect downstream targets for the homeodomain transcription factor labial. when ubiquitously expressed in the embryo, lab caused a significant transcriptional response among a wide variety of genes belonging to all functional classes represented on the array (table 1). the functional class with the highest absolute number of differentially regulated genes was ' transcriptional regulation ' (n = 20). other functional classes with high numbers of differentially regulated genes were ' metabolism ' (n = 13), ' proteolytic systems / apoptosis ' (n = 12), ' cell - surface receptors / cell adhesion molecules (cams)/ion channels ' (n = 12), and ' rna binding ' (n = 7). relative to the number of genes represented on the array within a given functional class, the highest relative percentage of differentially regulated genes was found in the functional classes ' proteolytic systems / apoptosis ' (19.4%), ' cell cycle ' (13.5%), ' transposable elements ' (11.4%), ' chromatin structure ' (11.1%), ' rna binding ' (11.9%), and ' transcriptional regulation ' (7.6%). figure 2 shows the lab - regulated genes and presents a quantitative representation of the change in expression levels for these genes. of the 96 genes that were differentially regulated, 48 showed increased expression levels and 48 showed decreased expression levels the gene with the highest increase in expression level (26-fold) was lab itself, in accordance with our experimental procedure. increases in expression levels above 10-fold were also observed for bicaudal c (bicc), swallow (swa) and oskar (osk), all encoding proteins involved in rna binding, as well as for the wings apart - like (wapl) gene belonging to the functional class ' chromatin structure '. the increased expression levels in bicc, swa, and osk are surprising, as all these genes are known to function as maternal control genes during early embryogenesis [41, 42 ]. as lab activity is normally only observed from gastrulation onwards, this suggests that high levels of widespread ectopic lab expression are able to activate genes which under wild - type conditions show spatio - temporal expression domains that do not overlap with that of lab. one encodes the enzyme ubiquitin carboxy - terminal hydrolase, whose mammalian homolog has also been found to be differentially upregulated by ectopic overexpression of the lab ortholog hoxa1. increased expression levels in the 1.5 - 5-fold range were prominent in several functional classes. for example, in the functional class ' proteolytic systems / apoptosis ', 12 of 13 differentially regulated genes were upregulated and most of these showed increased expression levels ranging between 1.5 and 5. strikingly, in the functional classes ' cell cycle ' and ' transcription / replication / repair ' all the differentially regulated genes were upregulated. thus, differentially expressed genes such as twine (twe), cyclin b (cycb) and cyclin d (cycd), belonging to the functional category ' cell cycle ', were all upregulated following lab overexpression. it is notable in this respect that recent experiments carried out on mammalian cell lines showed that ectopic overexpression of the lab ortholog hoxa1 also causes differential upregulation of cell - cycle regulatory proteins. decreases in expression levels in the 10-fold and above range were not observed, and decreases in the 5 - 10-fold range were only seen for the transposable r2 rdna element gene. decreased expression levels in the 1.5 - 5-fold range were, however, prominent in the functional class ' transcriptional regulation ' and in the functional class ' cell - surface receptors / cams / ion channels '. thus, almost three - quarters of the differentially regulated genes encoding transcription factors showed significant decreases in expression levels following lab overexpression. for example, the genes prospero (pros), distal - less (dll), tailup / islet (tup), mirror (mirr), huckebein (hkb) and abrupt (ab) were all downregulated. interestingly, it has been shown that distal - less is a direct target of homeotic gene control, and recent genetic studies demonstrated that tailup / islet expression in the lab - specific territory of the embryonic drosophila brain is dependent on lab gene action. as with the functional class ' transcriptional regulation ', 10 out of 12 genes representing the functional category ' cell - surface receptors / cams / ion channels ' were downregulated, including the genes derailed (drl), frizzled 2 (fz2), neurotactin (nrt), neurexin (nrx), rhomboid (rho) and 18 wheeler (18w). as is the case for tailup / islet, neurotactin expression in the lab - specific territory of the embryonic drosophila brain is dependent on lab gene action. the 18w locus has been identified as a binding site of the homeotic protein ubx in polytene chromosomes. to verify the differences in gene expression level after heat - shock - induced overexpression of lab as compared to heat - shocked wild - type embryos, quantitative rt - pcr was performed on selected candidate target genes. changes in expression levels were determined for eight genes that were differentially regulated following lab overexpression, namely lab, swa, ubiquitin conjugating enzyme 4 (ubcd4), twe, cycb, ubiquitin carboxy - terminal hydrolase (uch), scratch (scrt) and phosphoenolpyruvate carboxykinase (pepck). the gene squid (sqd), whose expression level remained unchanged under both experimental conditions, served as a control. as indicated in table 2, these experiments showed that the changes in relative expression level, as measured by rt - pcr, are consistent with the data obtained with the oligonucleotide arrays. we have used a novel combination of manipulative genetics and functional genomics to gain further insight into homeotic gene action in drosophila from a genomic perspective. using inducible overexpression and quantitative transcript imaging through oligonucleotide arrays, we have identified 96 genes (only 6.3% of the 1,513 identified genes represented on the oligonucleotide array) whose expression levels change significantly following lab overexpression. these findings suggest that lab regulates a limited and distinct set of candidate downstream genes. this appears to contrast with previous reports indicating that in late embryogenesis the majority of drosophila genes are under control of homeoproteins [23, 44 ]. it should be stressed, however, that a number of features of our functional genomic analysis prevent a direct comparison with these reports, which are based on dna - binding studies. first, although our analysis can quantify gene expression accurately and simultaneously for many identified genes, the temporal and spatial resolution of our analysis is low. this is because our experimental design averages gene expression throughout the embryo and during several embryonic stages. in consequence, our analysis may fail to detect genes that are only expressed in a small subset of cells or during a very restricted time period in embryogenesis. second, our overexpression protocol makes it difficult to control the level of lab protein as well as the temporal dynamics and stability of this protein. as different levels of a given homeoprotein can have different functional consequences in terms of developmental specificity [29, 45 ], the high level of lab protein may bias the set of candidate downstream target genes identified. third, in our studies lab overexpression is not accompanied by concomitant overexpression of cofactors, which are thought to act together with homeotic proteins to determine their in vivo target specificity [34, 46 ]. it should be noted that the gene mirror, which has been proposed to be an additional cofactor for homeoprotein specificity, was detected as downregulated following lab overexpression. although the question of the total number of target genes that are regulated by homeoproteins in vivo must await further analysis, our genomic perspective of lab gene targets does reveal several specific features of homeoprotein action. first, our results demonstrate that the homeodomain transcription factor lab acts on numerous candidate target genes that also encode transcription factors. the category ' transcriptional regulation ' comprises one of the largest sets of differentially regulated genes following lab overexpression. this is consistent with the idea that homeobox genes establish developmental patterns by acting through a cascade of transcription factors which regulate the expression of their own subset of downstream genes [1, 2, 9, 15 ]. second, our data indicate that upregulation of gene expression is prominent in several functional classes. thus, virtually all of the lab - regulated genes in the functional classes ' cell cycle ', ' transcription / replication / repair ', and ' proteolytic systems / apoptosis ' show increased expression. third, our results show that lab overexpression causes not only widespread activation but also widespread repression of gene expression. thus, of the 96 genes that are potential targets of lab, half are downregulated by overexpression of this homeobox gene. this widespread repression is especially pronounced in the functional classes of ' transcriptional regulation ' and ' cell - surface receptors / cams / ion channels '. for example, following lab overexpression, over 80% of the differentially regulated genes encoding cell - surface receptors / cams / ion channels showed decreased expression. taken together, our results identify a large number of novel candidate downstream genes of the homeodomain transcription factor lab. to our knowledge, most of these 96 identified and sequenced genes have not been previously shown to be lab targets. at present, we do not know which genes are direct targets (regulated directly by lab protein binding to dna regulatory sequences) or indirect targets of lab gene action. furthermore, our results demonstrate that oligonucleotide arrays are useful tools for analyzing, at a genome - wide level, the number, identity and quantitative expression levels of candidate downstream genes differentially regulated in vivo by developmental control genes. this confirms the general utility of microarrays for studying diverse molecular and cellular processes in drosophila [48, 49, 50 ]. considering the evolutionary conservation of gene structure, expression and function [1, 35 ], we propose that these results obtained in drosophila will also be valid for lab orthologs in other animals, including vertebrates. it will now be important to determine which of the detected candidate downstream genes in drosophila are direct targets and how they exert the developmental genetic programs imposed by lab gene action. overexpression of lab, we used the line p(whs - lab) with a heat - shock lab construct homozygous on the x chromosome. all fly stocks were kept on standard cornmeal / yeast / agar medium at 25c. embryos were collected overnight for 12 h on grape juice plates, further kept for 4 h at 25c and then subjected to a 36c heat shock for 25 min, followed by a recovery period of 25 min at 25c before rna isolation. therefore, at the time of rna isolation these embryos were at embryonic stages 10 - 17 (stages according to). embryos younger than embryonic stage 10 were not used, as heat shock in these earlier stages results in lethality. for in situ hybridization, digoxigenin - labeled sense and antisense lab rna probes were generated in vitro, with a dig labeling kit (roche diagnostics) and hybridized to whole - mount embryos following standard procedures. hybridized transcripts were detected with an alkaline phosphatase - conjugated anti - digoxigenin fab fragment (roche diagnostics) using nitro blue tetrazolium (nbt) and 5-bromo-4-chloro-3-indolyl phosphate (bcip) (sigma) as chromogenic substrates. the histochemical staining was performed using the vectastain elite abc kit (vector laboratories). embryos were mounted in canada balsam (serva) and photographed with a prog / res/3008 digital camera (kontron electronic) on a zeiss axioskop microscope with differential interference contrast optics. gene expression analysis was performed as described, using a custom - designed drosophila oligonucleotide array (roez003a ; affymetrix). the genes represented on the array and considered in this study correspond to 1,513 sequenced drosophila genes encoding open reading frames deposited in swiss - prot / trembl databases as of spring 1998. for a complete list of these genes each gene is represented on the array by a set of 20 oligonucleotide probes (25mers) matching the gene sequence. to control the specificity of hybridization, the same set of probes, containing a single nucleotide mismatch in a central position, is represented on the array. the difference between the perfect match hybridization signal and the mismatch signal is proportional to the abundance of a given transcript and calculated as its average difference value (avg diff). drosophila genes that were not unambiguously represented by a probe set of 20 probe pairs on the array were excluded from further analysis (29 probe sets were not used in this study). initial experiments designed to determine the sensitivity and reproducibility of hybridization showed that the use of total rna versus poly(a) rna as a template for cdna synthesis and subsequent amplification (synthesis of crna) gave comparable results, despite the fact that we consistently detected 5s rna and histone genes present on the array with crna derived from total rna. on the basis of these findings, all experiments were carried out using a total rna protocol. total rna was isolated from 200 mg of embryonic tissue, using guanidinium isothiocyanate in combination with acidic phenol (ph 4.0) (fast rna tube green kit from bio101) in a fast prep homogenizer fp120 (bio 101). after precipitation, the rna was dissolved in depc - treated water (ambion) and spectrophotometrically quantified using a genequant rna / dna calculator (pharmacia biotech). cdna was synthesized upon total rna as a template, using the superscript choice system for cdna synthesis (gibco / brl) with a t7-(t)24 dna primer. this primer (5'-ggccagtgaattgtaatacgactcactatagggaggcgg-(t)24vn-3 ') was purified by page. for first - strand cdna synthesis, a typical 40 l reaction contained 25 g rna, 200 pmol t7-(t)24 primer, 500 m of each dntp and 800 units reverse transcriptase (amv superscript ii). second - strand cdna synthesis was carried out at 18c for 2 h in a total volume of 340 l, using 20 units escherichia coli dna ligase, 80 units e. coli dna polymerase i and 4 units rnase h in the presence of 250 m of each dntp. after second - strand cdna synthesis, 0.5 l rnase a (100 mg / ml) (qiagen) was added and the samples were incubated at 37c for 30 min. thereafter, 7.5 l proteinase k (10 mg / ml) (sigma) was added and the samples were further incubated at 37c for another 30 min. after cdna synthesis was completed, samples were phenol - chloroform extracted, using phase lock gel (5 prime-3 prime) and ethanol precipitated. biotinylated antisense crna was synthesized from the dsdna template, using t7 rna polymerase (megascript t7 kit : ambion.). a 20 l reaction volume contained between 0.3 - 1.5 g cdna, 7.5 mm of both atp and gtp, 5.6 mm of both utp and ctp and 1.8 mm of both biotinylated bio-16-utp and bio-11-ctp (enzo diagnostics) and 2 l 10x t7 enzyme mix. the reaction was incubated at 37c for 8 h. thereafter, the unincorporated ntps were removed by running the sample over an rneasy spin column (qiagen). samples were precipitated, taken up in 20 l depc - treated water and spectrophotometrically quantified. thereafter, 40 g of the biotinylated antisense crna was fragmented by heating the sample to 95c for 35 min in a volume of 25 l, containing 40 mm tris - acetate (ph 8.1), 100 mm potassium acetate, 30 mm magnesium acetate. after the fragmentation gene chips were pre - hybridized with 220 l hybridization buffer (1x mes (ph 6.7), 1 m nacl, 0.01% triton, 0.5 g/l acetylated bsa, 0.5 g/l sonicated herring sperm dna) for 15 min at 45c on a rotisserie (heidolph) at 60 rpm. hybridization was done in a final volume of 220 l hybridization buffer, containing 40 g fragmented biotinylated crna. hybridizations were carried out for 16 h at 45c with mixing on a rotisserie at 60 rpm. after hybridization, the arrays were briefly rinsed with 6x sspe - t (0.9 m nacl, 0.06 m nah2po4, 6 mm edta, 0.01% triton) and washed on a fluidics station (affymetrix). hybridized arrays were stained with 220 l detection solution (1x mes buffer, containing 2.5 l streptavidin - r phycoerythrin conjugate (1 mg / ml) (molecular probes)) and 2.0 mg / ml acetylated bsa (sigma) at 40c for 15 min and washed again. pixel intensities were measured with a commercial confocal laser scanner (hewlett packard) and expression signals were analyzed with commercial software (genechip 3.1 ; affymetrix). detailed data analysis was carried out using race - a (roche), access 97 and excel 97 (microsoft) software. for quantification of relative transcript abundance the normalized average difference value (avg diff) was used. for each of the three experimental conditions (wt, hs - wt, hs - lab), four replicates were carried out (for the experimental conditions wt and hs - wt see, including the supplementary data). for the difference of the means of the avg diff values over the four replicates between condition 1 (hs - wt) and condition 2 (hs - lab), a t - test was performed. moreover, for downregulation, the mean avg diff value of a gene had to be above or equal to 50 in condition 1 ; for upregulation, the mean avg diff value of a gene had to be above or equal to 50 in condition 2. genes which had a normalized avg diff below 20 obtained automatically an avg diff of 20 (race - a protocol). to obtain a comprehensive analysis of the number and identity of genes differentially regulated by lab, candidates that were already differentially expressed in heat - shocked wild - type embryos compared to non - heat - shocked wild - type controls, were excluded from further analysis (and data not shown). previously, we have used quantiative rt - pcr to confirm that relative expression level changes in the 1.5-fold and above range, as detected on this array, accurately reflect differences in mrna abundance in vivo in drosophila embryos. in consequence, in this report only relative expression level changes in the 1.5-fold and above range are presented. three hundred nanograms of poly(a) rna, isolated from heat - shocked wild - type embryos and heat - shocked hs - lab embryos (mrna isolation kit ; roche diagnostics), was reverse transcribed with amv - rt and random hexamers (first - strand cdna synthesis kit for rt - pcr ; roche diagnostics). pcr was performed with 100 pg of template dna and gene - specific primers (designed, using seq web, wisconsin package version 10.0 ; gcg) on a light cycler (lightcycler ; roche diagnostics). continuous fluorescence observation of amplifying dna was done using sybr green i (lightcycler - faststart dna master sybr greeni ; roche diagnostics). after cycling, a melting curve was produced by slow denaturation of the pcr end - products to check the specificity of amplification. to compare the relative amounts of pcr products, we monitored the amplification profile on a graph, displaying the log of the fluorescence against the number of cycles. relative fold changes for a given gene under both conditions (heat shock wt versus heat shock hs - lab) were calculated using the fit point method (lightcycler operator 's manual, version 3.0 ; roche diagnostics). the genes represented on the high - density oligonucleotide array were grouped into 14 functional classes according to the function of the gene product and currently available genetic data. for this, notations in flybase, interactive fly and swiss - prot / trembl databases were used. a comprehensive presentation of all the genes represented on the oligonucleotide array as well as their attribution to functional classes is given as supplementary data to. overexpression of lab, we used the line p(whs - lab) with a heat - shock lab construct homozygous on the x chromosome. all fly stocks were kept on standard cornmeal / yeast / agar medium at 25c. embryos were collected overnight for 12 h on grape juice plates, further kept for 4 h at 25c and then subjected to a 36c heat shock for 25 min, followed by a recovery period of 25 min at 25c before rna isolation. therefore, at the time of rna isolation these embryos were at embryonic stages 10 - 17 (stages according to). embryos younger than embryonic stage 10 were not used, as heat shock in these earlier stages results in lethality. for in situ hybridization, digoxigenin - labeled sense and antisense lab rna probes were generated in vitro, with a dig labeling kit (roche diagnostics) and hybridized to whole - mount embryos following standard procedures. hybridized transcripts were detected with an alkaline phosphatase - conjugated anti - digoxigenin fab fragment (roche diagnostics) using nitro blue tetrazolium (nbt) and 5-bromo-4-chloro-3-indolyl phosphate (bcip) (sigma) as chromogenic substrates. the histochemical staining was performed using the vectastain elite abc kit (vector laboratories). embryos were mounted in canada balsam (serva) and photographed with a prog / res/3008 digital camera (kontron electronic) on a zeiss axioskop microscope with differential interference contrast optics. gene expression analysis was performed as described, using a custom - designed drosophila oligonucleotide array (roez003a ; affymetrix). the genes represented on the array and considered in this study correspond to 1,513 sequenced drosophila genes encoding open reading frames deposited in swiss - prot / trembl databases as of spring 1998. for a complete list of these genes each gene is represented on the array by a set of 20 oligonucleotide probes (25mers) matching the gene sequence. to control the specificity of hybridization, the same set of probes, containing a single nucleotide mismatch in a central position, is represented on the array. the difference between the perfect match hybridization signal and the mismatch signal is proportional to the abundance of a given transcript and calculated as its average difference value (avg diff). drosophila genes that were not unambiguously represented by a probe set of 20 probe pairs on the array were excluded from further analysis (29 probe sets were not used in this study). initial experiments designed to determine the sensitivity and reproducibility of hybridization showed that the use of total rna versus poly(a) rna as a template for cdna synthesis and subsequent amplification (synthesis of crna) gave comparable results, despite the fact that we consistently detected 5s rna and histone genes present on the array with crna derived from total rna. on the basis of these findings, all experiments were carried out using a total rna protocol. total rna was isolated from 200 mg of embryonic tissue, using guanidinium isothiocyanate in combination with acidic phenol (ph 4.0) (fast rna tube green kit from bio101) in a fast prep homogenizer fp120 (bio 101). after precipitation, the rna was dissolved in depc - treated water (ambion) and spectrophotometrically quantified using a genequant rna / dna calculator (pharmacia biotech). cdna was synthesized upon total rna as a template, using the superscript choice system for cdna synthesis (gibco / brl) with a t7-(t)24 dna primer. this primer (5'-ggccagtgaattgtaatacgactcactatagggaggcgg-(t)24vn-3 ') was purified by page. for first - strand cdna synthesis, a typical 40 l reaction contained 25 g rna, 200 pmol t7-(t)24 primer, 500 m of each dntp and 800 units reverse transcriptase (amv superscript ii). second - strand cdna synthesis was carried out at 18c for 2 h in a total volume of 340 l, using 20 units escherichia coli dna ligase, 80 units e. coli dna polymerase i and 4 units rnase h in the presence of 250 m of each dntp. after second - strand cdna synthesis, 0.5 l rnase a (100 mg / ml) (qiagen) was added and the samples were incubated at 37c for 30 min. thereafter, 7.5 l proteinase k (10 mg / ml) (sigma) was added and the samples were further incubated at 37c for another 30 min. after cdna synthesis was completed, samples were phenol - chloroform extracted, using phase lock gel (5 prime-3 prime) and ethanol precipitated. biotinylated antisense crna was synthesized from the dsdna template, using t7 rna polymerase (megascript t7 kit : ambion.). a 20 l reaction volume contained between 0.3 - 1.5 g cdna, 7.5 mm of both atp and gtp, 5.6 mm of both utp and ctp and 1.8 mm of both biotinylated bio-16-utp and bio-11-ctp (enzo diagnostics) and 2 l 10x t7 enzyme mix. the reaction was incubated at 37c for 8 h. thereafter, the unincorporated ntps were removed by running the sample over an rneasy spin column (qiagen). samples were precipitated, taken up in 20 l depc - treated water and spectrophotometrically quantified. thereafter, 40 g of the biotinylated antisense crna was fragmented by heating the sample to 95c for 35 min in a volume of 25 l, containing 40 mm tris - acetate (ph 8.1), 100 mm potassium acetate, 30 mm magnesium acetate. gene chips were pre - hybridized with 220 l hybridization buffer (1x mes (ph 6.7), 1 m nacl, 0.01% triton, 0.5 g/l acetylated bsa, 0.5 g/l sonicated herring sperm dna) for 15 min at 45c on a rotisserie (heidolph) at 60 rpm. hybridization was done in a final volume of 220 l hybridization buffer, containing 40 g fragmented biotinylated crna. hybridizations were carried out for 16 h at 45c with mixing on a rotisserie at 60 rpm. after hybridization, the arrays were briefly rinsed with 6x sspe - t (0.9 m nacl, 0.06 m nah2po4, 6 mm edta, 0.01% triton) and washed on a fluidics station (affymetrix). hybridized arrays were stained with 220 l detection solution (1x mes buffer, containing 2.5 l streptavidin - r phycoerythrin conjugate (1 mg / ml) (molecular probes)) and 2.0 mg / ml acetylated bsa (sigma) at 40c for 15 min and washed again. pixel intensities were measured with a commercial confocal laser scanner (hewlett packard) and expression signals were analyzed with commercial software (genechip 3.1 ; affymetrix). detailed data analysis was carried out using race - a (roche), access 97 and excel 97 (microsoft) software. for quantification of relative transcript abundance the normalized average difference value (avg diff) was used. for each of the three experimental conditions (wt, hs - wt, hs - lab), four replicates were carried out (for the experimental conditions wt and hs - wt see, including the supplementary data). for the difference of the means of the avg diff values over the four replicates between condition 1 (hs - wt) and condition 2 (hs - lab), a t - test was performed. moreover, for downregulation, the mean avg diff value of a gene had to be above or equal to 50 in condition 1 ; for upregulation, the mean avg diff value of a gene had to be above or equal to 50 in condition 2. genes which had a normalized avg diff below 20 obtained automatically an avg diff of 20 (race - a protocol). to obtain a comprehensive analysis of the number and identity of genes differentially regulated by lab, candidates that were already differentially expressed in heat - shocked wild - type embryos compared to non - heat - shocked wild - type controls, were excluded from further analysis (and data not shown). previously, we have used quantiative rt - pcr to confirm that relative expression level changes in the 1.5-fold and above range, as detected on this array, accurately reflect differences in mrna abundance in vivo in drosophila embryos. in consequence, in this report only relative expression level changes in the 1.5-fold and above range are presented. three hundred nanograms of poly(a) rna, isolated from heat - shocked wild - type embryos and heat - shocked hs - lab embryos (mrna isolation kit ; roche diagnostics), was reverse transcribed with amv - rt and random hexamers (first - strand cdna synthesis kit for rt - pcr ; roche diagnostics). pcr was performed with 100 pg of template dna and gene - specific primers (designed, using seq web, wisconsin package version 10.0 ; gcg) on a light cycler (lightcycler ; roche diagnostics). continuous fluorescence observation of amplifying dna was done using sybr green i (lightcycler - faststart dna master sybr greeni ; roche diagnostics). after cycling, a melting curve was produced by slow denaturation of the pcr end - products to check the specificity of amplification. to compare the relative amounts of pcr products, we monitored the amplification profile on a graph, displaying the log of the fluorescence against the number of cycles. relative fold changes for a given gene under both conditions (heat shock wt versus heat shock hs - lab) were calculated using the fit point method (lightcycler operator 's manual, version 3.0 ; roche diagnostics). the genes represented on the high - density oligonucleotide array were grouped into 14 functional classes according to the function of the gene product and currently available genetic data. for this, notations in flybase, interactive fly and swiss - prot / trembl databases were used. a comprehensive presentation of all the genes represented on the oligonucleotide array as well as their attribution to functional classes is given as supplementary data to. we thank jan mous, adrian roth, michel tessier, monika seiler and reto brem for essential contributions and helpful advice. we thank clemens broger, martin strahm and martin neeb (f. hoffman - la roche) for allowing us to use their race - a chip analysis software and volker schmid and natalie yanze for help with the light cycler. this research was supported by grants from the snsf and the eu - biotech program (to h.r.) and by f. hoffmann - la roche. heat - shock - driven ubiquitous overexpression of lab monitored by in situ hybridization and immunocytochemistry. (a - d) rna in situ hybridization ; (e - h) immunocytochemical staining. expression of lab is shown in heat - shocked wild - type embryos (a, c, e, g) and in heat - shocked embryos carrying a hs - lab construct (b, d, f, h). (a, b, e, f) overview of stage 10 - 17 embryos. (c, d) higher magnification of a single stage 15 embryo and (g, h) a single stage 13 embryo ; lateral view, and anterior to the left. embryos were exposed to a heat shock at 36c for 25 min and were allowed to recover for another 25 min before fixation. genes differentially expressed in response to heat - shock - induced overexpression of lab, grouped according to functional classes. bars represent the fold change between differentially expressed genes in heat - shocked wild - type embryos and heat - shocked hs - lab embryos. positive values indicate that the relative expression level of a gene is increased (upregulated) following lab overexpression and negative values indicate a decrease (downregulated). absolute average difference (avg diff ; see materials and methods) values are given for the lab overexpression condition as follows : white bars represent avg diff 1,000. genes differentially expressed in response to lab overexpression genes that are differentially expressed following heat - induced ubiquitous overexpression of lab in stage 10 - 17 hs - lab embryos, grouped according to functional classses. the functional class ' heat - shock proteins ' was excluded from the analysis (see materials and methods). n, number of genes within a functional group present on the chip ; n, number of genes differentially expressed within a functional group following lab overexpression ; n / n 100, number of differentially expressed genes within a functional class following lab overexpression, given as a percentage of the total number of genes in this class present on the array ; downregulated, total number of genes within each functional class differentially downregulated following lab overexpression ; upregulated, total number of genes within each functional class differentially upregulated following lab overexpression. comparison of fold change between oligonucleotide arrays and rt - pcr rt - pcr was performed on cdna derived from heat - shocked wild - type embryos and heat - shocked hs - lab embryos. fold changes determined by rt - pcr are represented as the mean values of eight independent replicates, derived from two different cdna preparations.	background : homeotic genes are key developmental regulators that are highly conserved throughout evolution. their encoded homeoproteins function as transcription factors to control a wide range of developmental processes. although much is known about homeodomain - dna interactions, only a small number of genes acting downstream of homeoproteins have been identified. here we use a functional genomic approach to identify candidate target genes of the drosophila homeodomain transcription factor labial.results:high-density oligonucleotide arrays with probe sets representing 1,513 identified and sequenced genes were used to analyze differential gene expression following labial overexpression in drosophila embryos. we find significant expression level changes for 96 genes belonging to all functional classes represented on the array. in accordance with our experimental procedure, we expect that these genes are either direct or indirect targets of labial gene action. among these genes, 48 were upregulated and 48 were downregulated following labial overexpression. this corresponds to 6.3% of the genes represented on the array. for a selection of these genes, we show that the data obtained with the oligonucleotide arrays are consistent with data obtained using quantitative rt-pcr.conclusions:our results identify a number of novel candidate downstream target genes for labial, suggesting that this homeoprotein differentially regulates a limited and distinct set of embryonically expressed drosophila genes.
cervical cancer is the second most common cancer among women worldwide and the most common cancer among women in mexico. more than 100 hpv genotypes have been described, of which approximately 40 are responsible for genital infection. hpv is classified as low or high risk based on its association with premalignant and malignant lesions, respectively. low - risk hpv types include 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, and 81. of these types, 6 and 11 cause 90% of the external anogenital wart cases and low - grade changes in cervical cells. the high - risk types include 16, 18, 31, 33, 35, 45, 52, and 58, of which 16 and 18 cause approximately 70% of all invasive cervical cancer cases. some authors have suggested that geographical differences in hpv distribution may have an impact on the effectiveness of the hpv vaccine in different populations. in 2005, the international agency for research on cancer hpv reported the worldwide distribution of hpv types in women with normal cervical cytology. the most frequent type is hpv 16, followed by hpv 42, 58, 31, 18, 56, 81, 35, 33, and 45. in mexico, hpv types 16, 18, 31, 33, 39, 45, 53, 58, and 59 have frequently been found in normal cervical samples [915 ]. regional data on the prevalence and type distribution of hpv are essential for estimating the impact of vaccines on cervical cancer and developing screening programs. the goal of the present study was to determine the prevalence and distribution of hpv types in women from mexico city. this study was conducted in the clinica de especialidades de la mujer de la secretara de la defensa nacional in mexico city, mexico. a total of 929 women, aged between 18 and 76 years, were recruited for the analysis. the protocol and informed consent documents were approved by the human research ethical committee of the clnica de especialidades de la mujer de la secretara de la defensa nacional. two cervical samples were obtained from each patient using a cytobrush for cytological analysis and hpv detection. cervical smears were used for cytomorphological examination using conventional papanicolaou (pap). for hpv detection, cytobrushes with cervical scrapes were placed in phosphate buffered saline (pbs) and stored at 70c until analysis. pap smears were interpreted by the head of the cytopathology laboratory pathology unit at the clnica de especialidades de la mujer de la secretara de la defensa nacional. dna extraction was performed with the dneasy blood & tissue kit (qiagen, hilden, germany) according to the manufacturer 's protocol. hpv detection and genotyping were performed by multiplex pcr using the hpv4a ace screening kit (seegene) according to the manufacturer 's protocol.the kit is a qualitative, in vitro test for the identification of hpv 16 and 18 and for screening of 16 high - risk hpv (hr) types 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82 and the low - risk hpv types 6 and 11. significant differences between groups with or without hpv were determined by the mann - whitney rank sum test and the fisher 's exact test at p < 0.05. the majority of participants reported one lifetime sexual partner (97%) and only approximately 3% of women reported more than one partner in their sexual history. the age of first intercourse was younger than 18 years for approximately 8.6% of women in the study. conventional pap smears identified normal cytological results or inflammation in 916 of 929 samples (98.6%). only 13 (1.4%) cases displayed abnormal cytology : 10 (1.1%) had low - grade squamous intraepithelial lesions (lsil), 2 (0.2%) had atypical squamous cells of undetermined significance (asc - us), and 1 (0.1%) had squamous cell carcinoma (cc). the age - specific prevalence of hpv is summarized in figure 1, which shows the highest prevalences, 22% and 20%, in women aged less than 20 years and 7076 years, respectively. hpv incidence was significantly higher among women with a history of more than one sexual partner or who were current smokers. there were no differences in the frequency of hpv infection in women who had intercourse younger than 18 years old or those with histories of sexually transmitted diseases (table 1). of the hpv - positive women, 99% had high - risk hpv genotypes and 1% had low - risk hpv genotypes. hpv hr was detected in 43% of women screened in this study : 42% (18) were hpv positive, and 14% (16) were hpv positive, which includes those with both infections. abnormal cervical cytology was detected in only 15.3% of hpv - positive women, and normal cytology was found in the other 84.7% of those cases, as summarized in table 2. in this study, we identified a 9.1% prevalence of hpv : 84.7% of women had normal cytology and 15.3% of women had abnormal cytology. other hpv studies in mexico using an open population of women with normal cervical cytology reported an incidence of hpv between 4.8 and 43.6% [9, 13 ]. a possible explanation for the difference in the reported hpv prevalences includes variables related to hpv acquisition, such as age, age of first sexual intercourse, number of lifetime sexual partners, socioeconomic status, education level, parity, marital status, number of pregnancies, use of hormonal contraceptives, smoking, and interregional variation [2, 9, 15 ]. similar to previous age - specific studies, both in mexico and other regions of the world, we found two age groups with higher hpv prevalences, women less than 20 years old and women 7076 years old. the younger age group (< 20 years old) with a higher incidence of hpv infection may be an indicator of sexual transmission, as it coincides with the initiation of sexual activity. the older age group (7079 years old) of hpv - positive women may have been exposed to high rates of hpv transmission when they were young or may have reactivated latent hpv infections by factors associated with older age. the heterogeneity of hpv genotype distribution in mexico is evident in this study, with the major frequency of hpv hr at 43%, followed by hpv 18 at 42% (including coinfection). similar to previous studies, statistically significant differences in hpv incidence were found among women with a history of more than one sexual partner or who were smokers. no significant differences in hpv frequency were found in women who had intercourse younger than 18 years old or had histories of sexually transmitted diseases. these results could have important implications for future screening procedures to assist the prevention of cervical cancer in mexico. the differences in hpv prevalence and distribution identified in this study have a potential impact on the effectiveness of hpv vaccinations, which may be investigated in future studies.	introduction. cervical cancer is the most common cancer among mexican women. the goal of the present study was to determine the prevalence and distribution of hpv types in women from mexico city. methods. our study was conducted in the clinica de especialidades de la mujer de la secretara de la defensa nacional, mexico. random samples were taken from 929 healthy women requesting a cervical papanicolaou examination. detection and genotyping of hpv were performed by multiplex pcr, with the hpv4a ace screening kit (seegene). results. 85 of nine hundred twenty - nine women (9.1%) were infected with hpv. of hpv - positive women, 99% and 1% had high- and low - risk hpv genotypes, respectively. the prevalence of the 16 high - risk (hr) hpv types that were screened was 43% : 42% (18) were hpv positive and 14% (16) were hpv positive, which includes coinfection. multiple infections with different viral genotypes were detected in 10% of the positive cases. abnormal cervical cytological results were found in only 15.3% of hpv - positive women, while 84.7% had normal cytological results. conclusions. we found a similar prevalence of hpv to previous studies in mexico. the heterogeneity of the hpv genotype distribution in mexico is evident in this study, which found a high frequency of hpv hr genotypes, the majority of which were hpv 18.
previous studies have indicated that different skin types have different levels of skin hydration and barrier function based on bioinstrumental measures of conductance and transepidermal water loss (tewl). another study has indicated that skin hydration and tewl are age - dependent but linked to ethnic skin type. however, another major component of skin that has been less investigated due to its complexity, but still contributes significantly to hydration and barrier function, are the sebaceous lipids. to investigate this topic further, in vivo biochemical experiments on the skin from different ethnic populations living in the same environment are needed. several previous studies have focused on dissecting the relationship between sebum output and the pathophysiology of acne. most of the published studies assess the total sebum output by instrumental analysis (e.g., the sebumeter), self - evaluation or by using a sebum absorbing adhesive tape (sebutape), which is a specially designed tape that has been proven to be reproducible and convenient to estimate sebaceous gland output. additionally, sebutape is used for collecting sebum for further quantitation of its components. to our knowledge no study thus far has measured individual sebaceous lipids in search of specific sebaceous lipids that could be uniquely associated with ethnic groups or would be correlated with instrumental parameters. this is likely due to the cumbersome and lengthy tasks associated with the individual extraction, separation and quantitation of lipids and their corresponding subclasses. to date, a few studies have reported on the correlation of sebaceous lipids and acne status ; however, these studies were conducted in diseased (acne) skin and not in healthy skin. elevated sebum excretion is involved in the pathophysiology of acne, as body sites that are rich in sebaceous glands are where acne lesions are typically manifested. high levels of sebum are associated with acne in adolescence and may offer a possible benefit by lubricating the skin, contributing to a better skin barrier as well as better skin hydration. because acne is unique to humans it has been suggested that the unique sebaceous lipids could be associated with this human - specific disease. the accumulation of squalene and the presence of unique fatty acids and waxes are unique manifestations of sebum. our study aimed to identify possible lipid components with sebum that could correlate with ethnic skin hydration and barrier and might be associated with age or ethnicity. in the current study we conducted a series of in vivo assessments on the skin of women from three different ethnic populations living in the same environment (i.e., same locale). this study was conducted to determine what, if any, differences were quantifiable in skin lipid content across age and ethnic skin type composition between females aged 1825 and 3545 y with no acne or other chronic skin diseases. extracted lipids from each sebutape were subjected initially to thin layer chromatography and three major lipid classes were separated : free fatty acids, triglycerides and wax and cholesterol esters. subsequently, each lipid class was extracted separately from the silica plate and subjected to saponification and derivatization to be prepared for individual fatty acid analysis. this was performed by gas chromatography with fluorescence ionization detector (gc - fid), as previously described. we were able to estimate the individual fatty acid population of each of the lipid classes listed above. the initial pilot study involved male and females from three ethnic groups (n = 1721). skin hydration and tewl were assessed. results showed significant differences (p northern asian > caucasian (fig. 1). the results for tewl demonstrated that african americans and caucasians were again significantly different (p northern asian > african american, which would indicate a better barrier function for african americans with a lower tewl (fig. 1). correlation of hydration and tewl values among three ethnic groups of males and females aged 2045 y. hydration and tewl values were grouped per the total gender and age combined ethnic population examined. higher skicon were value associated with greater hydration (red arrow, left panel). greater tewl (blue arrow, right panel) is associated with worse barrier function (red arrow, right panel). a smaller cohort group due to the labor - intensive methodology was chosen, including females 2025 and 3545 y (n = 68) for all ethnic groups. figure 2 summarizes the quantitative results in micrograms of lipid and the comparative analysis of the sebaceous lipid classes among the three different ethnic groups, total (top panel), 1825 y (middle panel) and 3545 y (bottom panel). it is apparent that the total amount of lipid is higher in the african american groups (150170 ug of fatty acids / sebutape/30 min) than in the caucasian americans (90100 ug of fatty acids / sebutape/30 min) regardless of the age ranges. the sebum levels of asian americans (110130 ug of fatty acids / sebutape/30 min) lies between the two other ethnic groups. the greatest difference and with a statistical significant difference (p < 0.05) among the ethnic groups was observed in the wax ester fraction that is a sebum - specific lipid class. african american females in total had higher levels of wax esters (62 ug of fatty acid / sebutape/30 min) than caucasian american females (40 ug of fatty acid / sebutape/30 min) (fig. 2, top panel). results were expressed as micrograms of fatty acids and were grouped per individual class of lipid. ffa, tg, we and squalane were graphed as well as total lipid, which was expressed as the sum of all of the aforementioned classes (a) for the total group tested and (b) per age group. p < 0.05. abbreviations : ffa, free fatty acids ; tg, triglycerides ; sq, squalene ; we, wax esters. because the overall wax esters fraction analysis demonstrated a significant difference african american females had higher production than the other two ethnic groups we looked at the individual lipid classes and we correlated the lipid analysis data to the skicon values acquired from the same subjects. 3), there was a strong pattern / trend between the level of the wax ester fraction in sebum with skicon values that indicate conductivity and water content. correlation of wax ester fraction and hydration values among three ethnic groups of females aged 2045 y. wax ester results and hydration values were grouped by combining females from all ethnic groups and analyzed by one - way analysis of variance and subsequent t - tests. the wax ester fraction was further analyzed and six fatty acids were identified that were significantly different in quantity (p < 0.05) between african and caucasian americans (table 1). some are synthesized naturally in skin, as the 14:0, 16:1, n10 and 18:1, n9 ; some are acquired exclusively from diet, as the iso-18:2, whereas others may be a product of bacterial metabolism, as the 15:0 or 17:1 fatty acids, which would indicate flora differences on the surface of the skin between these ethnic groups. three of these fatty acids were at higher levels than 1 g / per sebutape/30 min (fig. results were grouped by combining females from all ethnic groups and analyzed by one way analysis of variance and subsequent t - tests. correlation of the most predominant fatty acids from the wax ester fraction among three ethnic groups of females aged 2045 y. results were grouped by combining data from women in all ethnic groups and analyzed by one way analysis of variance and subsequent t - tests. in this study we analyzed sebum, hydration and tewl of subjects from different ethnic and age groups. the class of lipids that was significantly higher in african american women was wax esters. the only mammalian cells that synthesize wax esters are the sebaceous cells ; therefore this class of lipids serves as a marker for sebaceous differentiation. one point of uniqueness in human sebum is that this non - polar lipid class accumulates in unusually high levels (~25%). waxes are long, highly hydrophobic molecules in nature that act as a barrier against excessive hydration or dehydration. waxes could potentially alter the rheological properties of sebum, as it is one of the most non - polar molecules in sebum as well as in nature. because waxes may serve as a lipid marker for sebaceous activity, they could be far more upregulated than the rest of the lipids in cases of oily skin. we also performed the comparative analysis of the lipid classes in females from three different ethnic groups in conjunction with other instrumental parameters such as hydration / conductivity and tewl. in this study, besides demonstrating that african american women have higher amounts of wax esters and sebum than caucasian american women, we coupled these results with skin hydration and barrier function measures as well as instrumental measures. this is the first report that links an individual class of lipids, the wax esters, to a better barrier and higher hydration among three ethnic groups. in fact, the notion that sebum may have a moisturizing or conditioning effect is highly supported by these higher wax ester levels that may act as another layer of protection that enhances the skin s barrier. moreover, we demonstrate that both elevated sebum and higher hydration may contribute to a better skin barrier that is manifested with lower tewl. indeed, evidence in the literature indicates that african americans have better barrier or oilier skin. qualitative analysis was also performed but did not reveal any noteworthy differences among the ethnic groups. significant differences in six fatty acids in the wax ester fraction among the ethnic groups were identified. the most prevalent fatty acid in sebum, sapienic acid (16:1, n10), is significantly higher in african americans and correlated with the higher sebum output in that ethnic group. also higher in the african american group are oleic acid (18:1, n9) and myristic acid (14:0), precursor of palmitic acid (16:0). sapienic acid is the most abundant fatty acid in sebum ; therefore it is normal to be at higher levels in individuals with higher sebum output. the same argument can apply to the second most abundant monounsaturated fatty acid, oleic acid, a known adipogenic fatty acid. the most intriguing differences found are two fatty acids that are in lower levels but do not belong to the mammalian fatty acid metabolism, as they have a chain with an odd number of carbons, 15:0 and 17:1. it is well known that propionibacterium acnes colonize within the philosebaceous hair canal, and they could contribute significantly in the lipid output ; however, the higher presence of these fatty acids in the wax esters of african american females may indicate that (1) some bacterial fatty acids could get incorporated into sebaceous wax synthesis and (2) different ethnic groups could potentially have differences in the skin microflora. the last intriguing result is that we identified significant differences at the level of an isomer of linoleic acid, which is simply a dietary fatty acid. although our analytical method could not give us the exact location of the two double bonds, it is certain that the observed differences could account for different nutritional habits of the different ethnic groups. it would be of interest to perform a blind analysis of this marker in larger cohorts and to retrospectively identify high sebum - producer individuals to correlate with differential wax ester secretion. better analytical techniques would help to increase our understanding of waxes and their precursor, plus their role in the induction or the maintenance of the skin s conditioning state and barrier function. is open for many new discoveries and is constantly enhanced by advances in analytical chemistry. consequent steps could be to repeat this study in another season to determine the impact the climate / environment has on skin hydration, barrier function and lipid production among the three ethnic groups. acne - free subjects of both males and females with no serious dermatological issues from skillman, nj, area were recruited (after signing an informed consent form) from two age groups (1825 and 3545) and three ethnic origins, caucasian, african american and northern asian. each subject underwent a 30 min acclimation period in a temperature- and humidity - controlled room and then was subjected to the following tests on their facial skin : surface hydration measurements with skicon 200ex (ibs co.) ; nova dermal phase meter (nova dpm) ; barrier function measurements (vapometer ; delfin technologies, ltd.) ; multi - modal high resolution digital clinical imaging ; self - assessments ; and skin surface lipid sampling with two pairs of sebutape adhesive patches (cuderm corporation) on the forehead. sebutapes were sent to an external lab (scri, scotland), where the lipid classes were extracted via thin layer chromatography (tlc) and subsamples underwent mass spectroscopy and gc - fid. two pairs of sebutapes were applied for 30 min (to the forehead areas above both eyebrows) after degreasing with a cotton swab soaked in 70% isopropanol alcohol. the dried samples were stored under nitrogen at -20c until processed for tlc and gc - fid analysis according to a method established by mylnefield lipid analysis. in brief, the sample (50 ml) was spotted on a glass tlc plate, which was developed with 80:20:2 isohexane / diethyl ether / formic acid until the solvent front is a short distance from the top. the plate was air - dried and subsequently sprayed with 0.01% primuline to visualize the ffa, tag and ce / we bands under uv light. each marked band was removed from the glass tlc plate and was placed into a test tube, where 1 ml toluene and 2 ml 1% sulphuric acid in methanol were added. 5 ml of 5% nacl solution was added and the samples were extracted with 2 2ml isohexane. extracts were transferred to fresh test tubes that were shaken with 3 ml of 2% potassium hydrogen carbonate and then passed through a prewashed (3 ml isohexane) sodium sulfate column and also subsequently post washed with 2 ml of isohexane. the solvent was removed by nitrogen and isohexane + bht (70 l) before transferring them to a gc vial and injected with 5 l of the sample. a c17:0 was used as an internal standard on a cp - wax 52cb (0.25 mm 25 min 0.2 m) column, flow rate 1 ml / min. the gc was agilent 6890 and the gc was performed using the method published by william christie. overall differences between ethnicity means were tested using one - way analysis of variance models. pairwise comparisons of ethnicities were tested using tukey s test to adjust for multiple comparisons. differences of means between males and females within an ethnic group were tested using t - tests. considering the small sample sizes, significant results were verified via nonparametric tests (kruskal - wallis test for overall difference between ethnicities and wilcoxon rank sum test for pairs). acne - free subjects of both males and females with no serious dermatological issues from skillman, nj, area were recruited (after signing an informed consent form) from two age groups (1825 and 3545) and three ethnic origins, caucasian, african american and northern asian. each subject underwent a 30 min acclimation period in a temperature- and humidity - controlled room and then was subjected to the following tests on their facial skin : surface hydration measurements with skicon 200ex (ibs co.) ; nova dermal phase meter (nova dpm) ; barrier function measurements (vapometer ; delfin technologies, ltd.) ; multi - modal high resolution digital clinical imaging ; self - assessments ; and skin surface lipid sampling with two pairs of sebutape adhesive patches (cuderm corporation) on the forehead. sebutapes were sent to an external lab (scri, scotland), where the lipid classes were extracted via thin layer chromatography (tlc) and subsamples underwent mass spectroscopy and gc - fid. two pairs of sebutapes were applied for 30 min (to the forehead areas above both eyebrows) after degreasing with a cotton swab soaked in 70% isopropanol alcohol. the dried samples were stored under nitrogen at -20c until processed for tlc and gc - fid analysis according to a method established by mylnefield lipid analysis. in brief, the sample (50 ml) was spotted on a glass tlc plate, which was developed with 80:20:2 isohexane / diethyl ether / formic acid until the solvent front is a short distance from the top. the plate was air - dried and subsequently sprayed with 0.01% primuline to visualize the ffa, tag and ce / we bands under uv light. each marked band was removed from the glass tlc plate and was placed into a test tube, where 1 ml toluene and 2 ml 1% sulphuric acid in methanol were added. 5 ml of 5% nacl solution was added and the samples were extracted with 2 2ml isohexane. extracts were transferred to fresh test tubes that were shaken with 3 ml of 2% potassium hydrogen carbonate and then passed through a prewashed (3 ml isohexane) sodium sulfate column and also subsequently post washed with 2 ml of isohexane. the solvent was removed by nitrogen and isohexane + bht (70 l) before transferring them to a gc vial and injected with 5 l of the sample. a c17:0 was used as an internal standard on a cp - wax 52cb (0.25 mm 25 min 0.2 m) column, flow rate 1 ml / min. the gc was agilent 6890 and the gc was performed using the method published by william christie. overall differences between ethnicity means were tested using one - way analysis of variance models. pairwise comparisons of ethnicities were tested using tukey s test to adjust for multiple comparisons. differences of means between males and females within an ethnic group were tested using t - tests. considering the small sample sizes, significant results were verified via nonparametric tests (kruskal - wallis test for overall difference between ethnicities and wilcoxon rank sum test for pairs).	this study was conducted to compare lipid components of sebum from persons from three ethnic backgrounds caucasian, african american and northern asian. men and women with no acne in two age groups (1825 y and 3545 y) were recruited. skin surface hydration (skicon 200ex and novameter), barrier function (delfin vapometer), high - resolution clinical imaging, self - assessments and two pairs of sebutapes on the forehead that extracted the lipids on the surface of their skin were used. significant differences (p northern asian > caucasian. transepidermal water loss (tewl) measurements demonstrated that african americans and caucasians were significantly different (p northern asian > african american, which would indicate better barrier function for african americans with a lower tewl. african american women had more total lipid production than northern asian or caucasian women. when analyzing the three lipid classes (free fatty acids, triglycerides and wax esters), the trend became significant (p < 0.05) in the wax ester fraction when directly comparing african americans with caucasians. additionally, six lipids were identified in the wax ester fractions that were significantly different in quantity (p < 0.05) between african americans and caucasians. these results identified significant differences in sebaceous lipid profiles across ethnic groups and determined that the differences correlated with skin barrier function.
allergic contact dermatitis (acd) is a common cutaneous eczematous disorder caused by contact (either direct or aeromediated) with a range of environmental substances. pathogenetically, acd results from an immune reaction involving both innate and adaptive immunologic mechanisms. in particular, hyperreactive response to small chemicals (haptens) penetrating the skin depends on a series of events, such as haptens capability to activate and mobilize cutaneous dendritic cells (cdc), generation of hapten - epitopes for t - cell recognition, and hapten - cdc complex ability to prime effector t cells with skin homing proprieties [13 ]. in sensitized individuals, skin or systemic challenge with the specific sensitizer determines rapid recruitment of effector t cells, along with natural killer lymphocytes, which mediate tissue damage through release of proinflammatory cytokines and through hapten - loaded keratinocytes killing. the causes for such variability in acd clinical aspects are many (table 1). according to our data (unpublished), considering > 30.000 patch tested individuals for contact dermatitis, noneczematous forms are slightly more common (52%) than the classic eczematous one (48%). various clinical patterns of noneczematous acd have been described : some are linked to topical use of specific haptens and others more often dependent on allergens systemic administration (table 2). of all noneczematous clinical variants, the erythema multiforme - like (or contact erythema multiforme) is the most common. it can be elicited by different substances, particularly exotic woods, medicaments, and ethylenediamine (table 3). brazilian rosewood (dalbergia nigra), pao ferro (machaerium scleroxylon), and eucalyptus saligna are relevant as occupational causes of erythema multiforme - like eruption in carpenters, foresters, and cabinet makers. antigens in pao ferro and brazilian rosewood are crossreacting quinones, respectively, r-3, 4-di - methoxy - dalbergione, and r-4-methoxy - dalbergione [7, 8 ]. literature also lists extraoccupational cases from wooden bracelet and pendants made of d. nigra. m. scleroxylon has been described to cause a similar eruption in hobbyists who handled this type of wood to build boxes. other reported causes of erythema multiforme - like reactions include artemisia vulgaris [12, 13 ], poison ivy [14, 15 ], hypericum erectum, and terpenes. tincture of capsicum caused an analogous reaction in a woman who used the concoction to treat her knee arthritis. inula helenium, contained in a mixture to treat back pain, has also induced erythema multiforme - like eruption, with positive patch tests to sesquiterpene lactone mix and alantolactone. we observed an erythema multiforme - like reaction in a plant nursery worker, who had handled plants of p. obconica. patch tests were positive to primin (0.01% in pet), leaves and flower. histology showed foci of hyperkeratotic orthokeratosis, mild spongiosis, exocytosis, and few isolated necrotic keratinocytes ; at the superficial and mid dermis, a largely perivascular lymphocytic infiltrate was present. numerous topical drugs are reported as cause of erythema multiforme - like contact dermatitis, the vast majority being antimicrobials. according to our observation other causative drugs include sulfonamide [27, 28 ], promethazine, neomycin, mafenide acetate, ethylenediamine [25, 30 ], and mephenesin [31, 32 ]. among nonsteroid anti - inflammatory drugs, phenylbutazone, bufexamac, and mofebutazone have been reported. among corticosteroids, erythema multiforme - like eruptions can be the expression of contact allergy to nickel [3841 ] and cobalt. 9-bromofluorene induced a skin acute reaction in several chemistry students, who were exposed to the product during its synthesis [42, 43 ]. finally, many other compounds have been associated to erythema multiforme - like reactions, although exceptionally [5, 6 ]. early lesions are eczematous in morphology and localized at the allergen contact site. after a 1 to 15 days delay, the erythema multiforme - like eruption follows, involving the area around the original lesions or rather extending to the whole cutaneous surface. the latter occurrence generally ensues systemic exposition to drugs which the patient had previously been sensitized to topically. resolution is slow - paced ; these manifestations persist usually much longer than the original eczematous lesions (or sometimes appearing after regression of the latter). patch tests generally elicit eczematous positive reactions, with the exceptional vesico - bullous or urticarial lesions. differential diagnosis is set out with true erythema multiforme (table 4), the latter showing almost all target - like lesions with typical acral distribution and crops - like onset. vacuolar degeneration of basal cells is rarely present, while epidermal necrosis is very mild or absent. when bullae are present, they are intraepidermal. the histopathology of true erythema multiforme shows frank epidermal necrosis and vacuolar basal cells degeneration, while bullae are subepidermal. this particular form of noneczematous contact dermatitis is of unusual observation, and many cases remain undiagnosed. the eruption evolves in several weeks after the withdrawal of the offending agent and resolves with more or less persistent pigmentation. the purpuric aspects of contact dermatitis, and the respective patch test reactions can be secondary to irritant, or more frequently allergic, mechanisms. first reported cases date back to 1968 : 9 women developed purpura from cloth elastic inserts ; in every instance patch tests turned positive to n - isopropyl - n - phenyl - paraphenylenediamine (ippd), a rubber antioxidant. other 2 cases, showing diffuse purpuric reactions with negative bloodwork, were associated to ippd and specifically to the use of rubber boots. fisher reported 3 cases, respectively, from rubber diving suit, elasticized shorts, and rubberized support leg bandage ; in all 3 patients patch tests turned positive to ippd [47, 48 ]. pppp syndrome, defined as an acd characterized by pruritus, petechiae, and purpura, caused by ippd. ippd also prompted similar eruption in a woman in the pattern of her brassiere and in a man at rubber boots contact sites. pppp syndrome has also been described following use of orthopedic elastic bandages and rubber gloves ; in the latter case patch tests were positive not just to ippd but to n - cyclohexyl - n-phenyl - paraphenylenediamine and n, n-diphenyl - para - phenylenediamine as well., osmundsen gathered 167 cases of purpuric reactions from an optical whitener contained in washing powders [53, 54 ]. the petechial and itchy dermatitis interested those areas which are typically subject to tighter contact with clothes (armpits, arms, upper limbs folds, neck and thighs). the offending agent was tinopal ch 3566, a mixture of 2 noncrossreactive pyrazolines (monochlorobiphenyl - pyrazoline and dichloro diphenyl pyrazole). tinopal ch 3566 was used to bleach nylon fibers and caused a similar epidemic outbreak in spain, where 103 were collected. from that time on, the product was discontinued with no more cases reported. as of today a sailor developed generalized purpuric lesion with pigmentary outcomes at sites of contact with the military blue uniform. patch tests evidenced positive reaction to disperse blue 85, while histology demonstrated schamberg disease sign. we directly observed a case of purpuric acd to disperse yellow 27 (serisol fast yellow 6dw), an azoic dye used in acetate and polyester fibers, a result of para - aminoacetanilide and paraphenylphenol. the dye was part of a pair of trousers inner lining, and the dermatitis, while interesting the whole skin surface, started from and was particularly manifest at the thighs. histology proved traditional aspects of acd, with lymphocytic infiltrate and intense perivascular edema, associated to noticeable erythrocyte extravasation. purpuric eruptions have also been described in a black hats vendor from paraphenylenediamine, in british soldiers from formaldehyde resins contained in kaki wool shirts, and in a man assigned to mixed wool - synthetic residues harvesting. plants. frullania induced a diffuse purpuric reaction ; histology showed signs of leukocytoclastic vasculitis ; however, circulating immune complex and complement deposition assays were also positive. agave americana l, of agavaceae family, can determine purpuric contact dermatitis with histological features of leukocytoclastic vasculitis. zea mais (corn) has been shown to induce irritant purpuric phytodermatitis some hours after contact to green leaves. patch, photopatch, and scratch tests with alcoholic extracts of different plant parts (leaves, trunk, efflorescences) all resulted negative. two - hour experimental exposition to 98% d - limonene resulted in a severe and several week persistent purpuric reaction 6 hours after contact. fiberglass can induce direct or aeromediated contact dermatitis, with pruriginous, 0,10,5 mm diameter, mostly follicular purpuric papules. exposed and nonexposed areas are both affected, since these fibers are able to pass through clothing [66, 67 ]. vasculitic purpuric eruptions to peru balsam [25, 69 ], ethylenediamine [70, 71 ], benzoyl peroxide, and proflavine have also been reported. as is well known among those who practice dermatoallergology, petechial reactions to cobalt patch test, without edema, vesicles, and infiltration, can be observed. schmidt., in a 4-year time span, observed 123 cases (4.7%) of cobalt petechial reactions out of a total of 2594 patch - tested patients. twenty - three patients were retested and developed new petechial responses in 60% of cases. based on these authors data, the incidence of positive allergic reactions to cobalt was lower (2.9%) than the incidence of primary irritant reactions. judging on our practice, cases of petechial nonallergic reactions to cobalt and chrome are indeed rather numerous and frequently reproducible. purpuric contact dermatitis can be either toxic or allergic in nature. from a clinical - morphological perspective, differential diagnosis is not straightforward : both present palpable purpuric elements, evolve slowly and are followed by variably intense and persistent pigmentation. at times, clinical extension represents a useful feature in differentiating the 2 forms, the irritant being strictly limited to contact sites. moreover, lesional elements resolve more rapidly and are less infiltrated in the irritant form compared to the allergic one. diffuse contact irritation from fiberglass must be discerned from scabies, eczema (prurigo - like), animal and vegetal acariasis, and if persistent, from hodgkin disease. the anamnestic data of epidemic bursts in industries or bureaus (fibers dispersed from defective air conditioners) greatly aid diagnosis. the allergic form of purpuric contact dermatitis generally features diffuse and polymorphic manifestations : papulopurpuric and papulovesicular lesions parallel classic eczematous foci. secondary distant lesions can also present polymorphic or vasculitic aspects, as we have directly observed. hemostasis or complement system alterations are not generally described in reported cases nor are immune complexes commonly isolated. in every case we observed, among which 3 severe cases from peru balsam with frankly vasculitic and bullous lesions and various cases from ethylenediamine (in which the rash had followed systemic administration of aminophylline), specific laboratory exams fell into normal range [25, 44 ]. since endothelial cells degeneration is evident at electron microscopy, a selective effect on these cells has been hypothesized. in detail, specific toxic or allergic substances as well as certain mechanical stimuli (fiberglass) would exhibit an affinity for vessels endothelium [47, 57, 58 ]. alternatively, a primary lymphocytic reaction in response to the antigen at the perivascular site would free toxic lymphokines, ultimately responsible of endothelial damage. histopathology has been described, with comparable results, in most reported cases. in the epidermis, spongiosis and lymphocytic exocytosis are constant features, along with possible bulla formation. in the upper dermis the signs of leukocytoclastic vasculitis (vessels fibrinoid degeneration, edematous endothelium, scarce perivascular lymphomonocytic and neutrophilic infiltrate, erythrocytes extravasation, and karyorrhexis) are visible. the same features are present when examining a patch test response lesion (table 6) [47, 74 ]. bloodwork, histologic and patch test examinations are valid to differentiate the condition from vascular, hemostatic, and idiopathic purpuric affections. a particularly uncommon form of noneczematous contact dermatitis presents with clinical features resembling those of lichen planus. color developers, substances derived from paraphenylenediamine, are the most common cause of allergic contact lichenoid eruption. among these compounds, kodak cd2 (4-n, n - diethyl-2 methylphenylenediamine), kodak cd3 (4-n - ethyl - n-2-methanesulfonylaminoethyl-2-methyl - phenylenediamine sesquisulfate monohydrate), kodak cd4 (2-amino-5-n - ethyl - n-(hydroxyethyl)-aminotoluene sulfate), ilford mi 210 (n - ethyl - n (5-hydroxy - amyl) para - phenylenediamine hydrogen sulphate), and agfa tss (4-amino - n - diethylaniline sulfate). other cases of lichenoid contact dermatitis have been reported by mandel, in 9 out of 11 workers with contact allergy to a color developer, and by fry in 7 out of 20 patients with analogous sensitization. high speed, black - and - white film processing implies the use of similar chemicals, which can induce lichenoid reactions. as a general rule cases from paraphenylenediamine in hair dyes, p. obconica, nickel, epoxy resins, aminoglycoside antibiotics, and methacrylic acid esters for industrial use have also been described. oral mucosae involving forms are due to copper, zinc, and mercury contained in dental restorations. lichenoid contact dermatitis has to be differentiated from lichen planus, with its characteristic papulous polygonal lilac lesions. the onset of lichenoid contact dermatitis is almost invariably acute and the eruption spreads rapidly.. systemic absorption of offending agents can elicit skin lesions far from the original site of contact. in 5 cases we observed (3 from color film developers and 2 from paraphenylenediamine), histology displayed lack of hypergranulosis, foci of moderate spongiosis, and focal basal stratum vacuolization. basal cell vacuolization is the cause of incontinentia pigmenti, which could explain skin lesions peculiar color, a blend of red from flogosis with blue from dermal melanin. this uncommon dermatitis manifests with the clinical features of plaque parapsoriasis or an early stage mycosis fungoides [1, 70 ]. there are no specific causing haptens [8995 ], the most frequently reported being paraphenylenediamine, para - tertyl - butyl phenol resin, gold, ethylenediamine, and nickel. patch test reaction to these is eczematous in nature and can persist for several days. lymphomatoid contact dermatitis and mycosis fungoides alike present with infiltrative patches ; the former, however, demonstrates a bright erythematous color and undefined margins. mycosis fungoides instead shows atypical lymphocytes in focal abscess - like aggregations (i.e., the pathognomonic pautrier 's microabscesses) and a band - like subepidermal infiltration of large lymphoid cells with cerebriform nuclei (table 8). described by osmundsen in 1970, it is a melanic primitive hyperpigmentation, usually observed in dark phototypes and mostly occupational. the author observed an intense and bizarre skin hyperpigmentation due to contact with an optical whitener (tinopal ch 3566) used in washing powders and made by a combination of two pyrazolone derivatives, as of now discontinued. clinically, involved sites were those of textile contact dermatitis, with brown - blue to grayish hyperchromia. an epidemic outburst from contact to naphthol as has been reported in a textile business. hyperpigmentation was noticeable in dark skinned individuals, while fair skinned ones showed the signs of classical eczema. sudan i, vacanceine red, and brilliant lake red r are other offending colorants which have been reported. isolated occupational cases from insoluble cutting oils, paraphenylenediamine, and other substances riehl 's melanosis is nowadays also considered a pigmented contact dermatitis, mostly from cosmetic sensitizing fragrances and chemicals. nevertheless, allergic pustular reactions are known from nitrofurazone, black rubber, and minoxidil. the latter has been described in a woman who developed a vesicopustular eruption on the forehead after applying 2% minoxidil solution. patch test was strongly eczematous in another case of pustular allergic contact dermatitis from isoconazole nitrate. pustules are sterile and transient and can displace subcorneally, as observed in a case from trichloroethylene. metal salts, particularly nickel, copper, arsenic, and mercury represent the most common causes of these reactions, which are irritant in nature [110, 111 ]. as a matter of fact, pustular responses to nickel patch test are widely observed when testing atopics on lesional skin, with follicular papules, erythema, or lichenification. this further supports the irritant nature of the phenomenon. in subjects affected by atopic dermatitis, we often observed such pustular follicular reactions when patch testing with nickel but also with potassium bichromate. histology, documented in various cases, has always evidenced intraepidermial aggregations of neutrophils, without signs of lymphomonocytic exocytosis or spongiosis. we have always considered these reactions we directly observed irritant in nature [113, 114 ]. certain authors include this condition among noneczematous allergic contact forms. in our opinion, this dermatitis retains frankly clinicohistologic eczematous aspects, and a proper differential diagnosis would have to be made with the endogenous eczema pompholyx. as per our observations, dyshidrosiform allergic contact dermatitis the latter is defined as a contact sensitivity which complicates a preexisting primitive palmoplantar pompholyx. the latter tends to a chronic recurrent course, thus constituting a predisposing factor to occupational and extraoccupational contact allergy [117, 118 ]. from studies we carried out on 354 subjects with pompholyx genuine lesions, observed during a 5-year period, incidence of relevant positive patch tests reactions was 29.6%. topical medicaments (used to treat the original pompholyx) and other substances among which paraphenylenediamine (31.5% positive reactions), chrome (25%), cobalt (10.2%), mercaptobenzothiazole (9.3%), nickel (6.5%), and para - tert - butylphenol formaldehyde resin (2.7%) were the most often implicated haptens. patch tests relevance was related to specific occupational activities, use of peculiar gloves rather than shoes. more recently, a study we conducted on 45 individuals affected by palmoplantar pompholyx confirmed an acd incidence of 31%. primitive dyshidrosiform acd is instead an expression of systemic contact allergy, of common observation in nickel sensitized patients. oral challenge test with nickel reproduces the dyshidrosiform eruption in these subjects [119122 ], although this phenomenon has not been widely confirmed [123, 124 ].	irritant or allergic contact dermatitis usually presents as an eczematous process, clinically characterized by erythematoedematovesicous lesions with intense itching in the acute phase. such manifestations become erythematous - scaly as the condition progresses to the subacute phase and papular - hyperkeratotic in the chronic phase. not infrequently, however, contact dermatitis presents with noneczematous features. the reasons underlying this clinical polymorphism lie in the different noxae and contact modalities, as well as in the individual susceptibility and the various targeted cutaneous structures. the most represented forms of non - eczematous contact dermatitis include the erythema multiforme - like, the purpuric, the lichenoid, and the pigmented kinds. these clinical entities must obviously be discerned from the corresponding pure dermatitis, which are not associated with contact with exogenous agents.
neutrophils, also known as polymorphonuclear leukocytes (pmns), are short - lived and highly specialized immune cells that form the first line of defense against bacterial and fungal infections. not only are neutrophils an essential part of the innate immune system, but activated neutrophils also secrete a number of cytokines and chemokines to help to shape lymphocyte - oriented adaptive immunity [2, 3 ]. the recruitment of neutrophils to inflammatory sites is through a cellular process known as chemotaxis, directional cell migration guided by extracellular chemoattractant gradients. rapid recruitment of neutrophils is crucial for host defense ; however, excessive recruitment of neutrophils into healthy tissues causes damage and inflammatory diseases such as asthma and arthritis [5, 6 ]. thus, neutrophil chemotaxis is tightly controlled in vivo through chemoattractants and their receptors. chemotaxing neutrophils display a polarized morphology in a chemoattractant gradient (figure 1(a)). they extend their leading edges by assembling a force - generating actin network beneath the plasma membrane [7, 8 ]. actin also collaborates with myosin to retract the rear of migrating cells and to prevent errant pseudopod extension. neutrophils detect and move toward chemoattractant gradients by g protein - coupled receptor (gpcr) signaling pathways. the most important gpcrs expressed in neutrophils include formyl - peptide receptors (fpr1/2/3), classical chemoattractant receptors (blt1/2, pafr, and c5ar), and chemokine receptors (cxcr1/2 and ccr1/2). the engagement of chemoattractants with their gpcrs triggers dissociation / activation of the gpcr - specific g subunit from the g dimer [10, 11 ]. both g and g activate downstream effectors, such as phospholipase c (plc). it has been shown that gi and g12/13 are involved in neutrophil chemotaxis [11, 13 ] although the coupling mechanism of gpcrs and their specific g/ remains unclear. over the last decade, multiple signaling pathways have been revealed to control gpcr - mediated organization of actin cytoskeleton in directional cell migration. at the leading edge, signaling pathways control the activity of arp2/3 complexes that initiate the formation of new branches of actin filaments. in neutrophils, gpcrs / g protein activation triggers multiple signaling pathways to activate the rho family of small gtpases (cdc42 and rac1/2) to promote the growth of actin filaments (f - actin) [12, 1419 ]. gpcr activation also regulates the activity of cofilin, the f - actin depolymerization factor, to facilitate the rapid growth of f - actin in the leading edge [2023 ]. spatial - temporal activation of different signaling pathways for precisely controlled cell migration has just begun to be revealed. plc activation is an early event in response to numerous extracellular stimuli. upon activation, plc produces two important second messengers : diacylglycerol (dag) and inositol trisphosphate (ip3). both dag and ip3 play important roles in many signaling pathways, including the activation of protein kinase c (pkc) and protein kinase d (pkd) and the induction of calcium influx [12, 21, 23, 25 ]. it was shown that the signaling axis of plc / pkc / pkd plays important roles in many signaling pathways. in this review, we summarize the novel functions of the plc / pkc / pkd signaling axis in gpcr - mediated chemotaxis of neutrophils. in response to various extracellular stimuli, plc produces dag and ip3, which mediate multiple downstream pathways. in mammals, there are 13 phosphatidylinositide - specific plcs that are divided into 6 subgroups : plc, plc, plc, plc, plc, and plc [24, 2732 ]. several excellent reviews have summarized the structures and activation mechanisms of plc isoforms [24, 29, 33 ]. mammalian neutrophils express plc2, plc3, plc2, and plc (figure 1(b)). in murine neutrophils early studies reported that murine neutrophils lacking both plc2 and plc3 are still able to chemotax. surprisingly, some leukocytes with a single plc2 deficiency actually have enhanced chemotaxis, indicating that plc signaling might not play essential role in neutrophil chemotaxis. however, a plc/pi3k/gsk3 signaling pathway has been shown to regulate the activity of cofilin phosphatase slingshot 2 (ssh2) and control neutrophil polarization and chemotaxis. the authors further investigated in vitro chemotaxis behavior of murine neutrophils with plc2 deficiency and suggested that the normal chemotaxis behavior of these murine neutrophils was rather context- and assay - dependent. a recent study has shown that when plc activity is inhibited with the plc inhibitor u73122, chemotaxis of human neutrophils is reduced, suggesting an essential role of plc signaling in neutrophil chemotaxis. thus, plc signaling appears to play a complicated role in neutrophil chemotaxis that is still not well understood. in a chemoattractant gradient, plc is recruited and activated at the leading edges of chemotaxing cells, suggesting its likely role in the remodeling of the actin cytoskeleton. in neutrophils, gpcrs activate plc through several mechanisms (figure 1(c)). first, chemoattractant stimulation may trigger plc activation through direct interaction with and activation by released heterotrimeric g proteins [12, 34 ]. structural insights into gpcr - mediated plc activation have been summarized in a recent review. although there are currently no reported structures of a g-plc complex that could shed light on the molecular basis for their interaction and activation, many studies have sought to map the interface of their interaction. gdp - bound gi can inhibit plc activation, suggesting a common protein interaction interface on g. beside direct activation by heterotrimeric the crystal structure of the rac1-plc2 catalytic core complex has shown that the ph domain is the sole rac1 binding site on plc2 and that the ph domain - mediated rac1 interaction is sufficient to activate plc2/3. in neutrophils, moreover, in a chemoattractant gradient, the activated rac1 localizes at the leading edge where plc2 is highly activated in chemotaxing neutrophils. rac1-mediated plc activation might provide an activation mechanism that is independent of gpcr or heterotrimeric g protein. it is intriguing to understand rac1-mediated spatiotemporal activation of plc and its possible function in neutrophil chemotaxis. plc2 also plays critical roles in integrin- and fc receptor - mediated neutrophil functions, such as respiratory burst, degranulation, and cell spreading in vitro. plc1 is ubiquitously expressed and is mainly activated downstream of growth factor stimulation, including stimulation by platelet derived growth factor (pdgf), vascular endothelial growth factor (vegf), epidermal growth factor (egf), and fibroblast growth factor (fgf). plc2 is predominantly expressed in hematopoietic cells and is activated by immune cell receptors such as b cell and fc receptors. recently, it has been shown that chemokine gpcrs also mediate the membrane targeting and subsequent activation of plc2 in a phosphoinositide 3-kinase- (pi3k-) dependent manner. this result is consistent with the finding that plc1 activation is a consequence of the binding between the ph domain of plc1 and pip3 produced on the membrane. it has been reported that plc2 is specifically activated by rac2 [41, 42 ]. in neutrophils, the engagement of chemoattractants with their gpcrs triggers the activation of rac2 [18, 38 ]. the rac1/2-mediated plc/ activation adds another layer of complexity to the existing signaling networks of plc signaling. gpcr - mediated plc2 activation in neutrophils might provide an explanation for the normal chemotaxis behavior in murine neutrophils with single or double plc2/3 deficiency. thus, it is difficult to evaluate the plc isoform, and its activation is more important for neutrophil chemotaxis. also, as a scaffold protein with numerous interacting partners, it is unlikely that gpcr- or rac2-mediated plc2 activation serves solely as the backup for plc2/3 in neutrophils. neutrophils also express a low level of plc, which is activated by gpcr and gpcr - regulated small gtpases, including ras and rap [24, 28, 43 ]. recently, it was shown that plc plays a crucial role in the neutrophil - associated inflammatory response. future work is needed to elucidate the temporal and spatial activation profiles of each plc isoform and their molecular mechanisms and subsequent effects on neutrophil chemotaxis. pkc isoforms share a similar overall structure consisting of an nh2-terminal regulatory domain joined through a flexible linker to a conserved cooh - terminal catalytic domain that binds atp and substrates. the regulatory domain of pkc contains a pseudosubstrate domain that maintains the enzyme in an inactive conformation and membrane - targeting modules that control the subcellular localization of the enzyme. pkc, pkc1, and pkcii are conventional pkcs and contain tandem c1a / c1b motifs that bind diacylglycerol (dag) or phorbol esters (such as pma), a c2 domain that binds anionic phospholipids in a ca - dependent manner, and a ser / thr kinase domain (figure 2(a)). pkc is a novel pkc that contains a nonfunctional c2 domain and therefore is insensitive to ca. various stimuli activate all four pkc isoforms, and the activation of pkc is required for the oxidative burst of neutrophils [12, 47, 48 ]. pkc, pkc, and pkc phosphorylate all phosphorylation sites on p47phox [47, 49 ]. however, it is pkc and pkc, but not pkc, that play essential roles in fmlp- and pma - induced superoxide generation in neutrophils or hl60 cells [48, 50 ], indicating that different pkc isoforms perform specific functions in neutrophils. the isoform - specific functions of pkcs have long been missing in the signaling pathways of neutrophil chemotaxis. pkc and pkc share remarkable similarities in molecular composition, structure, and activation mechanism (figure 2(b)). in resting neutrophils, both of them localize in the cytosol. uniformly applied chemoattractant induces membrane translocation and subsequent activation of pkc and pkc in a plc - dependent manner, indicating that the binding of dag to their c1a domain serves as the major determinant for membrane translocation and activation [21, 23 ]. however, pkc and pkc interact with and activate different effectors to regulate ssh2 activity. gsk3, a substrate of pkc, is active in resting neutrophils and phosphorylates ssh2 to decrease its cofilin phosphatase activity, in turn, leaving cofilin in an inactive phosphorylated state. upon fmlp stimulation, pkc phosphorylates gsk3 and inhibits its activity, consequently increasing ssh2 activity and the activity of its target cofilin. recently, it has been shown that pkc interacts with and activates pkd1, and pkd1 phosphorylates ssh2 and inhibits its cofilin phosphatase activity. by interacting with different effectors, both pkc and pkc regulate cofilin activity in order to regulate actin - based protrusion in the leading edge of chemotaxing cells. it was also reported that an mtorc2-specific activation of pkcii regulates myosin ii activity in the trailing edge of cells. point mutation of these sites resulted in impaired membrane translocation of pkcii upon fmlp stimulation, providing an alternative membrane - targeting mechanism in addition to plc signaling. the authors identified adenylyl cyclase 9 (ac9) as a downstream effector of pkcii activation. adenylyl cyclases (acs) are activated and produce camp upon chemoattractant stimulation in both d. discoideum and neutrophils [52, 53 ]. in chemotaxing cells, camp is spatially restricted to the back of the cells to specifically regulate trail retraction and contraction in a myoii - dependent manner [5154 ]. this finding might also provide an example of how neutrophils utilize one common upstream activation pathway to precisely coordinate actin - based protrusion in the leading front and myosin ii - based contraction in the trailing edge. pkc translocates to the plasma membrane through the binding of its c1a domain with dag or phorbol esters and is involved in the oxidative burst in neutrophils [7, 55 ]. recently, it has been reported that pkc is required for neutrophil transmigration mediated by il-1 and fmlp (integrin - dependent), but not il-8 (integrin - independent), by regulating adherence of neutrophils. in corneal epithelial cells, pkc mediates cap37 (neutrophil - derived granular protein) induced chemotaxis. in fibroblast migration and pulmonary fibrosis development, neutrophils express g12, which localizes and reinforces signaling networks at the trailing edge of cells protein kinase d (pkd) belongs to a family of serine / threonine kinases that play critical roles in many physiological processes, including cell growth, protein trafficking, and lymphocyte biology. pkd isoforms share a conserved structural motif, n - c1a - c1b - ph - kd - c, and display a high sequence homology, particularly in the catalytic domain and c1a and c1b domains (figure 3(a)). the c1a domain binds to dag for membrane targeting, while the c1b domain has a higher affinity for phorbol ester. this explains the fact that chemoattractant stimuli trigger very similar dynamics of membrane translocation and cellular localization for all three pkds in neutrophils. however, the differences in the n - terminal region and in the regions flanked by the c1 and ph domains may confer isoform - specific functions. pkd1 contains an alanine - proline - rich region (ap domain) at the n - terminus, while pkd2 has a proline - rich region (p domain). interestingly, there was a distinct expression profile of pkd isoforms in a panel of leukocyte cell lines. the expression pattern of these three isoforms is not affected by the knockdown of the other isoforms, excluding the possibility of functional compensation among the three isoforms. accumulating evidence demonstrates the involvement of pkds in a variety of cellular processes that contribute to cancer development. it has also been shown that specific pkd isoforms are misregulated in several cancer types, including leukemia. pkd is activated by direct phosphorylation of two conserved serine residues in its activation loop by dag - binding pkc isoforms. phosphorylation at both sites is severely inhibited by pkc inhibitor g6983, indicating that activation of pkd is directly through phosphorylation at the activation loop by pkc. the author identified that pkcii, a dag - binding pkc isoform, interacts with pkd1 and is essential for neutrophil chemotaxis. phosphorylation at ser916, an autophosphorylation site of pkd1, is also detected in response to chemoattractant stimuli, indicating that autophosphorylation of pkd1 also occurs in neutrophils following chemoattractant stimulation. membrane translocation of pkd1 is mediated through several mechanisms in response to various kinds of stimuli, such as growth factor, phorbol esters, and gpcr agonists. in resting, neutrophil - like hl60 cells, in contrast to its behavior in other mammalian cell lines, the kinase - inactive mutant pkd1 (k612w) also localizes in the cytoplasm of hl60 cells, indicating that kinase activity is dispensable for pkd 's cellular localization. uniformly applied chemoattractant stimulation triggers a robust membrane translocation of all three pkds. in a chemoattractant gradient, pkd localizes at the rear of the leading edge of chemotaxing cells (figure 3(b)) the kinase - inactive mutant of pkd is recruited to the leading edge of chemotaxing cells, indicating that kinase activity is not required for membrane targeting of pkd. instead, membrane targeting is completely abolished by either treatment with plc inhibitors or a mutation of the c1a domain (dag - binding domain), which results in a decreased affinity toward dag. this result indicates that the binding of c1a domain and dag is the major determinant for membrane targeting of pkds in neutrophils. dag also recruits pkc to the membrane, where pkc phosphorylates and consequently activates pkds. thus, translocation to the plasma membrane allows pkd1 to interact with its upstream activator, such as pkc, to be phosphorylated and subsequently activated. after being activated each isoform of plc, pkc, and pkd might have its own interacting partners in a separated signaling pathway in diverse functions of neutrophils. in the following two paragraphs, we are going to focus on the downstream effectors of the plc / pkc / pkd axis that are involved in the remodeling of f - actin - based cytoskeleton and the regulation of other crucial signaling components. the family of actin - depolymerizing factor (adf)/cofilin proteins is comprised of cofilin-1 (a nonmuscle type of cofilin), cofilin-2 (a muscle type of cofilin), and adf (also known as destrin) in mammals. cofilin also contributes to f - actin assembly by increasing the actin monomer concentration for polymerization and consequently increasing the turnover rate of actin filaments in cells. cofilin might also increase new barbed ends by its intrinsic nucleation activity. however, phosphorylation is the best - studied mechanism of regulating cofilin activity. lim kinases (limks) and testicular protein kinases (tesks) phosphorylate cofilin to deactivate it while slingshot proteins (sshs) and chronophin dephosphorylate p - cofilin to activate it. in neutrophils, chemoattractants mediate the rapid dephosphorylation of cofilin. a chemoattractant - mediated plc/pi3k/gsk3 signaling pathway has been found to increase the activity of ssh2, which dephosphorylates and activates cofilin (figure 3(c)). the activation cycle of cofilin is especially important at the leading front, where rapid polymerization and depolymerization of f - actin cytoskeleton are required. hirayama and his coworkers used hl60 cells to study the cofilin activation cycle and demonstrated a clear activation cycle. a recent study has identified ssh2 as the direct target of the plc / pkc/pkd signaling axis to regulate cofilin activity. taken together, gpcr activation triggers two pathways to control the cycle of cofilin activity. the cofilin activation cycle is essential for a rapid and coordinated cycling of f - actin polymerization and depolymerization at the leading edge of chemotaxing cells (figure 3(c)). however, the signaling pathways and kinases that phosphorylate cofilin are still not fully understood in neutrophils. future work is necessary to fully understand the regulation of cofilin activity upon chemoattractant stimulation. in the future, it will be particularly important to understand how spatiotemporally distinct signaling pathways control the rapid and precisely coordinated regulation of cofilin activity in the leading front of chemotaxing neutrophils. the plc / pkc / pkd1 signaling pathway might also regulate the localization and functions of other key components involved in neutrophil chemotaxis. pi3ks phosphorylate phosphatidylinositol(4,5)-biphosphate (ptdins(4,5)p2 or pip2) into phosphatidylinositol(3,4,5)-triphosphate (ptdins(3,4,5)p3 or pip3) and the phosphatase and tensin homolog (pten) converts pip3 back to pip2. leading - edge localization of pi3k and trailing - edge localization of pten are key features of gradient sensing and polarization and are essential requirements for chemotaxis in neutrophils and dictyostelium discoideum [15, 6365 ]. it has recently been shown that pkd1 directly phosphorylates the p85 subunit of pi3k to enhance its interaction with pten, leading to polarized pten activity and thereby regulating neutrophil migration. membrane localization of pten is required for its function in both d. discoideum and neutrophils. the c2 domain of pten is required but not sufficient to recruit d. discoideum pten to the plasma membrane. have shown that small gtpase rhoa / rock mediates pten membrane targeting in murine neutrophils. recently, nguyen. generated a library that contains green fluorescent protein (gfp) fused to randomly mutated human pten and expressed the library in d. discoideum cells. one cluster of mutations with an enhanced membrane association is located in the c - terminal tail phosphorylation sites. these results indicate that phosphorylation plays essential roles in pten membrane targeting [15, 68 ]. it is not clear whether pkd1 is responsible for the phosphorylation of these sites in pten. it is of great importance to understand whether pkd is the kinase responsible for the phosphorylation of these sites in pten, because both pten and pkd have substantial functions in various types of cancer. in this review, we strove to summarize recent findings regarding novel functions of the classic plc / pkc / pkd signaling axis in neutrophil chemotaxis. future research should focus on revealing isoform - specific functions of plc, plc, and plc in gpcr - mediated neutrophil chemotaxis, specifically plc isoform - specific activation and the function of downstream effectors such as pkcs.	chemotaxis, a directional cell migration guided by extracellular chemoattractant gradients, plays an essential role in the recruitment of neutrophils to sites of inflammation. chemotaxis is mediated by the g protein - coupled receptor (gpcr) signaling pathway. extracellular stimuli trigger activation of the plc / pkc / pkd signaling axis, which controls several signaling pathways. here, we concentrate on the novel functions of plc / pkc / pkd signaling in gpcr - mediated chemotaxis of neutrophils.
varicose veins are a common progressive medical condition with widely ranging estimates of prevalence that will steadily worsen1. the veins become dilated (greater than 3 mm) with twisting and bulging due to weakness of their walls or valves, which usually occurs in the superficial veins of the lower extremities2. half of the adult population has the stigmata of minor venous disease3, and about 25% of the population has lower extremity varicose veins4. the prevalence of varicose veins is greater in women (2632%) than in men (1040%)5. they often affect individual s physical appearance, and quality of life and may also result in lost time from work and lost wages. symptomatic varicose veins are not a life threatening condition, but the condition is usually progressive and may result in leg ulcer.. a non - healing ulcer can require prolonged primary care, and it s the odor may lead to social exclusion. it is very difficult to predict which patient with varicose veins will develop a leg ulcer. however, it has been estimated that about half of venous leg ulcers are the result of varicose veins7. varicose veins management can range from conservative treatment to surgical approaches, all of which have different advantages, disadvantages, and long - term outcomes1. the first is prevention of complications, such as edema, bleeding, eczema, lipodermatosclerosis, and leg ulcers1, the second is relief of complaints caused by varicose veins, such as heaviness, tired legs, and cramps, and the third is the importance of cosmetic appearance. many patients only find their way to a phlebologic clinic because their varicose veins are cosmetically disturbing, and this may affect patients quality of life8. chronic diseases such as varicose veins have an important impact on a patient s quality of life (qol)9, 10. the aberdeen varicose veins questionnaire (avvq) is a disease - specific questionnaire that measures qol for patients with varicose veins12. the questionnaire, designed in 1993, consists of 13 questions related to all aspects of the problem of varicose veins13, 14. vascular compression therapies that include elastic wraps, elastic stockings, and intermittent pneumatic compression devices are the mainstay of conservative treatment for varicose veins. intermittent pneumatic compression (ipc) devices composed of sleeve- or boot - shaped chambers that fill with air and electrical pumps with gauges that provide intermittent compression to the lower extremities. the compression force may be applied either uniformly to the calf using a single chamber device or through a series of chambers inflated in a sequential manner from the ankle to thigh to achieve venous emptying15 which is termed sequential pneumatic compression (spc). the efficacy of ipc in the treatment of varicose veins is well documented10, 16. it reduces venous stasis by promoting venous blood flow17,18,19 and it stimulates fibrinolytic activity18, 20. it had been found to be an effective modality for treatment for venous ulceration21, 22. measurement of qol is a comprehensive assessment of the effect of such a treatment intervention for varicose veins on patients to determine if the treatment results provides improvement. a new combined qol and clinical instrument is needed to validly assess and compare the outcomes of venous treatments23. this study was conducted to investigate the effects of spc therapy on venous blood flow, refilling time, pain level, and qol of women with varicose veins. the study protocol and designed were approved by the local ethics committee of the faculty of physical therapy, cairo university. fifty - five women with bilateral varicose veins were investigated at the vascular outpatient clinic of el kasr el ainy teaching hospital and national heart institute. patients were enrolled in the study if they had a symptomatic varicose veins (clinical severity class c2 according to the ceap classification, a comprehensive classification system) of the lower limb (pain, soreness, burning, aching, throbbing, heavy legs, cramping, muscle fatigue, and/or night cramps) over a period of at least six months24. patients were required to have an educational level sufficient to allow them to read and understand avvq in english independently without any help. patients were excluded if they were pregnant or breastfeeding ; had any local leg condition interferes with sleeve wear, such as dermatitis, vein ligation, gangrene, or recent skin graft ; had severe leg arteriosclerosis or any other ischemic vascular disease ; had massive leg edema or pulmonary edema from congestive heart failure ; had a suspecting existing or previous venous thromboembolism ; had an extreme leg deformity or size (thigh circumference 71 cm) ; or had medical disorders such as diabetes16. patients were excluded if they could not read and understand the avvq in english on their own. twenty - seven patients were excluded from the study because of venous thromboembolism (9%, 5/55), size of the leg (16.3%, 9/55), and diabetic limitations (23.6%, 13/55), and the remaining 28 patients were enrolled. the patients who fulfilled the inclusion criteria and provided a written informed consent were randomly divided into two groups by using sealed envelopes. thirteen patients were allocated to the control group, and fifteen patients were allocated to the experimental group. a power analysis with =0.05 and power=80% determined that a group size of 15 was adequate to demonstrate a 25% change in the venous blood velocity, refilling time, and pain level between the groups. flow chart for design of study the venous system of the lower extremities of the patients was examined by duplex ultrasound (prosound ssd-4000, hitachi aloka medical, ltd., japan) in order to measure the maximum and mean venous blood velocities in centimeters per second (cm / sec) and the refilling time in seconds. a visual analog scale (vas) was used to measured pain level, and the avvq was used to measured qol of all patients in both groups before and after six weeks of treatment. all measurements were performed in a similar fashion at the same time of the day for both groups to reduce variability. each subject was placed in the supine position for a minimum of five minutes before the evaluation and baseline venous velocities were recorded in cm / sec. maximal and mean venous blood velocities were obtained from the common femoral vein cephalad to the saphenofemoral junction, and the refilling time was also measured. the angle of insonation of the ultrasound scan beam with the vein was 60 degrees25. all measurements were repeated three times, and the mean was used for analysis. the vas is 10 cm horizontal line with one end described as no pain (0 cm) and the other end described as the worst pain (10 cm). the distance between the extreme left of the scale (no pain) and the subject s mark was measured to the nearest millimeter. the avvq consists of questions relating to all aspects of the problem of varicose veins13, 14. the questionnaire has a section in which patients can indicate diagrammatically the distribution of their varicose veins. there are other questions relating to pain severity, ankle edema, ulcers, compression therapy use, and limitations on daily activities, as well as questions on the cosmetic effect of varicose veins. the questionnaire is scored from 0 (no effect) to 100 (severe effect)27, 28. the avvq therefore fulfilled all attributes necessary for an instrument to be used as a measure of health outcome and qol23. the patients were requested to fill in a copy of this questionnaire on day zero and then again at the end of the six weeks. patients in both groups received a physiotherapy program that consisted of lower extremity exercises, which included gluteal and quadriceps isometric exercises, active hip and knee flexion / extension, ankle dorsiflexion/ plantar flexion, and straight leg raising. the patients were instructed to perform ten repetitions of each exercise three times a day, five time per week for six weeks29. in addition, patients in the experimental group were treated with an spc device (eureduc tpo5, japan). each of the lower extremities was treated for thirty minutes daily, five days a week for six weeks. the device had three separate chambers, one over the distal calf, one over the proximal calf, and one over the distal thigh, that inflated sequentially. it was adjusted to apply a pressure of 65, 55, and 45 mmhg respectably in a distal to proximal direction for 12 seconds followed by 2.4 seconds of non - compression to allow venous refilling. statistical analyses were carried out using pasw statistics for windows, version 18.0 (spss inc., normality of the data was assessed using graphical methods, including the frequency distribution on a histogram and normal q - q plot. the kolmogorov - smirnov and shapiro - wilk tests were used as a confirmation. descriptive data were expressed as the mean standard deviation (sd) for demographic characteristics of patients. the independent t - was used to test for significant difference between mean values of two groups of normally distributed variables. mann - whitney u test was used to test difference between mean values of two groups of non - normally distributed variables. the paired t - test (for normally distributed data) and wilcoxon signed - rank test (for non - normally distributed data) for paired data were applied with 95% confidence intervals (95% ci). the baseline characteristics of the patients are summarized in table 1table 1.baseline characteristics of the patientscharacteristiccontrol group(n=13)experimental group(n=15)p - valueage (years)43.4 4.3540.3 4.800.390weight (kg)83.6 6.4784.5 6.570.627height (cm)160.0 5.02158.9 2.920.518bmi there were no statistically significant differences between the two groups with regard to age, weight, height, and body mass index. n : number ; bmi : body mass index at baseline, there were no statistically significant differences between the groups with regard to maximum blood flow velocity, mean blood flow velocity, and refilling time (table 2table 2.statistical analysis of venous blood flow variables within and between groupsmeasuregroupsprepostmaximum blood flow velocity (cm / sec)control (25)10.3 2.5415.6 3.33experimental (26)10.6 3.5719.0 3.93mean blood flow velocity (cm / sec)control (25)5.6 0.979.5 3.25experimental (26)6.2 1.2611.3 2.31refilling time (sec)control (25)29.3 3.1920.8 2.67experimental (26)30.5 4.0622.5 3.65data represent the mean standard deviation. experimental group, sequential pneumatic compression. p<0.05 statistically significant difference within the group before and after the intervention. p<0.05 statistically significant difference between groups at baseline and after the intervention period.). in both groups, maximum blood flow velocity, and mean blood flow velocity were significantly increased (p=0.000 in both groups). refilling time was significantly decreased in both groups (p=0.000 in both groups) (table 2). comparison of posttreatment measurement values between groups showed that maximum blood flow velocity, and mean blood flow velocity were significantly higher in the spc group compared with the control group (p=0.002, and p=0.024 respectively) ; while for refilling time, there was no significant difference between the groups (p=0.065) (table 2). p<0.05 statistically significant difference between groups at baseline and after the intervention period. the results of the vas and avvq for both groups are summarized in table 3table 3.statistical analysis of visual analogue scale and aberdeen varicose veins questionnaire within and between groupsmeasuregroupsprepostvas (mm)control (n=13)8 (78)5.5 (4.56)experimental (n=15)8 (79)4.0 (34)avvqcontrol (n=13)65.1 (61.2067.05)57.2 (53.6061.91)experimental (n=15)67.1 (61.8068.50)39.3 (32.1256.20)data are presented as the median (interquartile range). vas : visual analog scale ; avvq : aberdeen varicose veins questionnaire ; experimental : sequential pneumatic compression. p<0.001 statistically significant difference within the group before and after the intervention. p<0.001 statistically significant difference between groups at baseline and after the intervention period.. before treatment no significant difference was found between the groups for either the vas or avvq (p=0.120, and p=0.460 respectively). both groups demonstrated significant reductions in both the vas and avvq (p=0.001 in both groups). after treatment there was significant difference between the groups in the vas and avvq. participants in the spc group had greater reductions in both the vas and avvq compared with those in the control group after the treatment period (table 3). vas : visual analog scale ; avvq : aberdeen varicose veins questionnaire ; experimental : sequential pneumatic compression. p<0.001 statistically significant difference within the group before and after the intervention. p<0.001 statistically significant difference between groups at baseline and after the intervention period. varicose veins are widely seen as medically unimportant and deserving low priority for treatment30. on the other hand, they are one of the most common symptoms of chronic venous disease, causing a dramatic impact on the quality of patient life31. for accurate evaluation of treatment outcomes, there was no available published research about the impact of spc on clinical aspects and qol of varicose veins patients. this study was designed as an initial study to investigate the effects of spc therapy on venous blood flow, pain level, and qol in women with varicose veins. it is ideal for optimal visualization of anatomy, hemodynamics, the diameter of the veins, and reflux time which can be measured accurately32. varicose veins patients previously showed a reduction in qol compared with the general population, and this discrepancy was significantly improved at 6 weeks after surgey23. the avvq is a valid measure of quality of life for patients with varicose veins12. the national health service in the united kingdom, has been collecting avvq scores for varicose vein surgeries since 2009, and the results have been correlated with both euroqol five dimensions questionnaire (eq-5d) and eq vas scores ; as a result, the avvq has been found to be a sensitive tool for assessing disease burden in varicose vein patients33. in the netherlands, a similar study matched avvq scores with 36-item short form health survey (sf-36) scales and showed a strong correlation between the avvq and generic qol outcomes34. the data obtained in this study revealed that the measured parameters, maximum, mean blood flow velocity, refilling time, pain level, and qol were improved significantly in both the control and experimental groups. comparison of the posttreatment measurement values between the groups revealed that the maximum and mean blood flow velocity, pain level, and qol were significantly higher in the spc group compared with the control group, while no significant difference was observed between groups for refilling time. the significant results found in the control group could be related to the effect of exercise on the venous system. regular exercise such as walking, biking, and swimming has beneficial effects that maximize calf pump function. the calf musculature functions as a peripheral heart, and a poor ejection fraction associated with poor leg conditioning results in progressive edema and increased varicose veins symptoms. a regular exercise program can improve the overall ejection fraction, resulting in symptom improvement35. in this study, all patients performed lower limb exercises ; these exercises result in blood being pumped back to the heart from the thigh, calf muscles, and veins in the arch of the foot. a strong calf and thigh muscles promote healthy blood circulation and minimizes vein disease29, 36, 37. ipc is generally a painless and non - invasive technique with proven efficacy as a valuable adjunct in the management of patients with venous, lymphatic, and arterial disease38. the current study proved that ipc has significant effects on venous blood flow based on the following physiological effects. ipc increases the velocity of venous return and reduces the amount of blood inside the veins at any time through stimulation of endothelial cell production of nitric oxide and creates shear stress on the walls of blood vessels, which is the probable physiologic mechanism for enhanced nitric oxide production. increased nitric oxide production inhibits platelet aggregation and neutrophil adherence, both of which play important roles in the creation of secondary hypoxic injury. nitric oxide is also a neurotransmitter that can influence vascular tone, thereby increasing blood flow39, 40. delis., found that ipc applied to thefoot and calf produced maximal venous emptying from the leg41. the spc therapy used in this study delivers sequential graded compression ; that is, at any time, the pressure in any chamber is higher than the adjacent most proximal one. there is evidence of distal blood trapping and incomplete calf vein evacuation as a result of uniform compression42. spc allows greater venous blood clearance or less stasis and prevents reversed pressure gradient and blood trapping during uniform compression. pain develops in varicose vein patients as a result of increased tissue tension caused by swelling. compression therapy counteracts venous hypertension by facilitating venous return toward the heart, improving venous pump function and lymphatic drainage. it reduces edema by increasing the local hydrostatic pressure and lowering the superficial venous pressure, preventing the leakage of fluids and macromolecules, improving cutaneous blood flow, and therefore decreasing pain43. these results are in agreement with the study of kakkos.42 who revealed the hemodynamic superiority of sequential compression compared with other compression device16. figueiredo.44, found that use of ipc improves blood flow when applied in legs or thighs. other studies have consistently concluded that all intermittent compression systems produce changes in femoral vein velocity. at pressures of around 40 mmhg, the typical maximum velocities achieved with calf and/or thigh compression would be 3560 cm / sec with augmentations (maximum velocity during compression compared with maximum velocity at rest) of around 50250%45, 46. assessment of the qol status at baseline and on completion of the six - week active study period using the avvq showed that spc can significantly improve the qol of patients by improving venous blood flow and reducing pain. physical functioning, bodily pain, vitality, and social functioning all improved significantly within six weeks of treatment with spc. the aspects of qol measured by the avvq are known to be impaired in patients with varicose vains12. a limitation of this study was its small sample of patients due to the required educational level for patient who had to be able to read and understand the avvq in english independently without any help. as there are no valid arabic versions of the avvq available, it is necessary to conduct translation, cultural adaptation, and validation to produce an arabic version of the avvq. in this study, women with varicose veins were treated with spc along with regular lower limbs exercise, five days / week for six weeks. spc therapy emerged as an effective treatment in the management of varicose veins, offering a clinically significant improvement in both venous blood flow and pain. these benefits were paralleled by significant improvements in all evaluated aspects of quality of life.	[purpose ] the aim of this study was to investigate the effects of sequential pneumatic compression therapy on venous blood flow, refilling time, pain level, and quality of life in women with varicose veins. [subjects and methods ] twenty - eight females with varicose veins were selected and randomly allocated to a control group, and experimental group. maximum and mean venous blood velocities, the refilling time, pain by visual analog scale and quality of life by aberdeen varicose veins questionnaire were measured in all patients before and after six weeks of treatment. both groups received lower extremity exercises ; in addition, patients in the experimental group received sequential pneumatic compression therapy for 30 minutes daily, five days a week for six weeks. [results ] all measured parameters improved significantly in both groups, comparison of post treatment measurements between groups showed that the maximum and mean blood flow velocity, the pain level, and quality of life were significantly higher in the experimental group compared with the control group. on the other hand there was no significant difference between groups for refilling time. [conclusion ] sequential pneumatic compression therapy with the applied parameters was an effective modality for increasing venous blood flow, reducing pain, and improving quality of women life with varicose veins.
acquired jak2v617f somatic mutation is a hallmark of philadelphia negative myeloproliferative neoplasm (mpn). in myelodysplastic syndromes (mds), jak2v617f mutation is seen in less than 5% of the cases. both 2001 and 2008 who classifications include among myelodysplastic / myeloproliferative (mds / mpn) overlap syndromes a provisional entity, refractory anemia with ringed sideroblasts associated with sustained thrombocytosis (rars - t) that appears to have a relatively favorable prognosis especially if jak2v617f mutation, detected in about 60% of the cases, apart from rars - t, however, the impact of jak2v617f mutation in mds remains unknown [68 ]. we conducted a retrospective study of mds patients with jak2v617f mutation, after exclusion of rars - t cases, where patients were compared with matched unmutated mds without jak2v617f mutation. after approval by the clermont - ferrand university hospital ethics committee, a questionnaire was sent to all french centers of the groupe francophone des mylodysplasies (gfm) and of the french intergroup of myeloproliferative neoplasms (fim) to collect clinical, laboratory and follow up data in mds patients with known jak2v617f mutation status detected in blood by real - time polymerase chain reaction. we also obtained, from the gfm registry of french mds, data from patients where jak2v617f mutation had been tested. in the absence of specific recommendations, jak2v617f mutation in those patients had been assessed systematically at diagnosis in a few centers, or later in the disease course, mainly because of features with potential predictive value for jak2v617f mutation, like thrombocytosis. patients with chronic myelomonocytic leukemia, rars - t and mds following mpn were excluded. descriptive statistics were used to characterize the population on parameters collected at the time of diagnosis. overall survival (os) was defined from diagnosis to death or censored at latest contact with the patient. time to acute myeloid leukemia (aml) progression was defined from diagnosis to date of aml. patient characteristics and outcome according to jak2v617f mutation status were analyzed by univariate analysis using the chi - square test for qualitative analysis and kruskal wallis test for quantitative analysis and log - rank test for os comparison. the primary endpoint of this retrospective analysis was to assess the impact of jak2v617f mutation on mds. in an attempt to reduce the effect of potential bias in this cohort, this method allows balancing of all measured relevant variables between the 2 groups (jak2v617f positive and negative). covariates included in the propensity - score model were sex, hemoglobin, wbc, anc, platelet, ipss, karyotype, marrow blasts and age known to be clinical relevant in mds. statistical analysis was performed using stata 10.0 software (statacorp, college station, tx, usa). after approval by the clermont - ferrand university hospital ethics committee, a questionnaire was sent to all french centers of the groupe francophone des mylodysplasies (gfm) and of the french intergroup of myeloproliferative neoplasms (fim) to collect clinical, laboratory and follow up data in mds patients with known jak2v617f mutation status detected in blood by real - time polymerase chain reaction. we also obtained, from the gfm registry of french mds, data from patients where jak2v617f mutation had been tested. in the absence of specific recommendations, jak2v617f mutation in those patients had been assessed systematically at diagnosis in a few centers, or later in the disease course, mainly because of features with potential predictive value for jak2v617f mutation, like thrombocytosis. patients with chronic myelomonocytic leukemia, rars - t and mds following mpn were excluded. descriptive statistics were used to characterize the population on parameters collected at the time of diagnosis. overall survival (os) time to acute myeloid leukemia (aml) progression was defined from diagnosis to date of aml. patient characteristics and outcome according to jak2v617f mutation status were analyzed by univariate analysis using the chi - square test for qualitative analysis and kruskal wallis test for quantitative analysis and log - rank test for os comparison. the primary endpoint of this retrospective analysis was to assess the impact of jak2v617f mutation on mds. in an attempt to reduce the effect of potential bias in this cohort, this method allows balancing of all measured relevant variables between the 2 groups (jak2v617f positive and negative). covariates included in the propensity - score model were sex, hemoglobin, wbc, anc, platelet, ipss, karyotype, marrow blasts and age known to be clinical relevant in mds. statistical analysis was performed using stata 10.0 software (statacorp, college station, tx, usa). data from 132 mds patients with known jak2v617f mutation status were collected in 19 centers of the fim and gfm, including 37 jak2v617f positive (jak2 +) and 95 jak2v617f negative (jak2) cases. the jak2v617f mutation burden was available in only 7 patients, showing a median level of 16% (range, 150%). there were no significant differences between jak2 + and jak2 cases in terms of age, gender, hemoglobin level, who 2008 classification, cytogenetic findings and ipss. in jak2 + patients, the white blood cells (wbc) count, absolute neutrophil count (anc), platelet count and mean corpuscular volume (mcv) were significantly higher, while the marrow blast percentage was significantly lower than in jak2 cases. treatments used (table 1) were similar in both patient groups, especially with regards to erythropoietic stimulating agents, chemotherapy and hypomethylating agents. seventeen jak2 + (45%) versus 36 (37.8%) jak2 patients were red blood cell transfusion - dependent (p=.77). median follow up was 44 months (range 18349) in jak2 + and 69 months (range 32185) in jak2 patients (p=.95). os was significantly longer in jak2 + patients (median not reached) than in jak2 patients (median 66 months) (p=.011) (fig. the estimated 5-year os was 86% in jak2 + patients versus 57% in jak2 patients. the 10 year cumulative incidence of aml progression was significantly lower in jak2 + (20%) than in jak2 patients (47%) (p<.001). when the analysis was matching with propensity score, os was still significantly longer in jak2 + patients (median not reached versus 73 months, p=.031) whereas jak2 status had no significant prognostic value for os in ipss intermediate-2 and high patients (p=.18). in this series, which is to our knowledge the first to focus on mds with jak2v617f mutation other than rars - t, we found jak2 + cases to have higher wbc, anc and platelet counts and fewer marrow blasts than jak2 mds, but no significant difference in cytogenetics and ipss. jak2+cases had significantly lower risk of aml progression and better os than jak 2 patients, an os advantage which persisted in a matched case control analysis based on age, gender, marrow blast percentage and ipss. recently, in a large series of 439 mds, bejar. found no prognostic value to jak2v617f mutation but only 13 (3%) of the patients had jak2v617f mutation. in addition, whether some of those patients had rars - t was not indicated. finally, only 64% of the jak2 + cases had lower risk ipss in bejar. indeed, we found the favorable prognosis of jak2v617f mutation to be mainly restricted to lower risk ipss. thus, we found that mds other than rars - t with jak2v617f mutation were associated with laboratory features (higher wbc, anc, platelet count) with better outcome. however, prospective studies precisely evaluating the impact of this mutation are required to confirm our findings.	while in rars - t, jak2v617f mutation is common and associated with good prognosis, the clinical and prognostic impact of this mutation in other mds is unknown. we collected data from 132 non - rars - t mds with known jak2v617f mutation status. jak2v617f mutation was significantly correlated with lower progression to aml (p<.0011) and better overall survival (os, p=.011). os difference persisted after matching on age, sex, ipss and % marrow blast (p=.031). thus, in mds other than rars - t, jak2v617f mutation may be associated with favorable outcome.
after repeated previous consultations, a 73-year - old woman suffering for several months from itchy plaques and nodules on the face and neck presented for her follow - up visit (figures 1, 2). a biopsy had been performed during previous consultations and the histopathologic diagnosis lay between lymphoma and pseudo - lymphoma. dermoscopy of the lesions revealed a perfectly demarcated burrow and, at its end, a scabies mite (figure 3). the accuracy of dermoscopy for the diagnosis of scabies has been assessed as at least equal to microscopic examination after skin scraping. in fact, dermoscopy has the advantage of rapidly screening several lesions of the patient, in contrast to the microscopic examination which is performed by scraping only one or two lesions. given that in otherwise healthy individuals, no more that 45 mites exist on the skin at on time point, scanning all the lesions of the patient minimizes the possibility of a false negative result. in our patient, the atypical clinical manifestation and, especially, the distribution of the eruption on the face, misled the clinicians on repeated visits, whereas dermoscopy was not performed during the initial visits, because scabies was not even included in the differential diagnosis. this resulted in unnecessary surgical procedures and histopathologic exams, which might have been continued if dermoscopy was not eventually performed. this case highlights a basic principle of dermoscopy application, namely, that dermoscopy should be applied on every skin lesion and not only in clinically pre - selected cases.	dermoscopy is already considered a fairly established method for diagnosing scabies. this is because dermoscopy enables the visualization both of the burrow and the mite itself, forming the so - called jet with a contrail structure. in the present report we present an extraordinary case of a patient with scabies lesions on the face and neck, which was misdiagnosed during sequential visits and underwent unnecessary surgical diagnostic procedures. finally, the diagnostic problem was solved when dermoscopy was applied.
in the united states, the original older americans act (oaa) created a system of community - based social support services for persons age 60 and older. an updated oaa included provisions for access to these services by persons with intellectual and developmental disabilities (i / dd) and supports for older carers of persons with i / dd. many state and local initiatives have been initiated for use of the oaa as a means for promoting greater integration of older persons with i / dd into existing community - based social services for the elderly by targeting primarily the nation s network of senior centres and neighbourhood congregate meal sites. to help these efforts a national training program underwritten by a federal grant was undertaken in which workshops, networking building, and technical assistance efforts were conducted by a team of government and university colleagues. of the needs and wants of older people with i / dd, an opening of programs, and greater collaboration on sharing resources and providing supports and services. although obstacles remain, often linked to enmity toward people with disabilities, unwillingness to share funds and resources, or age - peer ignorance, the initiatives generally have proved to be productive resulting in enhanced social integration and the elimination of barriers to planning cooperative community services.	introductionin the united states, the original older americans act (oaa) created a system of community - based social support services for persons age 60 and older. an updated oaa included provisions for access to these services by persons with intellectual and developmental disabilities (i / dd) and supports for older carers of persons with i / dd. many state and local initiatives have been initiated for use of the oaa as a means for promoting greater integration of older persons with i / dd into existing community - based social services for the elderly by targeting primarily the nation s network of senior centres and neighbourhood congregate meal sites.descriptionto help these efforts a national training program underwritten by a federal grant was undertaken in which workshops, networking building, and technical assistance efforts were conducted by a team of government and university colleagues.conclusionthe outcome was an enhanced understanding among workers in community ageing programs of the needs and wants of older people with i / dd, an opening of programs, and greater collaboration on sharing resources and providing supports and services. although obstacles remain, often linked to enmity toward people with disabilities, unwillingness to share funds and resources, or age - peer ignorance, the initiatives generally have proved to be productive resulting in enhanced social integration and the elimination of barriers to planning cooperative community services.
on june 10, 2012, a previously healthy 4-year - old boy in hong kong had fever, cough, and rhinorrhea. he was admitted to pamela youde nethersole eastern hospital in hong kong, cardiac asystole developed, and he died 81.6% nt and > 96.9% aa identities with the cva2 prototype strain fleetwood (cva2f), suggesting that these strains belonged to the same serotype as cva2. the 5-utr and nonstructural regions (p2 and p3) of the 4 cva2 strains had highest sequence identities with those of other human enterovirus a strains but not with cva2f. cva2f, coxsackievirus a prototype strain fleetwood ; utr, untranslated region ; na, not applicable ; p1, capsid protein 1 ; vp, viral protein ; p2, nonstructural protein 2 ; p3, nonstructural protein 3. phylogenetic trees were constructed by using nucleotide sequences of the 5-utr and p1, p2, and p3 regions of the 4 cva2 strains and other human enterovirus a strains with complete genome sequences (figure 1). sequence alignment was performed by using clustalx version 2.0 (6). the best evolutionary model (general time reversible + invariant sites) for each dataset was determined by using modelgenerator (7). maximum - likelihood phylogenetic trees were constructed by using phyml version 3.0 (8), and bootstrap values were calculated from 1,000 trees. phylogenetic analysis showed that the 4 cva2 strains were most closely related to cva2f in the 5-utr and p1 region. the 4 cva2 strains clustered with ev71 subgenotype b3 strain sar / sha66 in the p2 region but with cva4 strain sz / chn/09 in the p3 region. phylogenetic trees of a) 5-untranslated region (utr), b) capsid protein (p1), c) nonstructural protein 2 (p2), and d) nonstructural protein 3 (p3) regions of 4 coxsackievirus a2 (cva2) strains, hong kong, 2012 and other human enterovirus (ev) a strains with complete genome sequences. trees were inferred from data by using the maximum - likelihood method with bootstrap values calculated from 1,000 trees. sequences for 758-nt positions in each 5-utr, 2,595 nt positions in each p1 region, 1,734 nt positions in each p2 region, and 2,259 nt positions in each p3 region were included in the analysis. only bootstrap values scale bars indicate estimated number of nucleotide substitutions per 5 (b and d) or 10 (a and c) nucleotides. virus strains (genbank accession nos.) used were cva2 fleetwood (ay421760), cva3 olson (ay421761), cva4 high point (ay421762), cva4 sz / chn/09 (hq728260), cva5 swartz (ay421763), cva6 gdula (ay421764), cva7 parker (ay421765), cva8 donovan (ay421766), cva10 kowalik (ay421767), cva12 texas-12 (ay421768), cva14 g-14 (ay421769), cva16 g-10 (u05876), cva16 sz / hk083 (gq279368), ev71 brcr (u22521), ev71 nagoya (ab482183), ev71 ms/7423/87 (u22522), ev71 sar / sha66 (am396586), ev71 9/97/sha89 (aj586873), ev71 5511-sin-00 (dq341364), ev71 804/no/03 (dq452074), ev71 tainan/4643/98 (af304458), ev71 03-kor-00 (dq341356), ev71 sz / hk085 (gq279369), and ev71 e2005125-tw (ef063152). multiple sequence alignment of genomes of representative cva2 strain 430895 and other human enterovirus a strains was generated by using clustalx version 2.0 and edited manually. once aligned, similarity plot and bootscan analyses were conducted by using simplot version 3.5.1 (window size 400 bp, step 20 bp) (9), with the genome sequence of cva2 strain 430895 as the query sequence. recombination analysis of complete genome of coxsackievirus a2 (cva2), hong kong, 2012. b) bootscanning and c) similarity plot analyses were conducted by using simplot version 3.5.1 (http://sray.med.som.jhmi.edu/scroftware/simplot/) (kimura distance model ; window size, 400 bp ; step, 20 bp) on a gapless nucleotide alignment generated with clustalx 2.0 (8) with the genome sequence of cva2 strain 430895 as the query sequence. in panel b, green line indicates cva2 prototype strain fleetwood, red line indicates ev71 strain sar / sha66 of subgenotype b3, yellow line indicates ev71 strain sz / hk085 of subgenotype c4, and blue line indicates cva4 strain sz / chn/09. p1, capsid protein 1, p2, nonstructural protein 2 ; p3, nonstructural protein 3 ; utr, untranslated region ; vp, viral protein. results showed high bootstrap supports for clustering between cva2f and the 4 cva2 strains at nucleotide positions 7003400, between ev71 strain sar / sha66 subgenotype b3 and the 4 cva2 strains at nt positions 37004030, between ev71 strain sz / hk085 subgenotype c4 and the 4 cva2 strains at nt positions 40304300, and between cva4 strain sz / chn/09 and the 4 cva2 strains at nt position 5700 to the 3 end of the genome. these findings indicated that recombination events might have occurred between nt positions 3400 and 3700 (corresponding to the 2a region), at nt position 4030 in the 2b region, and between positions 5400 and 5700 (corresponding to the 3c region). several possible recombination events were detected in other regions of the cva2 genome but with lower bootstrap supports. because noncapsid regions of enteroviruses are not correlated by serotype (10), results of phylogenetic and recombination analyses were based on the highest sequence similarity between enterovirus strains. although recombination was evident in the 4 cva2 strains, lack of comparative sequences indicated that the timing of recombination events was unknown, and low overall similarity to comparison sequences suggested that these events could be distant in time. as in many studies of human enterovirus a strains, lack of complete genome sequences for most serotypes, with the possible exception of ev71, limits interpretation of results for recombination analysis (10,11). in the present study, only 2 complete genome sequences of cva4 strains (prototype strain high point and strain sz / chn/09) were included in recombination analysis. therefore, sequencing and analysis of more complete genome sequences of human enterovirus a strains, particularly cva strains, from a wider geographic area over a longer period will provide a clearer picture of the role of recombination in this species. we report a novel enterovirus isolated from or detected in 4 young children with severe upper respiratory tract infections in hong kong, 2 of whom died. this virus was characterized by complete genome sequencing as a recombinant virus of at least 3 enteroviruses (cva2, ev71, and cva4), and had the capsid of cva2. although it could not be determined whether this virus was the cause of the deaths, this report might serve to alert other investigators of circulation of a more pathogenic enterovirus.	a natural recombinant of coxsackievirus a2 was found in 4 children with respiratory symptoms in hong kong, china, during the summer of 2012. two of these children died. vigilant monitoring of this emerging recombinant enterovirus is needed to prevent its transmission to other regions.
recently, fragment - based drug design has emerged as a popular method for screening small chemical structures against a known drug target. modifications to the lead compound serve to improve the target selectivity and administration / absorption, distribution, metabolism, excretion / elimination, toxicology (admet) profile. the resulting modifications aim to increase the chance of discovering a novel drug candidate. the probability of discovering a novel drug candidate is increased if the modifications to the lead compound are informed. one concept broadly applied to help medicinal chemists make informed modifications to lead compounds is that of bioisosterism. a bioisostere is a substituent or a fragment of an active compound with similar physical or chemical properties. bioisosteric modifications can be made to lead compounds with the aim being to improve the admet profile of the lead. only the properties important to the biological activity in each specific lead optimization need to be enhanced. however, the properties that are enhanced to improve the admet profile of one lead might unfortunately deliver contrasting results in the case of a different lead. therefore, successful modifications of a lead are unique and specific to the lead itself. the unique combination of modifications that are specific to each successful lead optimization is often unknown. as the specific modifications are unknown for each lead, a number of broad properties have been outlined that can be modified practically in the lead optimization process. these properties relate to both the drug molecule and its environment, and serve as a starting point when considering bioisosteric substitutions. the properties are size, shape, electronic distribution, lipid solubility, water solubility, pka, chemical reactivity, and hydrogen - bonding capacity. hydrogen bonding is an important noncovalent interaction between a biological molecule and its local environment. hydrogen bonding may influence binding affinity and solvation, membrane permeability, and adsorption onto surfaces. hydrogen bonding influences both the distribution of a drug to its target and the binding of a drug to its target. after the drug is administered and absorbed into the body, it must be delivered to the target. as the drug is absorbed into the body, it interacts with the biological fluids that transport the drug around the body. any exposed polar residues of the drug are capable of forming hydrogen bonds with the biological fluids as the drug becomes dissolved. the drug must then shed the solvent to bind the target. the hydrogen - bonding network involved in the displacement of the solvent with the gibbs energy of a drug interaction can be improved by focusing on hydrogen bonding. there is a favorable enthalpy change as the drug forms hydrogen bonds with the target. however, there is also an unfavorable enthalpy change as hydrogen bonds are broken as the drug sheds the layer of solvent upon binding. there is a favorable entropy change as the layer of solvent is shed upon binding. during binding, there is, however, an entropic penalty due to the loss of translational and rotational freedom. favorable enthalpy and entropy changes must be maximized while minimizing unfavorable changes to improve the binding profile of a lead compound. one way in which this may be achieved is to modify the lead in such a way that the strongest hydrogen bonds are formed between the drug and its respective target. knowledge of relative hydrogen - bond strengths is therefore important to medicinal chemists in lead optimization. quantum chemical topology is a method that extracts chemical information from wave functions by using the language of dynamical systems. qct generalizes the original quantum theory of atoms in molecules (qtaim) and builds on the idea of partitioning quantum mechanical property densities by means of a (gradient) vector field. this approach defines finite volumes in 3d space that express the partitioning of the quantum system. in this article, we only use topological properties retrieved from the electron density (for details and definitions, see section 3). bond critical points (bcps), also denoted (3,1), are featured in this study, together with the quantum mechanical functions evaluated at them. these local properties are complemented by global properties, which are obtained by 3d integration over the volume of a topological atom. these concepts have been reviewed many times before. despite the importance of hydrogen bonding in biological systems, there is a general lack of understanding of the relative strengths of hydrogen - bond basicity. the first scales of hydrogen - bond basicity were set up by taft and co - workers, who investigated linear free energy relationships (lfer) between the reference acid 4-fluorophenol and a series of oh hydrogen - bond donors (hbds). the equilibrium constants of these complexes were used to define the relative basicities of the hydrogen - bond acceptors (hbas). second, due to the nature of the lfers, pkhb values are limited to (oh) hbds. when the lfers were repeated using 5-fluoroindole instead of 4-fluorophenol as the reference acid, family - dependent behavior was observed. therefore, the pkhb scale covers only a limited proportion of all possible hydrogen bonds that may be formed, and has been made little use of by medicinal chemists. the log kbh scale, established by abraham and co - workers, was constructed using 34 reference acids complexed to a series of bases in a 1:1 ratio. the log kbh scale was intended to establish an overall generality of hydrogen - bond basicity. the log kbh values were obtained from 34 lfer equations in the form of eq 1:1where the index i refers to the base, a represents the acid, la and da are the gradients, and log k is the intercept, respectively, of the 34 straight line equations. the 34 lfer equations were constrained to intersect the log kbh axis at a magic point of 1.1 log units. this constraint allowed an equivalent but more convenient linear transform of the log kbh scale to be set up. the linear transform of the log kbh scale was called the 2h scale. adding the 1.1 log k units to the log kbh scale, and dividing by 4.636, the 2h scale conveniently ranges from 0 to 1. therefore, a 2h value of 0 represents no hydrogen bonding, and a 2h value of 1 represents the strongest hydrogen bonding. once the constants la and da in eq 1 are known for a particular acid, secondary log kbh values not included in the original data can be obtained. however, due to the statistical treatment of 34 such log kbh values to obtain 2h values, the determination of secondary 2h values not included in the original data set is not straightforward. many organic bases and potential drug molecules have multiple hydrogen - bonding sites. these bases with multiple hydrogen - bonding sites are known as polyfunctional hydrogen bond bases. as the 2h scale of hydrogen - bond basicity was considered to be the most general, it was adapted to account for polyfunctional hydrogen - bond bases. the 2h scale is based on equilibrium constants, but values are not directly calculated from equilibrium constants. instead, the 2h values are based on the general solvation equation in the form of eq 2:2 in this equation, sp relates to a set of solute water solvent partition coefficients in a given system, r2 is the excess molar refraction, 2h is the dipolarity or polarizability, and 2h and 2h are the hydrogen - bond acidity and hydrogen - bond basicity. the parameters of eq 2 can be determined experimentally with the exception of the acidity or basicity terms. the solutes were restricted either to give an acidity term of 0 or to monoacidic solutes for which the acidity term is known. the 2h values were estimated applying 2h values to eq 2 and back - calculating to return successive 2h values in an iterative process until a self - consistent set of equations and therefore 2h were obtained. it was therefore claimed that the 2h scale of overall hydrogen - bond basicity was more useful than the 2h scale when considering biological properties. despite being the most quoted scale of hydrogen - bond basicity, and despite the authors claim that it is more useful than 1:1 scales in the interpretation of biochemical and physiochemical properties, there are problems in the interpretation of 2h values. the 2h scale assigns only one 2h value to the whole molecule, and is based on partition coefficients rather than binding. although medicinal chemists may make use of 2h values to predict permeability, the 2h scale offers no thermodynamic binding information. it is clear that there is a need for a measure of hydrogen - bond basicity that targets the needs of medicinal chemists. the requirement for this scale is that it should combine the simplicity of the pkhb scale and its thermodynamic relevance with the analysis of polyfunctional bases as in the 2h scale. the pkbhx scale is essentially an extension of the pkbh scale in that it is based on equilibrium constants of 1:1 complexes in the solvent ccl4 where 4-fluorophenol is the reference acid. an important difference between the pkbhx scale, on one hand, and the pkbh and 2h scales on the other hand, is the experimental method used to calculate the equilibrium constants. the difference in experimental methods allows polyfunctional bases to be included in the analysis. fourier transform infrared (ftir) spectrometry is used in the determination of the pkbhx scale rather than f nmr, uv, and dispersive ir techniques, which are mostly used for pkbh and 2h. the advantage of using ftir is that multiple significant hydrogen - bond acceptor sites of polyfunctional bases can be analyzed. during the formation of a hydrogen bond, the stretching vibration of the xh bond is shifted to a lower wavenumber, giving rise to peaks in a ftir spectrum. the change in the vibrational frequency of the xh bond can be translated into a pkbhx value through family - dependent pkbhx ir relationships correlate the change in ir vibrational frequency of the xh bond to pkbhx values for a family of hbas and a reference hbd in 1:1 complexes. the ftir spectroscopy method allows equilibrium concentrations of these 1:1 complexes to be measured in a certain frequency range, therefore allowing a pkbhx value to be obtained. it is clear that the pkbhx scale is able to analyze polyfunctional bases as well as the 2h scale. an important difference, however, is that pkbhx values are thermodynamically significant because each basic site is considered separately. on the other hand, the 2h scale does not consider each hydrogen - bond acceptor site individually. this feature is illustrated by the fact that the pkbhx database contains 1338 pkbhx values taken from 1164 bases. this large data set is advantageous to medicinal chemists as most drugs contain several hydrogen - bond acceptor sites consisting of mainly o, n, s, f, cl, and sp hydridized carbons. it is therefore a possibility that the pkbhx scale could be used as a descriptor characterizing hydrogen bonding from which bioisosteric replacements could be found. the 2h scale considers the entire molecule to be thought of as the hydrogen - bond acceptor, whereas the pkbhx scale considers each individual hydrogen - bond acceptor site independently. the 2h scale depends on the entire molecule being intact to return a 2h value. the pkbhx scale, however, allows for each hydrogen - bond acceptor site to be assigned a pkbhx value. therefore, a fragmented drug - like molecule may be assigned pkbhx values but not 2h values. there have been a number of computational models used to predict hydrogen - bond basicity. the minimum value of the electrostatic potential at the hydrogen - bond acceptor atom has been used to obtain a family - dependent model for the hydrogen - bond basicity parameter used in linear solvation energy relationships. a more general model displaying a reasonable family - independent correlation, r = 0.81, between log khb and the minimum value of the electrostatic potential has also been established. basicity is typically more difficult than acidity to model due to the variety of different hba sites. by considering the nitrogen hbas separately, the correlation can be improved when correlating pkhb to the minimum of the electrostatic potential, and refined further by also including the interaction energy, giving r = 0.99. platts developed a model to predict hydrogen - bond basicity based on dft calculations on a series of bases taken from abraham s list of 2h values. theoretical properties were calculated for both the isolated base and the base complexed with hf. once again, strong family - dependent relationships were observed. the atomic multipole moments of nitrogens occurring in the isolated bases gave the best correlations, r = 0.786. the combination of the minimum electrostatic potential at the nucleus of the hba and an atomic charge (according to qct) of the isolated base gave a better correlation, yielding r = 0.887 for oxygen hbas. however, with the use of a hf probe, improvements were made with the oxygen model yielding r = 0.939. the binding energy calculated from the supramolecular complex correlated reasonably well, r = 0.901, to give a good family - independent model. it could be argued that the 2h scale might not be the best measure of basicity for the medicinal chemist to predict. this is due to the lack of hba site - specific information and thermodynamic information offered by the scale. a recent study repeated the supramolecular approach of platts, this time using phenol as the hbd, and found good general correlations (r = 0.94) between log k values and the hydrogen - bond binding energy for nitrogen and oxygen hbas. platts later refined his initial model correlating free energy of formation with pkhb values. fits of r = 0.969 were observed for a family - independent model using hf as the hbd. the final refinement to this model was made by combining the minimum of the electrostatic potential at the hba and properties of the hydrogen - bond critical point (bcp), leading to r = 0.97. devereux. suggested this model to be too expensive computationally for use in qct - aided drug design. these authors constructed a model by combining the minimum, median, and mean of the electrostatic potential at the hba with the minimum of the local ionization energy on the hba surface and correlating with 2h values. they observed family - dependent models with non - nitrogen bases producing fits of r = 0.931, while nitrogen bases gave r = 0.871. however, the results were found to be less accurate than platts model, which correlated properties of hydrogen - bonded complexes with 2h values. it would therefore seem that the most accurate models used to predict hydrogen - bond basicity must be constructed from properties of the hydrogen - bonded complex. also, by using 2h values, the model is limited to the 1:1 hydrogen - bonded complexes only. however, in drugtarget interactions, 1(hba):n(hbd) complexes are likely to occur, and an improvement to the model could be made by correlating to a measure of hydrogen - bond basicity that accounts for all major hydrogen - bond acceptor sites of a particular molecule. however, drug design warrants the inclusion of polyfunctional bases and therefore needs a model that goes beyond the 1:1 ratio. such a refined model must keep the accuracy of platts model by considering properties of the hydrogen - bonded complex, while taking into account polyfunctional hydrogen - bond acceptors. a good candidate to fulfill the advantage of using pkbhx values over 2h values is that a pkbhx value is listed for each hydrogen - bond acceptor site. this important feature allows for the correlation between a single value of a particular property of a specific hydrogen bond and a single value relating to the basicity of that hydrogen bond. the bases were chosen to include a wide range of pkbhx values for nitrogen, oxygen, and sulfur acceptor atoms. the strength of the nitrogen bases ranges from weaker nitrile and aniline acceptors, to stronger pyridine and amines. the oxygen bases include weaker phenol and alcohols, and stronger carbonyl and amide acceptors. a small set of sulfide and thiol sulfur acceptors also formed part of the training set. the distribution of the pkbhx values within the training set was chosen to roughly match that of the entire pkbhx database. hydrogen - bond complexes (1:1) were formed between the 41 training bases and 6 possible hbds. the latter consist of 4 oh donors (water, methanol, 4-fluorophenol, and serine), complemented by methylamine and hydrogen fluoride. the pkbhx scale was set up using 4-fluorophenol as the reference hbd, and is thought to be applicable to all oh, nh, and nh donors. therefore, the chosen hbds in this study, with the exception of hydrogen fluoride, should give complexes that the pkbhx scale can be applied to. a set of hydrogen - bonded complexes were set up, based on those investigated by platts. they were generated by placing a hydrogen atom of the hbd approximately 2.0 away from the primary hba site. the essence of the hydrogen - bond system is then conveniently denoted by d the hydrogen - bonded complexes were geometry optimized at the b3lyp/6 - 311++g(2d, p) level of theory. properties of the complexes were obtained and correlated with their respective pkbhx values. the properties include the hydrogen - bond length r(ah), the change in the hbd bond length r(h d), and the hydrogen - bond energy e, the details of which are explained below. note that the symbol denotes the difference of the complex and the constituent monomers forming it. atomic properties are obtained by a volume integral over an atomic basin. the kinetic energy densities g(r) and k(r), and the atomic energy e(), are defined in eqs 3, 4, and 5:345where g(r) is one type of kinetic energy density, while k(r) is another type. the symbol d refers to the integration over the spatial and spin coordinates of all electrons except one, dr denotes a three - dimensional integration over the basin of the topological atom, while r represents the virial ratio correction factor. it is an important property of a topological atom that its kinetic energy is well - defined (which is not true for an arbitrary subspace). indeed, g() = k(). the atomic charges are defined in eqs 6 and 7:67where z is the charge of the nucleus inside the topological atom. all atomic integrations used to analyze atomic properties were calculated with the aimall suite. the atomic properties used here are e(h), or the change (going from the monomers to the complex) in the hbd energy, and q(h), or the change in charge of the hydrogen atom in the hbd. bond properties were evaluated and analyzed only at bcps associated with ha hydrogen bonds and bcps of the covalent hbd bonds (d h), both in the hydrogen - bond system d ha. properties analyzed at the hydrogen bcp are the electron density (ah), the laplacian of the electron density (ah), the kinetic energy density at the bcp g(ah), and the kinetic energy density g(r) at the bcp, divided by the electron density g/(ah). properties of the hbd critical point are the change in the electron density (h d), and the change in the laplacian of the electron density (h d). this program also generated figure 1, which shows an atomic basin of a hbd and all bcps of a hydrogen - bonded complex. the purple dots are the bond critical points (bcps), and the atomic basin of the hydrogen in the hydrogen bond is marked in wireframe (where the electron density has been cut off at = 10 au at the outer surfaces). basis set superposition error (bsse) calculations were carried out to accurately estimate the hydrogen - bond binding energy for the water series only. as the bsse of hydrogen - bond energy turned out to be very small, bsse calculations were not repeated for complexes without water. the method used to perform the bsse calculations is that of handy and co - workers. equations 8, 9, and 10 show the details:8910equation 8 shows how to calculate the hydrogen - bond energy without considering the bsse. equation 9 calculates the bsse of the hydrogen - bond energy using the counterpoise correction method. in eq 9, the asterisk refers to the geometry of the hba and hbd in the optimized complex, and cp stands for counterpoise and refers to the energy of the hba and hbd in the presence of ghost hbd and hba gaussian functions, respectively. equation 10 calculates the counterpoise corrected hydrogen - bond binding energy from eqs 8 and 9. criticism of the b3lyp functional led to several investigations showing how the mpwb1k functional consistently performs better for weak interactions such as hydrogen bonding. to investigate this criticism in the context of the current work, a further set of calculations using the mpwb1k/6 - 311++g(2d, p) level was performed for the training set of bases where water was used as a hbd. to account for polyfunctional bases, calculations were performed at the b3lyp/6 - 311++g(2d, p) level on a set of 11 bases with 2 hydrogen - bond acceptor sites giving 22 acceptor sites in total. the properties calculated from the 1:1 complexes have been compared to properties calculated from the same set of bases in 2:1 complexes. table 1 list the names of the 41 bases (i.e., hbas) and their corresponding pkbhx values. each of the hbas listed in table 1 has been used to form a hydrogen - bonded complex with each of the six hbds, which are water, methanol, 4-fluorophenol, methylamine, serine, and hf. examples of these complexes are given in figure 2, except for hf, illustrating each possible element (s, o, and n) as an acceptor. five examples of hydrogen - bonded complexes in their optimized geometry : (a) waterpyridine, (b) methanolphenol, (c) 4-fluorophenoldimethyl sulfide, (d) methylamineacetamide, and (e) serinedimethyl amine. table 2 summarizes the correlations of calculated properties (energetic, geometric, atomic, and bcp) with the experimental pkbhx values. with the exception of the hydrogen fluoride set, the quantity e(h) consistently outperforms all other properties, giving r values of 0.96 for water, 0.95 for methanol, 0.91 for 4-fluorophenol, 0.93 for serine, and 0.97 for methylamine. particular attention was given to the electronic energy of the hydrogen bond, e. however, poor correlations across the board of oh and nh donors were observed. the bsse corrected energy offered no improvement to the correlations. however, the plot of e against pkbhx where hydrogen fluoride was used as the probe gave a correlation of r = 0.60. although 0.60 is a weak correlation, it is significantly better than any of the values obtained for oh and nh donors (which are generally about 0.30 with the exception of 4-fluorophenol with r = 0.52). lamarche and platts obtained a value of r = 0.91 for the correlation between computed e and taft s pkhb values using hydrogen fluoride as the hbd. taking into account the stronger correlations of e with pkbhx and the observations of lamarche and platts when hydrogen fluoride is used as the hbd, it could be said that any relationship between e and basicity is highly questionable due to the use of hydrogen fluoride as a probe. indeed, the computed e of a hydrogen fluoride complex correlates reasonably well to basicity values, but a link can not be made between theory and experiment as the basicity scales are not applicable to hydrogen fluoride complexes. the e and e(h) values in brackets in the water set are taken from mpwb1k calculations. the data set is as in table 1, minus 3-chloropyridine, dimethyl sulfide, ethyl thiol, ethylmethylsulfide, 4-fluorophenol, and diethyldisulfide. these hbas were omitted due to computational timing constraints. as a direct comparison, r = 0.97 for e(h) when the reduced data set is used for b3lyp calculations. the mpwb1k density functional has been shown to perform better than b3lyp when computing the interaction energies of hydrogen - bonded complexes. table 2 reveals that hydrogen - bond binding (interaction) energy correlates with pkbhx for mpwb1k calculations but not b3lyp calculations. however, as the binding energies of the complexes used here do not correlate with pkbhx values as strongly as e(h), it is adequate to use b3lyp. furthermore, table 2 shows how e(h) calculated from a mpwb1k wave function performs equally as well as e(h) calculated from a b3lyp wave function when correlating e(h) to pkbhx. correlation of the qct e(h) with pkbhx values for the bases listed in table 2 with hbds : (a) water, pkbhx = 197.34e(h) 3.749, r = 0.96, se = 0.30, f = 829.49 (se = standard error) ; (b) methanol, pkbhx = 180.94e(h) 3.7074, r = 0.95, se = 0.31, f = 740.62 ; (c) 4-fluorophenol, pkbhx = 170.44e(h) 3.8052, r = 0.91, se = 0.42, f = 391.90 ; (d) serine, pkbhx = 179.19e(h) 1.6701, r = 0.93, se = 0.37, f = 522.46 ; (e) methylamine, pkbhx = 194.73e(h) 2.3995, r = 0.97, se = 0.26, f = 1089.93. figure 3 shows the plots of e(h) against pkbhx for water, methanol, 4-fluorophenol, serine, and methylamine. however, pkbhx values did not correlate with e(h) when hf was used as the hbd, as made from the very low correlation coefficient r = 0.04. it is claimed that the pkbhx scale is applicable to hydrogen - bonded complexes provided the hbd s functional group is either oh, nh, or nh. therefore, hydrogen fluoride was chosen as a control probe because hydrogen - bonded complexes, where hydrogen fluoride is the hbd, have no link to the experimental pkbhx scale. the relationship between e(h) and pkbhx values gains credibility as it holds only for hbds that the pkbhx scale may be applied to. therefore, a strong link between theory and experiment has been established through the partitioned atomic energy of the hbd. essentially, the energy assigned to a theoretically derived atomic basin is related to a molecular free energy quantity that is derived from experiment. it is also astonishing that e(h) is able to predict the pkbhx of an external data set. an external data set of 41 compounds has been chosen to demonstrate that the qct property e(h) can predict pkbhx values of hbas chosen from the pkbhx database. figure 4 plots the predicted versus experimental pkbhx values for the hbas listed in table 3. predicted pkbhx values are calculated from eq 11:11which corresponds to the least - squares line in panel b in figure 3. this panel plots e(h) against pkbhx, where methanol is the hbd, complexed to the hbas of table 2. methanol was chosen as the probe to use in the model for three reasons : (i) its simplicity, (ii) its use in obtaining secondary lfer derived experimental values, and (iii) the strong correlation of r = 0.95 between the e(h) of these complexes and their pkbhx values. although the correlation between e(h) and pkbhx for the methylamine set (r = 0.97) is stronger than the methanol set, it is challenging to model specific hydrogen bonds with methylamine due to the presence of an additional hydrogen attached to the nitrogen as compared to oh donors that have a single hydrogen. this additional hydrogen may cause the molecule to rotate during optimization and form interactions other than the desired one, a characteristic often observed in this work due to no geometrical constraints placed upon the optimization. therefore, it is easier to describe pkbhx in terms of e(h) on water or methanol hbds. an f - test was performed, and the variances between the water and methanol models were not found to be significantly different. following, a paired t test assuming equal variances was performed. again, the water and methanol models were not found to be significantly different. therefore, there is no statistical advantage to choosing methanol as the probe over water. however, methanol has been chosen due to its usefulness for obtaining secondary pkbhx values from lfers. the advantage that methanol can be used in such a way justifies the slightly lengthier calculations. therefore, by using methanol as the probe, the computation is kept in close proximity to the experimental procedure. correlation of the predicted pkbhx values with experimental pkbhx values for the bases listed in table 3. predictions are based on the straight line equation for methanol complexes (figure 3b). experimental = 0.8503 predicted 0.0011 ; r = 0.90, se = 0.75, f = 1.80. figure 4 shows a relatively strong correlation, r = 0.90, for the plot of predicted versus experimental pkbhx values. the only constraint put on the compounds in table 3 is that the hba must be either an oxygen, a nitrogen, or a sulfur atom. not only does the model based on e(h) show impressive external predictability, but also good generality. there seems to be no need to separate the hbas into atomic groups (i.e., families) nor build individual models for each hba type. the results discussed so far are for 1:1 hbd : hba complexes where the bases have only one major hba site. the attractiveness of the pkbhx scale to the medicinal chemist lies in its inclusion of data for the more realistic cases of polyfunctional bases with two or more major hba sites. table 4 shows a list of 11 hbas, each with two major hba sites (so leading to 22 entries). two approaches were used to test the validity of the e(h) model for polyfunctional bases. the first approach simply involved generating a 1:1 complex for each hba site. the second approach aimed to mimic experiment in which the bases are surrounded by an excess of acid allowing each hba site to reach equilibrium with a hbd. the way in which the experiment was mimicked consisted of generating 2:1 hbd : hba complexes where both hba sites are occupied in the same calculation. the first approach in which 1:1 complexes were generated marginally outperforms the second approach in which 2:1 complexes were computed. however, the fact that 1:1 complexes slightly outperform 2:1 complexes is beneficial to the computational chemist because 1:1 complexes are computationally less expensive than the 2:1 complexes. the symbol in brackets indicates the hba site where (n) is a nitrogen atom, (o) is an oxygen atom, (nit) is a nitrogen atom on a nitrile functional group, and (pyr) is a nitrogen atom on a pyridine functional group. correlation between the qct e(h) value and the pkbhx values for the 11 polyfunctional bases (each with two hba sites) listed in table 4 with the hbd methanol common to all. (a) 1:1 complex, (b) 2:1 complex. (a) pkbhx = 178.72e(h) 3.9521, r = 0.93, se = 1.39, f = 279.50 ; (b) pkbhx = 166.86e(h) 3.4887, it has been shown that the energy, e(h), assigned to a theoretically derived atomic basin of a molecule in the gas phase is related to a molecular free energy quantity that has been derived from experiment, pkbhx. furthermore, the relationship breaks down when hydrogen fluoride is used as the hydrogen - bond donor. therefore, the relationship between computation and experiment is only observed with computations that use hydrogen - bond donors that the experimental scale is applicable to. it has also been shown that pkbhx values outside the initial data set are accurately predicted by the model set up with the initial data set. therefore, e(h) is able to predict the pkbhx values of an external data set. the attraction of the pkbhx scale is that a pkbhx value is listed for each hydrogen - bond acceptor site on polyfunctional bases. it has also been found that e(h) relates to pkbhx values of polyfunctional bases. this is an important observation when considering site - specific thermodynamic data and could help to improve computational drug design and development.	hydrogen bonding plays an important role in the interaction of biological molecules and their local environment. hydrogen - bond strengths have been described in terms of basicities by several different scales. the pkbhx scale has been developed with the interests of medicinal chemists in mind. the scale uses equilibrium constants of acidbase complexes to describe basicity and is therefore linked to gibbs free energy. site specific data for polyfunctional bases are also available. the pkbhx scale applies to all hydrogen - bond donors (hbds) where the hbd functional group is either oh, nh, or nh+. it has been found that pkbhx can be described in terms of a descriptor defined by quantum chemical topology, e(h), which is the change in atomic energy of the hydrogen atom upon complexation. essentially the computed energy of the hbd hydrogen atom correlates with a set of 41 hbas for five common hbds, water (r2 = 0.96), methanol (r2 = 0.95), 4-fluorophenol (r2 = 0.91), serine (r2 = 0.93), and methylamine (r2 = 0.97). the connection between experiment and computation was strengthened with the finding that there is no relationship between e(h) and pkbhx when hydrogen fluoride was used as the hbd. using the methanol model, pkbhx predictions were made for an external set of bases yielding r2 = 0.90. furthermore, the basicities of polyfunctional bases correlate with e(h), giving r2 = 0.93. this model is promising for the future of computation in fragment - based drug design. not only has a model been established that links computation to experiment, but the model may also be extrapolated to predict external experimental pkbhx values.
clinical manifestation of superior vena cava syndrome (svc syndrome) results from complete or partial obstruction of the svc. the svc carries approximately one - third of the venous return to the heart and collects blood from the arms, head, and upper chest to the heart. when the svc is obstructed, blood flows through a collateral vascular network to the lower body and the inferior vena cava or the azygus vein. therefore, obstruction below or at the level of azygus vein results blood bypassing from superficial venous system. during several weeks however, if the obstruction occur above the level of azygus vein, svc bypasses and no clinical manifestation of syndrome occur. like other veins in body therefore, any compression of the vein results in increasing in pressure inside the vein. symptoms develop over a period of 2 weeks in approximately a third of patients, and over longer periods in other cases physical signs include plethora, swelling of neck or chest veins and tackypnea. rarely, cyanosis, horner 's syndrome and a paralyzed vocal cord may also be present. although infectious causes of svc syndrome included syphilitic and tuberculosis, but these etiologic factors are rare in these years. the most common etiology of svc syndrome is malignant disease and most common malignancies, including non - small - cell lung cancer (in 50% of patients), small - cell lung cancer (approximately 25% of patients), lymphoma, and metastatic lesions (each approximately 10% of patients). currently, obstruction of the svc caused by thrombosis or non - malignant condition 's accounts for approximately 35% of cases, reflecting the increased use of long - term central venous catheters or permanent pacing electrodes. a 30-year - old man was admitted to alzahra hospital, isfahan, iran in january, 2012, because of dyspnea and facial edema. the patient had been well until 4 days before admission, when sudden onset dyspnea and orthopnea developed. medications included brome hexin syrup, theophylin and antihistamins without any effects. on examination, the blood pressure was 120/80 mm hg, the pulse 80 beats per min, the temperature 37c, the respiratory rate 16 breaths per minute, the oxygen saturation 92% while the patient was breathing ambient air, and the weight 80 kg. results of a complete blood count blood levels of urea nitrogen, albumin, total and direct bilirubin, creatine kinase, prothrombin time (pt), activated partial thromboplastin time (aptt), international normalized ratio (inr), aspartate aminotransferase and arterial blood gas were normal. an electrocardiogram showed sinus rhythm at a rate of 90 beats per min, without any problem. in echocardiography left ventricular ejection fraction was 55% and other finding was normal. in analysis, thoracic computed tomography (ct) with intravenous contrast performed as illustrate in figure 2. in order to reducing his respiratory symptoms and improving respiratory distress, the patient received intravenous corticosteroid (dexamethasone 8 mg 3 times a day for 3 days.). immunohistochemistry revealed leukocyte common antigen (lca) and terminal deoxynucleotidyl transferase (tdt) positive. chest x - ray of patient in arrival day chest ct scan of patient treatment with hyper cyclophosphamide, vincristin, adriamycine, dexamethasone (cvad) regimen done and respiratory symptoms resolved after 5 days completely. patient was selected for bone marrow transplantation. the svc syndrome, occurs in approximately 15,000 persons in the united states each year. if the syndrome occurs after the use of intravascular devices, diagnosis of the cause is straightforward. in other situations imaging with ct scanning of chest with intravascular media made diagnosis correctly. initial management of patient with svc syndrome includes supportive measures such as providing supplemental oxygen and elevating the head of the bed. other treatments like diuretic therapy for reducing the intravascular volume, or a short course of parenteral steroids (dexamethasone, 4 mg every 6 h) to decrease edema and tumor burden are controversies. several treatments have been introduced for svc syndrome depending on the underlying disease like thrombolytic therapy or stenting in thrombosis of svc in patients that svc damaged in introducing intravascular devices. mainstem of treatments in svc syndrome induce malignancies is chemotherapy and radiotherapy after definite tissue diagnosis via ct scan - guided biopsy of mediastinal mass. surgical bypass grafting is infrequently used to treat the svc syndrome. in malignant cause of svc syndrome, untreated symptom reduces life expectancy less than 30 days. however, treated cases expectancy depends on type of malignancy and applying proper therapy like chemotherapy. in our case, after chemotherapy introduced, symptoms and signs of disease disappear and patient arrives to complementary treatments.	when superior vena cava (svc) compress or obstructed by internal or external pressure, we encounter to svc syndrome. the cause of this compression is malignant or benign. although the widespread use of permanent central venous access catheters coupled with the improved success of chemotherapy has increased the incidence of svc syndrome not caused by direct tumor infiltration (non - malignant svc syndrome) but svc syndrome may be a sign of advanced malignancy. in this report, we present a 30-year - old man with lymphoma that present with svc syndrome at presentation. with chemotherapy, patient was recovered from signs and symptoms.
our understanding of cerebellar function has historically been based on the premise that the cerebellar cortex is the site of information processing, or at least memory storage and retrieval. this corticocentric viewpoint assumes that cerebellar output is mainly carried by the projection neurons of the deep cerebellar nuclei (dcn). however, evidence clearly suggests that the dcn are not merely a relay station, but rather constitute a site of signal integration if not signal generation. indeed, a role in adaptive information processing and perhaps pattern generation may be deduced from the presence of specific forms of synaptic plasticity within the dcn [24 ]. understanding the information - processing capabilities of a neuronal circuit several excellent studies have made an effort to examine the diversity of dcn neurons using morphological staining [5, 6 ], either alone or in combination with electrophysiological recordings. these studies have revealed considerable diversity of dcn neurons but have found only modest correlations between electrophysiological features and neuronal morphology, indicating that these neurons can not be classified solely based on morphometric criteria. to overcome this challenge, our laboratory took advantage of transgenic mouse lines that express fluorescent proteins under the promoters for glutamic acid decarboxylase (gad67) and a glycine transporter (glyt2), which are often used as markers for gabaergic and glycinergic neurons, respectively [11, 12 ]. a careful examination of cerebellar structures in gad67-egfp mice confirmed that all gabaergic (gaba - containing) neurons in the dcn express egfp. since we did not directly identify the transmitters released by these egfp - expressing cells, and in keeping with the terminology applied in the original publications, we have addressed the various neuronal subtypes based on the presence of these markers (e.g., gad67-positive, gad+) rather than the most probable neurotransmitter (e.g., gaba). neurons that were not identified by either of the two markers are likely to be glutamatergic (for a more detailed discussion see). the mouse dcn comprises three distinct nuclei (medial, interposed, and lateral), each of which features a specific composition of neurons of various sizes. there are significant differences in gross anatomy and neuronal morphology between the three dcn especially the lateral dcn, also known as the dentate nucleus in primates [5, 14 ]. however, all of the dcn engage in two pathways of information flow (fig. 1) : the olivo - cortico - nucleo - olivary (ocno) loop, and the pathway formed by mossy fibers, granule cell axons, purkinje cells, and dcn projections to premotor nuclei [1, 15, 16 ]. these pathways are organized in compartments that follow the zonal topography of the cerebellar cortex, each of which is likely to be related to different modes of cerebellar function. 1schematic drawing of the main neuron groups in the dcn including afferent and efferent connections, emphasizing the two topographically distinct routes of information flow : the ocno loop (left) and the mossy fiber - cortico - nucleo - dcn pathway that mediates cerebellar output to premotor nuclei. color coding and naming of dcn neurons follow a scheme presented in and, which will be followed throughout this review. red arrows denote glutamatergic (excitatory) axons ; green arrows denote gabaergic (inhibitory) axons ; and blue arrows denote glycinergic or mixed glycinergic / gabaergic axons. arrows with dashed lines denote connections that are of uncertain strength or have not been unequivocally demonstrated. note that local axons of dcn neurons are not shown for the sake of illustrative clarity. gadnl large gad67-negative cells, gadns small gad67-negative cells, gad+ gad67-positive (gabaergic) cells, gly - i non - spontaneous glyt2 + (glycinergic) cells, fn fastigial nucleus, gly - f projecting glyt2 + neurons of the fastigial nuclei, gad+io small gabaergic neurons projecting to the inferior olive schematic drawing of the main neuron groups in the dcn including afferent and efferent connections, emphasizing the two topographically distinct routes of information flow : the ocno loop (left) and the mossy fiber - cortico - nucleo - dcn pathway that mediates cerebellar output to premotor nuclei. color coding and naming of dcn neurons follow a scheme presented in and, which will be followed throughout this review. red arrows denote glutamatergic (excitatory) axons ; green arrows denote gabaergic (inhibitory) axons ; and blue arrows denote glycinergic or mixed glycinergic / gabaergic axons. arrows with dashed lines denote connections that are of uncertain strength or have not been unequivocally demonstrated. note that local axons of dcn neurons are not shown for the sake of illustrative clarity. gadnl large gad67-negative cells, gadns small gad67-negative cells, gad+ gad67-positive (gabaergic) cells, gly - i non - spontaneous glyt2 + (glycinergic) cells, fn fastigial nucleus, gly - f projecting glyt2 + neurons of the fastigial nuclei, gad+io small gabaergic neurons projecting to the inferior olive this review will compile work from our three original studies of the neuronal types found in the lateral cerebellar nucleus of the mouse as well as complementary information from other previous and concurrent studies, without attempting to comprehensively review the available literature on other features of the cerebellar nuclei. we will therefore focus primarily on four types of neurons identified in the mouse lateral dcn based on gad67 and glyt2 promoter activity, with the data from each neuron type indicated by color code : black, large gad67-negative (putative non - gabaergic) dcn cells (gadnl) ; orange, small gad67-negative dcn cells (gadns) ; green, gad67-positive (gad+, gabaergic) cells ; and blue, non - spontaneously active glyt2 + cells (gly - i). the gad+ neurons include both local and inferior olive - projecting gabaergic neurons. our own data from the gabaergic cells are strongly biased towards the larger (soma size > 12 um) cells, among which we have observed no evidence for axons projecting outside the nucleus. the gabaergic neurons that project to the inferior olive (io) have been described as very small [18, 19 ], and for this reason, we believe that they constitute a separate cell type, which we refer to as gad+io. to our knowledge, there are no studies that have specifically examined the electrophysiology of these small io - projecting neurons. to acknowledge the undoubtedly great functional importance of this cell population, we have included the gad+io cells in our schematic representations while noting that a more detailed examination remains a goal for future studies. in vitro and in vivo recordings from unidentified dcn neurons have revealed that the vast majority of these neurons are spontaneously active at action potential (ap) firing rates of 3555 hz driven by tonic cation currents [2024 ]. our recordings have confirmed that the major populations of dcn neurons that are likely to have been encountered in these previous recordings namely gadnl, gadns, and gad+ cells are intrinsic ap generators, while a small population of large glyt2-positive cells do not exhibit spontaneous activity (fig. because of their inactivity, we have termed these glycine inactive (gly - i). like the other three cell types, gly - i cells can be driven to high - frequency firing by extrinsically imposed depolarization (fig. however, gly - i cells could not be made to fire continuously at high frequencies like the other cell types ; instead, they tended to fire short bursts of fast aps. the gad+io neurons have not yet been carefully examined by intracellular recordings, but our few recordings suggest that they do exhibit spontaneous firing behavior that is similar to the local gabaergic neurons (mean firing frequency 710 hz at 32c ; m. uusisaari, unpublished observations). fig. a example traces from cell - attached voltage - clamp recordings. with the exception of gly - i cells, b example traces showing voltage responses to depolarizing current injections (1-s, 1.5 pa / pf for gadnl, gadns, and gad+ ; 0.9, 1.5, and 2.4 pa / pf for gly - i). note the higher firing rheobase in gly - i cell. c peak - aligned, averaged action potentials ; width of the trace represents sem. d example traces showing voltage responses to hyperpolarizing current injections (1-s, 1.5 pa / pf). gadnl, gadns, and gad+ cells displayed ih - indicating voltage sags (single arrowhead) and pronounced rebound depolarization and associated firing (double arrowhead). e statistical summaries of key electrophysiological parameters. i spontaneous firing frequency under cell - attached conditions. ii action potential half width, measured from repetitively fired action potentials at room temperature (rt ; 24c). note that in order to enable meaningful comparison of i - f curves in cells of significantly different sizes, the injected current is given in relative units of pa / pf. v spiking frequency adaptation during a step depolarization that evoked ~40 hz (gadnl, gadns, and gad+) or ~30 hz (gly - i) firing. panels from e were modified from the following sources : i and iii from, ii from, and iv and v from a combination of with. asterisks denote statistical significance : p 100 pf) and shorter aps and another being small in somatic size (cm < 100 pf) with broader aps. these cell groups were respectively classified as gadnl and gadns populations, divided by a (somewhat arbitrary) boundary value of 100 pf. one attractive hypothesis is that these different ap widths are a consequence of different expression levels of delayed - rectifier type potassium channels, particularly, kv3 subtypes [25, 26 ]. our own studies [27, 28 ] and subsequent investigations by joho and coworkers [29, 30 ] do not indicate a strong correlation between neuronal subtypes and kv3 channel expression. however, it seems that kv3 channel expression levels are somewhat lower in neurons containing gaba (local and/or io - projecting) than in the large glutamatergic projection neurons, which would be consistent with gabaergic cells showing broader aps than glutamatergic ones. ap width is not the only clearly distinguishing feature between gadnl and gadns cells ; gadns cells also exhibit a stronger late component in the afterhyperpolarization (fig. 2c, double arrowhead) and somewhat longer rebound depolarization and associated spiking after release from a hyperpolarizing step (fig. 2d ; [7, 31, 32 ]). with the exception of gly - i cells, which have not yet been thoroughly characterized in this aspect, all cell types consistently show a pronounced sag in their response to hyperpolarizing current steps, indicative of hyperpolarization - activated cation current (ih) and rebound bursting that may involve the activity of t - type calcium currents (fig. as recently reviewed by turner and coworkers, differences in rebound burst behavior may reflect distinct expression patterns of t - type calcium channel - forming cav3 isoforms. however, this study focused on spontaneously active large - diameter neurons, and their data pool is unlikely to contain a significant fraction of the sparse, gly - i neurons that are also characterized by lack of spontaneous activity. our preliminary examinations of gly - i cells suggest that these intrinsically silent neurons are also less prone to rebound bursting than the other, pacemaker - like neurons of the dcn (m. uusisaari, unpublished observations). finally, these four cell types differ in spike frequency versus injected current density and their spike frequency accommodation. most notably, gadnl neurons can be driven to the highest firing frequencies with a nearly linear current frequency (i - f) relationship while also exhibiting the smallest amount of spike frequency adaption during sustained firing (fig. cells respond to depolarization with saturating values of firing frequency, and show a significant amount of frequency adaptation. again, this is in contrast to the situation in the cerebral cortex, where gabaergic interneurons typically fire at high and sustained frequencies. the gly - i cells exhibit the strongest accommodation, to the extent that they tend to stop firing after a few initial spikes. it would thus seem that the dcn neurons cover a range of signal transduction modes, from tonic firing and rate - coding (gadnl) to burst - firing and onset or time - interval coding (gad+, gly - i). classical and more recent studies [68 ] have attempted to classify dcn neurons on the basis of their morphology. these cells vary considerably in terms of dendritic arborization and axonal branching, and these efforts have revealed no strong correlation between simple dendritic morphologies and electrophysiological characteristics. our own set of ~100 biocytin - filled gad+, gad, and glyt2 + cells included neurons with axons that either coursed locally or projected to more distant targets. in general, neurons with larger cell bodies exhibited more complex dendritic morphology (fig. 3), and this complexity undoubtedly enables these cells to perform non - linear integration of synaptic information. the concentration of synaptic contacts from the collaterals of mossy and climbing fibers on the dendrites of these cells is in keeping with their elaborate computational properties. representative examples of the five dcn neuronal classes stained with biocytin and comparison of cell body sizes between cells responding to three different neurotransmitters. note that despite the differences in average cell body size between cell types, these values overlap considerably between groups. asterisks denote statistical significance : p < 0.001 variability in dcn neuron morphology. representative examples of the five dcn neuronal classes stained with biocytin and comparison of cell body sizes between cells responding to three different neurotransmitters. note that despite the differences in average cell body size between cell types, these values overlap considerably between groups. asterisks denote statistical significance : p < 0.001 despite the cell - to - cell variability of dendritic structures, the above - described subtypes could not be readily identified by their morphology alone, even with information about gad67 expression. indeed, quantitative analysis of several measures of dendritic morphology and, surprisingly, cell body size revealed clearly overlapping distributions. importantly, gad+ cells could not be reliably differentiated from gad cells on the basis of cell body size, and the gly - i cells described in also significantly overlap with gad cells in terms of cell body size. since many of the electrophysiological recordings found in literature are reported to originate from large dcn neurons, these data are likely biased towards glutamatergic cells, but presumably also include gabaergic or glycinergic cells. since the electrophysiological differences between gad+ and gad cells are subtle, it is not surprising that most reports did not find clearly distinguishable cell types. in this context, it should be noted that the gly - i cells might have been omitted in previous slice studies as unhealthy cells, as they are not necessarily spontaneously active. this is unfortunate, as our targeted staining of these silent, glyt2 + cells revealed that they project directly to the cerebellar cortex, unlike any other cell type that we have encountered in the dcn so far, making further elucidation of their behavior mandatory. the actual synaptic targets within the cerebellar cortex remain unknown for these putatively glycinergic and thus, inhibitory neurons, but are likely to be cerebellar golgi cells as these are the only neurons in most areas of the cerebellar granule cell layer that express glycine receptors. purkinje neuron (pn) axons from the cerebellar cortex provide the main efferent input to the dcn, and this is their main target. based on counts of purkinje and dcn neurons (estimates for mouse are ~200,000 pns and ~30,000 neurons in all three dcn), signal transmission from the cortex to the nuclei shows overall convergence, but the true extent of this convergence at the cellular level is not known. based on the count of purkinje axon terminals on large dcn cell bodies, and taking into account various estimates of how many terminals a single axon will form on a given neuronal body and the spread of pn axons within the nuclei, estimates of the number of pns contacting a single large dcn neuron range between several tens and several hundreds [3941 ]. despite anatomical evidence [5, 42 ] suggesting that the number of pn synapses on dcn cells is variable across different types of dcn cells, it is generally assumed that all dcn neurons are equally controlled by pn activity. however, since most work examining gabaergic synaptic currents in dcn [4347 ] has focused on large - diameter neurons, which presumably correspond primarily to gadnl neurons, some of the above conclusions will need to be revisited with regard to different neuronal subtypes. to clarify whether pn input is comparable across different dcn neuronal types, we have studied the properties of spontaneously occurring inhibitory postsynaptic currents (ipscs) in dcn cells, taking advantage of the fact that spontaneous release does not require intact purkinje neuron - dcn connections, which are rare in slice preparations. whole - cell voltage - clamp recordings using high - chloride intracellular solution (erev cl ~ 0 mv) revealed a striking difference between gad+ and gad neurons. both gadnl and gadns cells were under constant bombardment of spontaneous, tetrodotoxin - insensitive ipscs with kinetics similar to those described previously for purkinje - dcn synapses, but gad+ neurons received only very infrequent ipscs with slower kinetics and much smaller amplitudes (fig. our initial hypothesis was that these differing kinetics were simply the result of differential expression of gabaar subunits in these cell types. experiments using gabaar subunit - specific agents revealed that there are, indeed, differences in gabaar subunit composition between the dcn cell types. however, these differences are not the only cause for the observed differences in ipsc kinetics as ipscs in both gad+ and gadns cells proved sensitive to selective up - modulators for the alpha1 and alpha3 subunits. these experiments revealed an additional and rather intriguing cause for the difference in ipsc kinetics : whereas ipscs in gad neurons could be recorded immediately upon breaking into whole - cell mode, ipscs in gad+ cells usually became apparent only tens of seconds after establishing a whole - cell configuration (fig. 4c, upper left). this led to the proposition that gabaergic synapses on gad+ cells are predominantly formed on distal dendrites rather than cell bodies ; on the other hand, pns form large numbers of synapses on the bodies of large glutamatergic dcn neurons, to the extent that gad cell bodies seem to be entirely covered by gabaergic presynaptic terminals [5, 11 ]. notably, large dcn neurons (gadnl) are also most likely to exhibit distal gabaergic inputs that generate slower and smaller - amplitude synaptic events, although those may be masked by the larger events triggered by proximal synapses. taken together, it appears that the slower ipsc time course seen in gad+ cells arises from a combination of distal dendritic localization of synapses and a specific receptor subtype composition, as well as possible differences in the spatiotemporal dynamics of gaba release [3, 45, 49 ]. all recordings in this figure were obtained using a high - cl internal solution (erevgaba ~0 mv). i example traces of ipscs in control conditions (upper panel) and with added ttx (lower panel). b ipsc kinetics in gad+ cells are significantly slower than in gadnl and gadns cells. i comparison of ipsc occurrence frequency between the cell types in control (solid color bars) and ttx (hatched bars). asterisks denote statistical significance : p < 0.05, p < 0.001. iii iv comparison of 1090% rise time and decay time constant distributions at 32c. i normalized frequency of ipscs as a function of time since breaking into whole - cell configuration. ii iv suggested mechanism behind the delayed appearance of ipscs. upon breaking into whole - cell configuration, the reversal potential of gaba - mediated cl currents (erev gabaa) near somatic gabaars on gadnl cells is quickly shifted to 0 mv, providing inward driving force for chloride and thereby permitting ipsc measurements. on the other hand, the erev gabaa near gabaars located in distal dendrites is shifted more slowly, requiring diffusion of cl from the soma to the dendrites. c (i) are modified from spontaneous ipscs in gadnl, gadns, and gad+ cells. all recordings in this figure were obtained using a high - cl internal solution (erevgaba ~0 mv). i example traces of ipscs in control conditions (upper panel) and with added ttx (lower panel). b ipsc kinetics in gad+ cells are significantly slower than in gadnl and gadns cells. i comparison of ipsc occurrence frequency between the cell types in control (solid color bars) and ttx (hatched bars). asterisks denote statistical significance : p < 0.05, p < 0.001. iii iv comparison of 1090% rise time and decay time constant distributions at 32c. i normalized frequency of ipscs as a function of time since breaking into whole - cell configuration. ii iv suggested mechanism behind the delayed appearance of ipscs. upon breaking into whole - cell configuration, the reversal potential of gaba - mediated cl currents (erev gabaa) near somatic gabaars on gadnl cells is quickly shifted to 0 mv, providing inward driving force for chloride and thereby permitting ipsc measurements. on the other hand, the erev gabaa near gabaars located in distal dendrites is shifted more slowly, requiring diffusion of cl from the soma to the dendrites. a c (i) are modified from unfortunately, we have only limited data on the properties of the synaptic inputs to gly - i cells. preliminary results obtained in our laboratory suggest that these cells resemble gadns cells more than gad+ cells with respect to ipsc kinetics and frequency (m. uusisaari, unpublished observations), which suggests that gly - i cell bodies are being targeted by purkinje neuron axons. as gly - i cells seem to be endowed with the capability to control cerebellar cortical activation via their putative inhibitory synaptic contacts in the granule cell layer, elucidation of their afferent inputs is a pressing open task. working from our present understanding of these various functionally different classes of neurons, a partial picture on the ins and outs to the dcn emerges (fig. 5). based on histological evidence, the pns are known to synapse on gadnl and gad+io neurons [18, 42 ], but the extent to which pns innervate other cell types is less well established. our electrophysiological data suggest that gadns cells are contacted by pn axons to the same extent as gadnl cells, while gad+ cells are innervated to perhaps a lesser degree, based on the different properties of spontaneous ipscs in these cells. bagnall and coworkers have reported that the large, glycinergic projection neurons of the fastigial nucleus appear to be under pn control, even though it is unclear whether pn synapses are present on the gly - i neurons in the lateral nucleus. conversely, it is known that climbing fiber (cf) collaterals contact both gad+io and gadnl dcn neurons and that mossy fiber (mf) collaterals synapse on gadnl cells, but the relative strength of these connections and whether mf or cf synapses occur on other cell types is unknown [5, 19 ]. unpublished observations from our laboratory suggest, however, that both gad+ and gadns cells receive glutamatergic extracerebellar synaptic afferents (m. uusisaari, unpublished observations). notably, the roles and targets of inhibitory dcn interneurons are entirely unknown, even though non - purkinje gabaergic presynaptic terminals have been described on both gabaergic and glutamatergic dcn neurons. fig.. arrows with dashed lines denote axons of local dcn neurons or collaterals of efferent axons. pn purkinje neuron axon, mfc mossy fiber collateral, cfc climbing fiber collateral, cctx cerebellar cortex, glyr glycine receptor, gabaar gabaa receptor, ampar / nmdar ampa and/or nmda receptor schematic drawing of the putative synaptic connections for the different dcn neuron types. arrows with dashed lines denote axons of local dcn neurons or collaterals of efferent axons. pn purkinje neuron axon, mfc mossy fiber collateral, cfc climbing fiber collateral, cctx cerebellar cortex, glyr glycine receptor, gabaar gabaa receptor, ampar / nmdar ampa and/or nmda receptor clearly, we presently lack even a basic description of all of the various cell types in the dcn and are far from compiling a circuit diagram that reflects an understanding of their functional properties. fundamental issues that need to be addressed in future work include the examination of the neurons that project from the dcn to the io, determining the extent to which our observations in the lateral nucleus can be generalized to the other two nuclei, and the completion of a careful histological examination of the spatial distribution of cell types within the different nuclei. following on the identification of these different cell types, we also need to examine their afferent and intrinsic connections. notably, previous studies aimed at characterizing the properties of mf and cf synapses were specifically biased towards large neurons, whereas there is clear anatomical evidence for afferent connections targeting different cell types. even less is known about the functional role of the connectivity within the dcn, which is likely to consist of local gabaergic, glutamatergic, and glycinergic synaptic communication [5, 51, 52 ]. it seems likely that the dcn is organized in a modular fashion, since afferent and efferent connections segregate according to their extra - nuclear sources. there is a possibility that synaptic crosstalk may occur between these modules in a manner analogous to the parallel fibers that provide information flow across the zones of the cerebellar cortex. finally, future research efforts should explore the possible function of inhibitory nucleo - cortical synaptic connections formed by gly - i cell axons [5456 ]. our growing toolbox of mouse strains in which neuronal subtypes are conveniently identifiable via cell type - specific expression of fluorescent protein reporters, in conjunction with techniques for neural circuit labeling via transsynaptic gene delivery, should enable us to effectively address these issues in the years to come.	the deep cerebellar nuclei (dcn) are at the center of the cerebellum not only anatomically but also functionally. classical anatomical studies have described different types of dcn neurons according to their expression of various marker proteins, but only recently have we begun to characterize these different cell types according to their electrophysiological properties. these efforts have benefited greatly from the availability of transgenic mouse lines that express green fluorescent protein under the control of the glutamic acid decarboxylase (gad67) and glycine transporter (glyt2) promoters, which are markers for gabaergic and glycinergic neurons, respectively. these studies have identified several types of neurons within the lateral cerebellar nuclei, each of which exhibits distinct active membrane properties. in addition to their differential use of neurotransmitters (glutamate, gaba, or glycine), these cell types also receive and provide synaptic information from different sources and to different targets.
functional gastrointestinal disorders (e.g., functional dyspepsia and irritable bowel syndrome) are frequent conditions encountered in clinical practice,. according to a recent systematic review and meta - analysis, the prevalence of functional dyspepsia was 27% and that of irritable bowel syndrome was 16%. people with functional gastrointestinal disorders experience gastrointestinal symptoms despite the absence of morphological or anatomical abnormalities. while these disorders are not life - threatening, it was noted that quality of life was markedly impaired and productivity was decreased in such patients. one of the major factors for these diseases is abnormal gastric motility. the principal functions of the stomach are reservoir, mixing, and emptying of gastric contents. the fundus acts as the reservoir, and the gastric corpus and gastric antrum facilitate mixing and emptying. contraction and dilatation of the stomach are related to these functions, and abnormal gastric motility leads to depression of these functions. for functional assessment of gastrointestinal motility, the barostat technique is the current standard,. this system estimates the gastric tone by the change in volume or pressure of air in a balloon placed in a hollow organ. scintigraphy and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ^{13}{\rm c}$\end{document } breath tests are also important methods. these methods track the motion of a marker, included in a meal, from outside of the body. although these methods are non - invasive, they involve indirect assessment of gastric motility, because the system can only assess the gastric flow produced by the gastric motility. it can estimate the volume of the stomach or detect gastric contraction waves generated for the mixing and emptying functions by measuring the stomach shape. it has been elucidated that gastric contraction waves are different in healthy humans and patients with functional gastrointestinal disorders through the use of mri,. however, this system can only obtain two - dimensional cross - sectional images of the stomach. therefore, functional quantitative analyses by mri would be untrue. on the other hand, an endoscope can be used to look inside the body, including observation of the gastrointestinal tract. for such examinations recently, three - dimensional endoscopes have been developed and put into practical use. a three - dimensional endoscope is currently almost always used as a surgical navigation system. however, it is expected that they will become useful as diagnostic instruments, because it is possible to obtain the three - dimensional shape of an organ from inside of the body. this paper presents a gastric contraction imaging system using an endoscope, as shown in fig. 1. by using the three - dimensional shape of the stomach obtained from the endoscope, contraction waves are measured to assess the gastric motility. this system has low invasiveness and can assess the motility of the stomach wall directly in a three - dimensional manner. in addition, an endoscope is usually used for examination of the stomach. in other words, we evaluate the proposed gastric motility imaging system using a three - dimensional endoscope. in section ii, we evaluate the performance of a prototype three - dimensional endoscope and the effectiveness of the proposed method in an experiment with a wave simulated model. we previously developed a compound eye system called the tombo (thin observation module by bound optics). this system has a compact structure and can take close images because it uses a micro - lens array. this feature is an advantage because the optical system involves a flexible endoscope that allows bending. these images are available for use in wide - field, high - resolution, and three - dimensional measurements. a schematic diagram of the compound optics system is shown in fig. 2. the compound optics system consists of a micro - lens array, partition, and cmos image sensor. each micro - lens focuses optical signals on the image sensor. the images obtained by this system are presented with slightly different information for the view. based on triangulation, three - dimensional information about the imaged object can be computed. an area - based method with the sum of the squared difference is used to obtain the corresponding points in two images. a sub - pixel estimation with a parabolic function and cross - checking by replacing the left and right images is also used to improve the accuracy of measurement. searching for corresponding points for all pixels on one image, the three - dimensional profile of the stomach is mixed with the stomach geometry itself and the contraction profile, as shown in fig. 1. therefore, the contraction waves must be separated from the three - dimensional profile of the stomach wall for analysis. the geometry of the stomach is obtained as the most frequent value in a time series, and the other value reflects the contraction profile. the shape of the contractions is obtained by measuring the distance between the geometry of the stomach and the measurement profiles. the three - dimensional data obtained from the three - dimensional endoscope include noise or outliers, and missing areas caused by masking could exist. to overcome these problems, we model the geometry of the stomach and the contraction waves. here, the shape of the gastric contractions is modeled by a one - dimensional gaussian function and the geometry of the gastric antrum is regarded as a circular tube, as shown in fig. the one - dimensional gaussian function on the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ x$\end{document}-axis can be expressed as follows:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ $ g(x)=ne^{\left(-{{x - x_{c}}\over{2\sigma^{2}}}\right)}\eqno{\hbox{(1)}}$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ n$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ x_{c}$\end{document } are the attitude and position of the wave, respectively, and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \sigma$\end{document } indicates the spread of the wave. from the gaussian parameters a custom - made rigid endoscope with a compound optics system was used to reconstruct three - dimensional images. the tip of the endoscope has a tool channel, compound optics, and illumination. the light source device used was clv - u20d (olympus co.). in this study, we used two images arranged on the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ y$\end{document}-axis for three - dimensional measurements. we defined a coordinate system for the endoscope as follows : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ z$\end{document}-axis parallel to the optical axis ; and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ x-$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ y$\end{document}-axes parallel to the image sensor. table ispecification of the prototypelens array\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ 3 \times 3$\end{document}focal length1.5 mmf - number7.5angle of field46 degreesdepth of field2050 mmpixel size\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ 2.2 \mu { \rm m}$\end{document}pixel count (per unit)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ 415\times 415$\end{document } first, we evaluated the accuracy of the three - dimensional measurements using the prototype system. a target with the simplest of geometric features was used to evaluate the accuracy of depth measurements. a rigid flat plane with a grid sheet the cross points of the grid were used as the features and the grid interval was 1 mm. images of the grid on the target were taken at distances from the endoscope tip starting at 10 mm and finishing at 50 mm with 10-mm increments. the means and standard deviations of the estimated distances from the target plane to the endoscope tip for the set distances of 10, 20, 30, 40, and 50 mm were \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ 9.8\pm 0.066$\end{document }, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ 19.9\pm 0.061$\end{document }, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ 30.0\pm 0.121$\end{document }, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ 40.0\pm 0.198$\end{document }, and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ 50.0\pm 0.264~{\rm mm}$\end{document }, respectively, in three trials for each distance. the percentage of measurement error for the distance between the endoscope tip and the target plane was less than 2% when the distance ranged from 10 to 50 mm. 5.reconstruction of the surface of a flat plane on the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ x\hbox{--}z$\end{document } plane at different distances from the endoscope tip. in the second experiment the target was fixed on a rotator that permitted the angle between the optical axis of the endoscope and the orientation of the flat plane to be changed. the angle between the flat plane and the optical axis was then changed in increments of 15 degrees up to 75 degrees. the means and standard deviations of the estimated distances between two different feature points separated by 1 mm for the set 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the percentage of measurement error for the distance between two different points was less than 6% when the distance ranged from 10 to 50 mm and the angle ranged from 15 to 60 degrees. for the angle of 75 degrees, the percentage of maximum measurement error was about 25%. finally, to evaluate the accuracy of the prototype system in the clinical situation, we conducted experiments with a dog. a 10-mm brass cube was put on the colon of the dog, as shown in fig. the mean value for the measured length was \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ 10.4\pm 0.35~{\rm mm}$\end{document } in three pairs of images obtained from different positions. this error could be caused by secretory fluid attached to the cover glass of the lens. the object used for the wave simulated model was a plastic spring cylinder, as shown in fig. 7. a wave was made on the spring 's surface by pushing the coil from the opposite surface. the wave was manually propagated with increments of approximately 3.2 mm along the central axis of the spring. in this case, the wave generated on the plane was mixed with the shape of the spring, and we thus used a rectangular coordinate system. we defined a coordinate system for the object as follows : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ z$\end{document}-axis parallel to the contraction ; \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ x$\end{document}-axis parallel to the central axis of the spring ; and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ y$\end{document}-axis parallel to another axis. after axial correction, the shape of the waves could be obtained by subtracting the paraboloidal surface and the reconstructed shape of the surface along the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ z$\end{document}-axis. the object was placed so that the vibration direction of the wave was along the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } 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\usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ x$\end{document}-axis). in fig. 11, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \rm s}_{i}~({\rm i}=1, 2, \ldots)$\end{document } means the surface that propagated the wave by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { + } { \rm 3.2}~{\rm mm}$\end{document } from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \rm s}_{i-1}$\end{document}. the mean measured value for the attitude was \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ 7.2\pm 0.3~{\rm mm}$\end{document}. the mean measured value for the wave movement along the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ x$\end{document}-axis was about \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { + } { \rm 3.2}~{\rm mm}$\end{document}. fig. gray dots show the raw data and red lines show the gaussian function - fitted raw data. (a) gaussian function - fitted wave and (b) position of the wave. the average wave attitude is 7.2 mm and the average wave movement is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { + } { \rm 3.3}~{\rm mm}$\end{document}. next, to evaluate the potential for clinical application, the wave simulated model was covered with thinly - sliced pork. 12 shows one of the captured images of a wave made on the pork surface. the mean measured value for the attitude was \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ 6.6\pm 0.3~{\rm mm}$\end{document } and the mean measured value for the wave movement was about \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { + } { \rm 3.3}~{\rm mm}$\end{document }, as shown in fig. 14.position and attitude of detected propagated waves extracted from the pork surface. in the same way, 15 shows typical results for an angle of 30 degrees between the central axis of the spring and the optical axis. 16 shows the position and attitude of the detected wave in the coordinates of the wave when the wave was manually propagated with increments of about \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { + } { \rm 3.2}~{\rm mm}$\end{document } along the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ x$\end{document}-axis. the mean measured value for the attitude was \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ 6.1\pm 0.3~{\rm mm}$\end{document}. the mean measured value for the wave movement along the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ x$\end{document}-axis was about \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { + } { \rm 3.0}~{\rm mm}$\end{document}. fig. 15.reconstructed shape of the pork surface with an angle of30 degrees between the cylinder axis and the optical axis. 16.position and attitude of detected propagated waves extracted from the pork surface with an angle of 30 degrees between the cylinder axis and the optical axis. the prototype system could measure the geometry of a flat plane with an error of less than 10 percent of the distance between two different points on images. subsequently, we conducted experiments with a dog to evaluate the accuracy of the prototype system for a clinical status using an object with known geometry. however, some errors occurred in the animal experiment, which were caused by secretory fluid. a secreted fluid is an obstacle for measurement, and a matching error occurs because the image is blurred by water droplets. since our system does not have equipment to remove any dirt present, the secretory fluid attached to the cover glass of the lens can not be removed. next, we demonstrated a three - dimensional endoscope system for assessment of gastrointestinal motility using a wave simulated model. the experimental results showed that the amplitude and position of a wave could be measured with 1-mm accuracy. unlike mri, an endoscope can not capture the whole shape of the stomach at once. the hidden surface of the contraction wave can not be used to reconstruct the three - dimensional profile. thus, the proposed system estimates the shape of peristaltic contractions through the use of a one - dimensional gaussian function. nevertheless, the wave surface opposite to the endoscope had low information when the angle between the central axis of the object and the optical axis was 30 degrees, and the estimated surface fitted by the gaussian function was quite well fitted with the raw data. from the data shown in figs. contraction waves were initiated every 20 s, and the width and speed of these waves were 18 mm and 2.5 mm / s, respectively. the speed of the contraction wave is sufficiently slower than the performance of the prototype system. consequently, the prototype system can detect contraction waves of the stomach. in a clinical status thus, the obtained images are recorded from different viewing positions in a time series and the contraction waves on images also move with the movement of the endoscope tip. this issue can be solved by performing three - dimensional registration using a portion that does not move, because contraction waves are sufficiently slower than the movement of the endoscope and occur partially. in this paper, we have proposed a gastric contraction imaging system for assessment of gastric motility using a three - dimensional endoscope. for analysis of gastric motility, we described a method that derived peristaltic contractions from the three - dimensional profile of the stomach wall. moreover, we evaluated the developed three - dimensional endoscope system using a flat plane. this system can measure the geometry of a flat plane with an error of less than 10 percent of the distance between two different points on images. subsequently, we conducted experiments with a dog to evaluate the accuracy of the prototype system for a clinical status using an object with known geometry. we then evaluated the validity of a prototype system using a wave simulated model. in that experiment, we demonstrated that the amplitude and position of the wave can be measured with 1-mm accuracy. these results suggest that the proposed system can measure the speed and amplitude of contractions. in the future	this paper presents a gastric contraction imaging system for assessment of gastric motility using a 3-d endoscope. gastrointestinal diseases are mainly based on morphological abnormalities. however, gastrointestinal symptoms are sometimes apparent without visible abnormalities. one of the major factors for these diseases is abnormal gastrointestinal motility. for assessment of gastric motility, a gastric motility imaging system is needed. to assess the dynamic motility of the stomach, the proposed system measures 3-d gastric contractions derived from a 3-d profile of the stomach wall obtained with a developed 3-d endoscope. after obtaining contraction waves, their frequency, amplitude, and speed of propagation can be calculated using a gaussian function. the proposed system was evaluated for 3-d measurements of several objects with known geometries. the results showed that the surface profiles could be obtained with an error of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { < } { 10\%}$\end{document } of the distance between two different points on images. subsequently, we evaluated the validity of a prototype system using a wave simulated model. in the experiment, the amplitude and position of waves could be measured with 1-mm accuracy. the present results suggest that the proposed system can measure the speed and amplitude of contractions. this system has low invasiveness and can assess the motility of the stomach wall directly in a 3-d manner. our method can be used for examination of gastric morphological and functional abnormalities.
estimates indicate that 67% of individuals will suffer neck pain at some stage of their life1. np is a significant health problem not only for adults but also for the young. the onset and course of np is affected by multiple factors with physical, psychosocial and individual factors interacting in the development of these disorders2, 3. the job nature of secondary school teachers involves a lot of head down posture, such as in frequent reading, assignment correction, and writing on the blackboard5. thus, secondary school teachers are at risk of developing occupationally related np and upper limb pain. high workload, low colleague support, high anxiety and some psychological factors have been found to be significant risk factors for the development of np in teachers3. the transition from conventional to modern methods of teaching is a major cause of teachers developing np. available studies of np and its causes among teachers are limited. according to one study back pain, neck pain and headache, are the most common ailments of the workers in the school environment20. another study of nursery school teachers found 26.5% of them complained of neck stiffness5. the learning environment for children is progressively being facilitated by new forms of information technology (it). traditional methods of teaching, such as school textbooks, are gradually being enhanced and replaced by new forms of it such as internet sources and interactive multimedia software. these initiatives have resulted in an increase in the number of classes that involve computing. several studies have shown associations between physical exposures and neck / upper extremity symptoms during computer work6, 7. long duration of computer work is associated with prolonged periods of holding a static posture2, which is most pronounced in the neck and shoulder region, resulting in increased forward neck flexion and increased static muscle tension in this region2, 8,9,10. increased forward neck flexion may result in increased tension in posture, stabilizing muscles as well as increasing compression forces in the articulation of the cervical spine, resulting in a higher risk of work related muscular disorders2. the deep cervical flexor muscles (dcf) are considered to be an important stabilizer of the head - on - neck posture. it has been theorized that when muscle performance is impaired, the balance between the stabilizers on the posterior aspect of the neck and the dcfs will be disrupted, resulting in loss of proper alignment and posture, which is then likely to contribute to cervical impairment10. therefore dcf training is recommended for increasing the endurance of these postural muscles, leading to improvement in np. research recommends that training that emphasizes the correct use of dcf, before introducing strengthening of the global cervical spine musculature, is more effective in the rehabilitation of the cervical spine than nonspecific strengthening of neck muscles. the craniocervical flexion test (ccft) regime appears to be an ideal strategy for specifically activating dcfs and reducing augmented activity of the scm muscle. there is evidence that restoration of the supporting capacity of dcf parallels reduction in neck pain and cervicogenic headache. hence, dcf muscle training is recommended for the clinical management of neck pain11. endorse a specific craniocervical flexion exercise (ccfex) protocol in the supine position. this program initially involves retraining a static holding contraction of the target muscles at a submaximal level to improve their tonic postural function. the type of training employed is based on the nature of the dysfunction presenting in these muscles9, 12, as well as their normal functional role1. the purpose of this study was to determine whether, there is an improvement in pain and disability of school teachers with np after 4 weeks of deep cervical flexor training with pressure biofeedback. in india, so, we need to make teachers physically, mentally and functionally better so that they can focus on the betterment of students. this study also emphasizes the role of dcf training in decreasing pain and disability among teachers. in india, the teacher is considered equivalent to god. thus future of students depends more or less on teachers. if teachers suffer, it directly affects the future of students as well. so, we need to make teachers physically, mentally and functionally better so that they can focus on the betterment of students. this study also emphasizes the role of dcf training in decreasing pain and disability among teachers. thirty teachers (24 females and 6 males) aged group 25 to 45 years participated in this study. the subjects were teachers of the central board of secondary education affiliated schools who had more than 5 years of experience. the teachers were included if they had a neck pain score on the numeric pain rating scale (nprs) of greater than 5, mild to moderate disability scores on the neck disability index (ndi) and poor ccf test results. subjects were excluded if they had undergone any cervical spine surgery or reported any neurological signs. subjects with a history of congenital or acquired postural deformity, spinal cord compression, tumor, instability, fracture, inflammatory disease or infection were also excluded. the proposal for the study was presented before the review committee of hamdard university and received approval. after selection, subjects were randomly allocated to one of the two groups with 15 subjects in each : group a : the experimental group received deep cervical flexor muscles training with pressure biofeedback and conventional exercises. group b : the control group received deep cervical flexor muscles training with only conventional exercises. the independent variable in the study was dcf muscles training and the dependent variables were pain (p) on nprs, and functional disability (d) on ndi. we used a pressure stabilizer, pressure biofeedback unit (stabilizer tm, chattanooga group, inc., chattanooga, tn), a height adjustable bed, markers and papers. a letter was sent to the principals of cbse affiliated schools with details of the research and their consent was obtained. subjects were informed about nature and procedure of study and all their questions were answered. baseline information of the dependent variables was taken at the beginning of study, on day 1 (p0, d0), before commencement of the training protocol. data were collected at the end of 2 weeks of training (p14, d14) and at the end of 4 weeks (p28, d38). nprs and ndi were used to evaluate the level of pain and functional disability, respectively. both experimental and control groups performed conventional exercises. in addition the experimental group also performed deep cervical flexor muscles training using pressure biofeedback. training was done for 4 weeks, 4 days a week with 2 minutes rest between sets. the ccft exercise program included 3 sets in a session 10 repetitions per set. the independent t test was used to compare age and baseline values of pain and disability between groups. the paired t test was used to compare pain on nprs and disability on ndi within groups. all dependent variables were compared between baseline, their values at 2 weeks and their values at end of 4 weeks. the independent t test was used to compare pain on nprs and disability on ndi between the groups. the values of both groups, group a and group b, were compared at baseline and for the difference from baseline, at the end of 2 weeks and at the end of 4 weeks. we used 95% ci and the results were accepted as significant if p 0.05) d 0 pretest reading on day 1, d 14 reading at the end of 2 weeks, d 28 posttest reading after 4 weeks a - experimental group, b- control group p < 0.05 ndi : in the comparison of disability, values between the baseline (d0), after 2 weeks (d14) and after 4 weeks (d28), significant improvement was noted in both groups a and b (p<0.05). the mean reduction in ndi scores in group a was 6.715 0.67 at p= 0.000, and the mean reduction in group b was 2.207 0.1059 at p = 0.000. the difference between d0 and d14 (d14d0) for both groups a and b was significant (p=0.015). the difference between d14 and d28 (d28d14) between the groups was significant (p=0.022). the difference between d0 and d28 (d28d0) between the groups was significant difference was obtained (p=0.003) (table 1). p 0 pretest reading on day 1, p 14 reading at 2 weeks and p28 posttest reading after 4 weeks a - experimental group, b- control group p < 0.05 nprs : in the comparison of pain, the values at baseline (p0), after 2 weeks (p14) and after 4 weeks (p28), showed significant improvements in both groups a and b (p<0.05). the mean reduction in nprs scores in group a was 3.137 0.41 and in group b was 1.797 0.1514. the difference between p0 and p14 (p140) for both groups a and b was significant (p=0.05). the difference between p14 and p28 (p28p14) between the groups was significant (p=0.04). the difference between p0 and p28 (p28p0) between the groups was significant (p=0.01) (table 2). this study was designed to determine the effects of deep cervical flexor muscles training using pressure biofeedback on neck pain and disability experienced by school teachers with neck pain. the results of our study show that there was a significant change in both pain and disability in both the groups after the intervention. however, the experimental group showed better results than the control group in terms of reduction of pain and disability. the amount of np and disability were not significantly different between the members of the two groups at the start of the study (baseline measurement). this could be attributed to the same nature of the duties performed by teachers at school. to our knowledge, this is the first study in which craniocervical flexion training was given to teachers having neck pain. therefore the lack of literature in this area limited the scope for direct comparison with other studies. the results of our study can be compared to other studies in a general way only, due to differences in treatment protocols, subject population, measures taken, and duration of treatment. the increase in endorphins that occurs after training and better neuromuscular control may decrease pain. afferent from these muscles cause endogenous options to be released and also the beta - endorphins from the pituitary gland. neck exercises may allow the musculotendinous proprioceptors to downgrade their stretch reflex responses using operant conditioning techniques and multiple practice sessions. the intrafusal fibers may be reset, discontinuing the cycle of muscle tension, impaired circulation with metabolite accumulation and pain associated with myogenic (myofascial) pain14. this result may be explained by the reduction in pain intensity which can bring about improvement in disability. herman.21 also proposed that the relationship between pain and neck disability index is quite strong as pain intensity is one of the ten areas addressed in ndi. literature on ndi reports that the minimal detectable score and the minimal clinically important difference is 5 points. moreover it is recommended that the ndi be used at baseline and for every 2 weeks thereafter to measure the progress of therapy. deep cervical flexor training as a treatment for neck pain and resulting disability is based on the rationale that deep cervical flexors, the longus colli muscle in particular, have a major postural function in supporting cervical lordosis, since in the functional mid - ranges of the cervical spine the lose their endurance capacity in patients with neck pain11, 15. therefore, it is thought that pressure biofeedback specifically targets dcf muscles and decreases neck pain. our results are supporting the initial hypothesis, are in agreement with those obtained in a randomized controlled trial conducted by jull.9, to determine the effect of 6 weeks of low - load craniocervical flexion exercise on cervicogenic headache patients. the results showed that the treatment significantly reduced the pain associated with neck movements and joint palpation. a single case study done by grant.10, also reported that the endurance of the deep cervical flexors improved and there was a reduction in mechanosensitivity of selected neural, muscular and articular structures of a screen based keyboard operator after four weeks of active stabilization training of the deep cervical flexors and lower scapular muscle group9. a possible explanation for our findings is that the factors influencing the school teachers are multifactorial. anthropometric variations, the possible adverse developmental effects of prolonged exposure to postural stresses, computer furniture, mental stress and reports of pain and vision factors can all influence school teachers posture17. most of the teachers complained that they did nt have the time to take care of themselves due to school work3. accordingly a dedicated 20 min for exercises at school itself as a part of their schedule for 4 weeks may have had a good psychological impact on them, indirectly resulting in pain relief apart from the physiological effect. therefore, a multidimensional approach may be needed if sustainable improvements are to be made including psychological assurance. the experimental group may have shown better results because doing exercises with constant feedback encourages patients doing the exercise to perform it correctly and gets them more involved in the treatment. there are two types of extrinsic feedback- knowledge of results (kr) and knowledge of performance (kp). pressure biofeedback is a type of kp, which is given during and after performance of a task and is related to how the task is performed. a therapist provides the information through the apparatus and by attending to the information the patient forms a closed loop. feedback helps in motor learning which is a set of processes associated with practice or experience leading to permanent changes in the capability of responding. biofeedback techniques are used to augment the patient s sensory feedback mechanisms through precise information about body processes that might otherwise be inaccessible. the treatment duration of the deep cervical flexor training protocol we used in our study was modified to four weeks, four days a week, once a day with every session monitored by a therapist following an original protocol prescribed by jull., which was performed by patients at home with pressure biofeedback, twice a day for 6 weeks and monitored by therapist only once a week19. the purpose of modification was to inculcate the treatment protocol in teachers school timetable and to suit their schedule. also conducting every session under supervision of therapist was considered more suitable for teachers than unsupervised performance with biofeedback at home. this study may help to prevent neck pain or arrest it in the early stages in the teaching population. once the teachers feel better this would make their job easier and more productive. given that computer use in schools is increasing day by day2, 6, the risk of development of musculoskeletal disorders within the teaching population has also increased resulting in a potentially greater number of disorders. musculoskeletal disorders have been associated with a decline in productivity in adults and seriously affect work performance. supposing that these symptoms now emerge earlier in a lifespan than in previous generations, we should expect increasing sick leave and early retirement. this study was performed on limited number of subjects, 15 subjects in each group. furthermore, subject and researcher blinding was not implemented, and teacher s posture was not included in the study. also the effect of dcf muscles training on posture and endurance of dcf muscles was not evaluated, and due to limitation of time we could not conduct a follow up of the patients. to our knowledge, this is the first study in which deep cervical flexor training was used for teachers having neck pain. to give this protocol a more grounded base of practice, further studies need to be carried out on this group with larger samples. further studies using 6 weeks or longer duration with subsequent follow up are recommended, and the relationship of forward head posture and np in this group also needs documentation. electromyography could be used to provide additional information on muscle activation associated with any observed postural changes. moreover, future studies could be designed to address the potential contribution of various factors, including musculoskeletal imbalances, that might possibly influence neck pain and forward head posture in this group. more dynamic, objective and functional outcome measures should be taken that better reflect the improvement in the postural endurance of deep cervical flexors resulting from therapy. the results of this study show that there was significant improvement in pain and disability after 4 weeks of deep cervical flexor training for school teachers with neck pain. moreover training using both pressure biofeedback and conventional exercises was found to be superior to training which only used conventional exercises.	[purpose ] the job of secondary school teachers involves a lot of head down posture as frequent reading, assignment correction, computer use and writing on a board put them at risk of developing occupational related neck pain. available studies of neck pain experienced by teachers are limited. the purpose of this study was to determine whether training of deep cervical flexor muscles with pressure biofeedback has any significant advantage over conventional training for pain and disability experienced by school teachers with neck pain. [subjects ] thirty teachers aged 2545 years with neck pain and poor craniocervical flexion test participated in this study. [methods ] a pretest posttest experimental group design was used in which experimental group has received training with pressure biofeedback and conventional exercises while control group received conventional exercises only. measurements of dependent variables were taken at baseline, and after 2 and 4 weeks of training. pain intensity was assessed using a numeric pain rating scale and functional disability was assessed using the neck disability index. [results ] the data analysis revealed that there was significant improvement in pain and disability in both the groups and the results were better in the experimental group. [conclusion ] addition of pressure biofeedback for deep cervical flexor muscles training gave a better result than conventional exercises alone. feedback helps motor learning which is the set of processes associated with practice or experience leading to permanent changes in ability to respond.
mosaicism can be defined as a condition in which at least two genetically distinct cell populations from the same differentiation lineage exist within the same tissue. the genes that are mutated and the time point at which these mutations occur determine the type of tissue that will be affected and the extent of involvement. in skin, several mosaic disorders have been described (itin and buechner 1999) and are characterized by islands of abnormal skin cells that presumably correspond to a clone of mutated cells that arose from a single cell in which the mutation initially occurred (happle 1993). it remains unclear how certain genetic defects lead to a mosaic disease, whereas others never become apparent in the same tissue. a candidate disease to analyze the mechanisms that determine how genetic mosaicism leads to a clinical phenotype is the keratin disorder epidermolytic hyperkeratosis (ehk), which is characterized by a generalized and a mosaic form (paller. 1994). keratins are members of the intermediate filament (if) gene family and are mainly expressed in epithelial cells. they form a cytoskeletal scaffold in keratinocytes of the epidermis to provide stability, thus contributing to the mechanical integrity of the epidermis (franke. 1981). keratins are grouped into two classes, the acidic type i (k9-k20) and the neutral - basic type ii (k1-k8) class (moll. keratins are obligate heteropolymers that require equimolar amounts of each type to assemble into ifs (steinert and liem 1990). the pair of keratins that is expressed is usually specific for the cell type and state of differentiation (franke. 1981 ; roop 1995). in the epidermis, if assembly is a dynamic process where newly synthesized filaments are integrated into the existing network. in the basal cell compartment, keratins k5 and k14 form the if cytoskeleton and defects in either keratin are associated with the autosomal - dominant blistering disease, epidermolysis bullosa simplex (ebs ; bonifas. keratins k1 and k10 are expressed in keratinocytes that are committed to terminal differentiation and point mutations in the corresponding genes have been identified in ehk (cheng. 1992 ; chipev. the majority of mutations in ehk are located in the same codon, affecting an evolutionary highly conserved arginine residue. this site, codon 156 of the keratin 10 gene, has consequently been found to be a hot spot for mutations due to cpg methylation and deamination (rothnagel. 1993). the clinical course of ehk is severe and is characterized by blistering and erythroderma (redness of the skin) at birth, and development of hyperkeratoses (thickening of the uppermost layer of the epidermis) later in life, predominantly over the flexural folds and in areas exposed to mechanical stress (digiovanna 1999). histologically, perinuclear vacuolar degeneration, lysis of the suprabasal keratinocytes and a thickened stratum corneum are characteristic findings (bale. 1993). at the ultrastructural level, clumps of keratin filaments are seen in a perinuclear distribution in suprabasal keratinocytes ; these clumps have been shown to contain keratins k1 and k10 (ishida - yamamoto. 1992). whereas mosaic forms have been reported for ehk where stripes of affected and unaffected skin alternate (paller. why do these two keratin disorders behave so differently ? to investigate this question and to study the molecular and cellular basis of mosaic diseases, we established a genetic mouse model for ehk that allows focal activation of a mutant k10 allele using a ligand - inducible cre recombinase (kellendonk. induction of the ehk lesions in a circumscribed area of the skin resulted in a phenotype characteristic of mosaic diseases, with patches of affected and unaffected skin. analysis of dissected keratinocytes from lesional areas demonstrated activation of the mutant allele in epidermal stem cells. we demonstrate that mutant ehk stem cells and wild - type stem cells can coexist in the basal layer of the epidermis, which may explain the persistent islands of phenotypic skin. furthermore, we show that the severity of the clinical phenotype in this mouse model correlates with the expression level of the mutant allele. these findings have important implications for gene therapy approaches for dominant diseases and these strategies can be tested in our model, as it mimics the human disease at the genetic level. finally, our results indicate that lack of selective pressure against certain mutations in epidermal stem cells could explain how genetic mosaicism results in clinical mosaicism. a bac clone containing the full - length mouse keratin 10 gene was isolated from a 129 svj genomic library (incyte genomics). two overlapping genomic fragments, a 4-kb ecori and a 6-kb bamhi fragment, were fused at an ecori site in intron 2 to generate a 4.8-kb bglii - xhoi fragment. the point mutation (cgcgc) was introduced by pcr - mediated mutagenesis in the context of a 400-bp bsteii - ndei fragment, which was then reinserted into the targeting construct, thus replacing the wild - type codon at position 154. a neo cassette (pgkneo) was inserted into a unique ndei site in intron 1 and this construct was introduced into the kpni and noti sites of the ppnt vector which contains a herpes simplex virus thymidine kinase gene under the control of the phosphoglycerate kinase promotor (pgktk ; tybulewicz. 1991). the procedures for embryonic stem (es) cell culture, electroporation, drug selection, and southern blot analysis of targeted clones have been described previously (ramirez - solis. 1993). in brief, the noti - linearized targeting vector (see fig. 1 b) was electroporated into ab2.2 es cells, which were maintained on snl76/7 feeder plates (bradley. es clones were selected in geneticin (g418) and fiau (1(1 - 2-deoxy-2-fluoro--darabinofuransyl)-5-iodouracil) and screened for homologous recombination by southern blot analysis, using probes derived from sequences not included in the targeting construct (see fig. 1 a) and an internal neomycin probe to confirm a single integration event (data not shown). to excise the neomycin resistance gene from the genome of targeted es clones, es cells were electroporated with pog231, a mammalian cre expression vector. the recombinant es cells were injected into c57bl/6 blastocysts, followed by transfer to pseudopregnant icr females. chimeric offspring were crossed with c57bl/6 mice and agouti offspring carrying the targeted alleles (+ /mut ; + /mut) were identified by pcr analysis of genomic dna obtained from tail biopsies. homozygous pups (mut / mut) were obtained from heterozygous (+ /mut) intercrosses. crepr1) pups were obtained by mating heterozygous + /mut mice to the previously described ru486-inducible cre transgenic line (berton. 2000). ru486 (0.5 mg / ml in ethanol / dmso) was topically applied onto the skin of newborn bigenic mice once a day for 45 d until blisters were visible. skin samples were processed as described and stained with hematoxylin and eosin (wojcik. samples for double - label immunofluorescence were prepared as described previously (bickenbach. 1996) and sequentially incubated with primary antibodies : rabbit anti mouse k10 and guinea pig anti mouse k14 (roop. 1984) ; then secondary antibodies : swine anti rna was isolated from the epidermis of heterozygous pups with rnazol b (tel - test), as described previously (wang. 1995). 2 g of rna were reverse transcribed using moloney murine leukemia virus (m - mlv) reverse transcriptase and random primers, according to the manufacturer 's recommendation (promega). a 280-bp fragment encompassing the point mutation was amplified from the cdna : 5-biotin ggattcggaggagatggtgg-3 and 5-biotin caatctgcagcagcacgttg-3. reverse transcription (rt)-pcr samples were digested with acii, analyzed by gel electrophoresis, followed by transfer to a positively charged nylon membrane (gene screen plus ; nen life science products). as the point mutation destroys an acii restriction site, the 280-bp pcr product is cleaved into fragments of 210 and 70 bp in dna from the wild - type allele, but is resistant to digestion in dna from the allele harboring the point mutation. the biotinylated dna fragments were detected using a nonisotopic chemiluminescent detection system (brightstar biodetect ; ambion inc.). skin biopsies from previously ru486-induced and -uninduced areas were fixed in 10% formalin overnight, dehydrated through graded ethanol, and embedded in paraffin. 5-m sections were deparaffinized in xylenes, rinsed in graded alcohols, and stained with nuclear fast red (vector laboratories). i laser capture microdissection (lcm) system (arcturus engineering). a thermoplastic polymer coating (ethylene vinyl acetate ; capsure) attached to a rigid support the transfer film is activated by a near infrared laser pulse which melts the film onto the targeted cells, thus forming a strong focal bond. 500700 cells from the stratum spinosum were captured per sample and dna was extracted in a buffer containing 1 mg / ml proteinase k, 0.5% nonidet p-40, 0.25% tween 20, 0.2 mm edta ph 8.0, and 10 mm tris - hcl ph 8.0 and analyzed by pcr with two primer pairs specific for the loxp (5-gggttattgaatatgatcgg-3 and 5-aggtaaggcgcttcagactc-3) and neomycin sequences (5-acagcaagggggaggattgg-3 and 5-tactattcctgcagccgacc-3), respectively. a bac clone containing the full - length mouse keratin 10 gene was isolated from a 129 svj genomic library (incyte genomics). two overlapping genomic fragments, a 4-kb ecori and a 6-kb bamhi fragment, were fused at an ecori site in intron 2 to generate a 4.8-kb bglii - xhoi fragment. the point mutation (cgcgc) was introduced by pcr - mediated mutagenesis in the context of a 400-bp bsteii - ndei fragment, which was then reinserted into the targeting construct, thus replacing the wild - type codon at position 154. a neo cassette (pgkneo) was inserted into a unique ndei site in intron 1 and this construct was introduced into the kpni and noti sites of the ppnt vector which contains a herpes simplex virus thymidine kinase gene under the control of the phosphoglycerate kinase promotor (pgktk ; tybulewicz. 1991). the procedures for embryonic stem (es) cell culture, electroporation, drug selection, and southern blot analysis of targeted clones have been described previously (ramirez - solis. 1993). in brief, the noti - linearized targeting vector (see fig. 1 b) was electroporated into ab2.2 es cells, which were maintained on snl76/7 feeder plates (bradley. es clones were selected in geneticin (g418) and fiau (1(1 - 2-deoxy-2-fluoro--darabinofuransyl)-5-iodouracil) and screened for homologous recombination by southern blot analysis, using probes derived from sequences not included in the targeting construct (see fig. 1 a) and an internal neomycin probe to confirm a single integration event (data not shown). to excise the neomycin resistance gene from the genome of targeted es clones, es cells were electroporated with pog231, a mammalian cre expression vector. the recombinant es cells were injected into c57bl/6 blastocysts, followed by transfer to pseudopregnant icr females. chimeric offspring were crossed with c57bl/6 mice and agouti offspring carrying the targeted alleles (+ /mut ; + /mut) were identified by pcr analysis of genomic dna obtained from tail biopsies. homozygous pups (mut / mut) were obtained from heterozygous (+ /mut) intercrosses. crepr1) pups were obtained by mating heterozygous + /mut mice to the previously described ru486-inducible cre transgenic line (berton. 2000). ru486 (0.5 mg / ml in ethanol / dmso) was topically applied onto the skin of newborn bigenic mice once a day for 45 d until blisters were visible. skin samples were processed as described and stained with hematoxylin and eosin (wojcik. samples for double - label immunofluorescence were prepared as described previously (bickenbach. 1996) and sequentially incubated with primary antibodies : rabbit anti mouse k10 and guinea pig anti mouse k14 (roop. 1984) ; then secondary antibodies : swine anti rabbit conjugate with fitc (dako) and rna was isolated from the epidermis of heterozygous pups with rnazol b (tel - test), as described previously (wang. 2 g of rna were reverse transcribed using moloney murine leukemia virus (m - mlv) reverse transcriptase and random primers, according to the manufacturer 's recommendation (promega). a 280-bp fragment encompassing the point mutation was amplified from the cdna : 5-biotin ggattcggaggagatggtgg-3 and 5-biotin caatctgcagcagcacgttg-3. reverse transcription (rt)-pcr samples were digested with acii, analyzed by gel electrophoresis, followed by transfer to a positively charged nylon membrane (gene screen plus ; nen life science products). as the point mutation destroys an acii restriction site, the 280-bp pcr product is cleaved into fragments of 210 and 70 bp in dna from the wild - type allele, but is resistant to digestion in dna from the allele harboring the point mutation. the biotinylated dna fragments were detected using a nonisotopic chemiluminescent detection system (brightstar biodetect ; ambion inc.). skin biopsies from previously ru486-induced and -uninduced areas were fixed in 10% formalin overnight, dehydrated through graded ethanol, and embedded in paraffin. 5-m sections were deparaffinized in xylenes, rinsed in graded alcohols, and stained with nuclear fast red (vector laboratories). sections were visualized using the pixcell i laser capture microdissection (lcm) system (arcturus engineering). a thermoplastic polymer coating (ethylene vinyl acetate ; capsure) attached to a rigid support was placed in contact with the tissue section. the transfer film is activated by a near infrared laser pulse which melts the film onto the targeted cells, thus forming a strong focal bond. 500700 cells from the stratum spinosum were captured per sample and dna was extracted in a buffer containing 1 mg / ml proteinase k, 0.5% nonidet p-40, 0.25% tween 20, 0.2 mm edta ph 8.0, and 10 mm tris - hcl ph 8.0 and analyzed by pcr with two primer pairs specific for the loxp (5-gggttattgaatatgatcgg-3 and 5-aggtaaggcgcttcagactc-3) and neomycin sequences (5-acagcaagggggaggattgg-3 and 5-tactattcctgcagccgacc-3), respectively. we have established a mouse model for the skin disease ehk by introducing a k10 mutation, which is found in the majority of human cases of ehk (rothnagel. the knock - in / replacement strategy was designed to replace the wild - type amino acid arginine encoded by codon 154 (cgc) with a sequence coding for cysteine (tgc) in es cells by homologous recombination (fig. 1). heterozygous + /mut and + /mut mice were derived from es clones that contain a neomycin (neo) cassette or in which neo had been deleted in vitro before injection into wild - type blastocysts, respectively. these two lines express the mutant allele at different levels, resulting in different phenotypes (see below). for the inducible system, heterozygous + /mut mice were used, as the neo cassette suppresses expression of the mutant allele. consequently, the animals develop a very mild phenotype which is mainly restricted to skin exposed to mechanical stress. two mouse lines are required for the inducible system, the first line harbors the target sequence flanked by loxp sites (floxed neo cassette), and the second line expresses an inducible form of cre recombinase (kellendonk. the cre transgene encodes a fusion protein consisting of cre recombinase and a truncated form of a progesterone receptor (pr1), which is sequestered in the cytoplasm until activated by topical application of an inducer to the skin (e.g., ru486). upon ligand binding, the fusion protein translocates to the nucleus, where cre exerts its effect by excising any sequence that is flanked by loxp sites in the same orientation. in regenerating tissues like the epidermis, the excision event will only be permanent if epidermal stem cells are targeted. therefore, the crepr1 construct was placed under the transcriptional control of an epithelial - specific promotor that drives transgene expression in the basal layer of the epidermis, where epidermal stem cells are located (berton. 2000). bigenic mice were generated by crossing these two lines (+ /mut, crepr1). topical application of ru486 to the ventral side of the trunk and extremities of newborn bigenic mice induced blistering at the site of induction after three to five treatments (fig. after the blisters ruptured, scaling developed around the site of the previous blisters. with the onset of hair growth, scaling diminished, leaving hyperkeratotic areas on the paws (fig. these hyperkeratotic areas persisted and developed into thick, brownish hyperkeratoses as seen in older ehk patients (digiovanna 1999). persistence of the phenotype for 6 mo after the initial ru486 application suggested that cre - mediated excision of the neo cassette had occurred in epidermal stem cells, as the epidermis is renewed every 810 d in mice (potten. this suggested that cre - mediated excision of the neo cassette and therefore activation of the mutant allele had occurred in epidermal stem cells and that these cells persisted and gave rise to daughter cells that expressed the mutant allele in the suprabasal layers of the epidermis. these results clearly demonstrate that keratinocytes containing the neo cassette did not migrate into lesional areas. persistent islands of phenotypic skin were also observed in chimeric mice derived from + /mut es cell clones, where the neo cassette had been excised in vitro before injection into wild - type blastocysts. these mice exhibited focal keratotic lesions on the paws at birth that developed into thick, brownish hyperkeratoses (fig. b). the focal lesions in both the inducible model system and the chimeras are equivalent to the linear, asymmetric hyperkeratotic areas in humans in the mosaic form of ehk, which is characterized by alternating stripes of affected and unaffected skin that follow the lines of blaschko (happle 1987). it has been shown that the mosaic form is caused by postzygotic k10 mutations that occur during embryogenesis (paller. our analysis of the inducible mouse model clearly suggests that in ehk, mutant epidermal stem cells exist side by side with wild - type stem cells. as the mutant k10 allele is not expressed in the basal layer, there is no selection against mutant ehk epidermal stem cells. in contrast, a selection process takes place against defective epidermal stem cells in a mouse model for ebs, when the mutant allele is focally activated in the basal layer (cao. an interesting aspect of the mouse models generated in this study (+ /mut, + /mut) is the correlation of a mild phenotype with reduced expression of the mutant ehk allele. heterozygous mice that contain the point mutation and the neo cassette (+ /mut) exhibit a very mild scaling phenotype due to suppressed expression levels of the mutant allele. rna analysis revealed that the mrna from the mutant allele was significantly reduced to 3540% of the levels of the wild - type allele (data not shown). to confirm that mutant k10 mrna was efficiently translated into protein, we mated heterozygous + /mut mice to obtain mice that were homozygous for the mutant allele (mut / mut). these mice showed a very severe phenotype at birth with extensive blistering and erosions, and died shortly thereafter (fig. skin biopsies showed a complete disintegration of the stratum spinosum in lesional areas (fig. 4 b). immunofluorescence microscopy revealed abundant k10 expression in the suprabasal layers of the epidermis (fig. as the wild - type k10 allele is not present in this mouse, all of the k10 protein detected is expressed from the mutant alleles. therefore, the mutant k10 mrna is translated into protein, but the presence of wild - type k10 in heterozygous + /mut mice is sufficient to overcome the effects of the reduced level of mutant k10. first, gene therapy approaches will be different for ehk and ebs. whereas repopulation of a phenotypic area by corrected stem cells is predicted in the case of ebs (cao. 2001), successful approaches for ehk will have to include a strategy that allows selection of genetically corrected epidermal stem cells and ablation of defective ehk stem cells. recent studies suggest that it may be possible to achieve selection of genetically modified epidermal stem cells by topical selection with cytostatic and antimitotic compounds, such as colchicine (pfutzner. second, in contrast to the general assumption that gene therapy approaches for dominant diseases must either correct the mutant allele or completely inhibit its expression, our data suggest that amelioration of the ehk phenotype may be achieved by partial suppression of the mutant allele or overexpression of the normal allele, thus altering the ratio of wild - type to mutant protein. as our previous transgenic studies have not revealed adverse effects from an approximate twofold overexpression of a wild - type k1 allele (bickenbach. 1996), it is intriguing to speculate that overexpression of the wild - type protein two- to threefold might be sufficient to improve the phenotype in ehk patients.	stem cells are crucial for the formation and maintenance of tissues and organs. the role of stem cells in the pathogenesis of mosaic skin disorders remains unclear. to study the molecular and cellular basis of mosaicism, we established a mouse model for the autosomal - dominant skin blistering disorder, epidermolytic hyperkeratosis (mim 113800), which is caused by mutations in either keratin k1 or k10. this genetic model allows activation of a somatic k10 mutation in epidermal stem cells in a spatially and temporally controlled manner using an inducible cre recombinase. our results indicate that lack of selective pressure against certain mutations in epidermal stem cells leads to mosaic phenotypes. this finding has important implications for the development of new strategies for somatic gene therapy of dominant genodermatoses.
as malaysia progresses both socially and economically, noncommunicable diseases have also fast become its public health concern. malaysia burden of dm continues to increase between 2009 and 2012 ; malaysia national diabetes registry registered a total of 653,326 patients diagnosed with t2 dm. the prevalence of diabetes in malaysia is projected to be 21.6% of its adult population by the year 2020. dm has been shown to be closely related to increased premature and preventable mortality, as well as macro- and microvascular complications such as heart disease, stroke, end - stage renal failure, blindness, and amputation. a study in malaysia showed that ministry of health malaysia spent a calculated amount of rm386,531.21 for a 6-month period to manage dm. the same study showed that estimated direct cost per patient was rm2,684.24 and for indirect cost rm1,062.88 annually. to reduce the complications of dm and in turn the cost of treatment, it is important for patient to achieve good glycemic control. a study found that a 1% reduction in hba1c was associated with a 37% decrease in risk for microvascular complications and a 21% decrease in the risk of death related to diabetes. evidence from many previous studies shows that self - management training in t2 dm is effective for short - term glycemic control. another study found that adherence to self - management is crucial in the overall management of diabetes and those who perform diabetes self - management (dsm) effectively achieve better short- and long - term health. sarawak, one of the states in east malaysia, registered a total of 43,333 patients with t2 dm during the period of 2009 to 2012. out of those who had hba1c test, 39.1% achieved the malaysian glycemic treatment target of hba1c < 6.5%. the percentage of those who achieved the glycemic treatment target would even be lower if those who did not have hba1c were included. thus, it is important to ascertain the reasons for low percentage of patients achieving glycemic control in order to plan and implement effective interventions. this was a cross - sectional study conducted in four randomly selected diabetes clinics in kuching and samarahan division of sarawak. the inclusion criteria were patients (a) with t2 dm for more than one - year duration, (b) aged 18 years to 65 years, (c) able to understand english or bahasa melayu, and (d) resident in the two divisions for at least six months. sample size was determined using the formula by naing., where n = zp (1 p)/d. based on the mean prevalence of good control of 38.9%, p was determined at 0.389. d was set as + 0.05, and the level of statistical significance,, was 0.05. ethical approval was obtained from research and ethics committee, universiti malaysia sarawak, and medical research and ethics committee, ministry of health, malaysia (nmrr-12 - 5 - 10829). data were collected via a face - to - face interview to ensure consistency as majority of the respondents were illiterate. section a was designed to obtain demographic data and the health profile of respondents (10 items). there were five behaviours involved : diet habit, exercise, medications compliance, self - monitoring of blood glucose, and foot care. two items performance of insulin injection and self - monitoring of blood glucose (2 items)were excluded in this study. this decision was made based on previous study that showed that only 12.9% of the malaysian diabetes patients were on insulin therapy and only 3.4% of patients with t2 dm performed self - monitoring of blood glucose. to respond, respondents were asked to recall their activities for the last seven days and stated the numbers of days they performed the dsm. section c of the questionnaire collected data on beliefs in treatment effectiveness which consisted of nine items. the questionnaire assessed two main aspects : (a) the belief that dsm activities were important in controlling the blood glucose items 1 to 4and (b) the belief that dsm activities were important in preventing the diabetes - related complications. five - point likert - type scale was used to score the items. for items 1 to 4, 0 indicated not important, while 4 indicated extremely important. items 5 to 9 asked the respondent 's belief in the dsm activities in preventing the diabetes complications ; the 5-point likert scale was from 0 (not possible) to 4 (extremely possible). higher score denoted greater perceived beliefs in the treatment effectiveness in controlling the illness and preventing the complications. it comprised seven items, and 5-point likert scale was used for the measurement with lowest score of 0 (definitely yes) to 4 (definitely not), the highest. in this part, respondents were asked how they perceived their capability in performing the dsm activities : diet, exercise, monitoring their blood glucose, foot care, and taking medication. section e has seven items measuring the perceived support received by the respondents ' in the past three months. the perceived support was measured according to the 5-point likert scale, from 0 (never) to 4 (always). section f has seven items that collected data on the healthcare team provider - patient communication. in this study, the word doctor from the original questionnaire was changed to healthcare provider because in local setting, diabetes patients were assessed by either the assistant medical officer or nurses during the follow - up visits. patients are referred to doctor only if there are complications. besides, the term healthcare provider provided a wider scope for respondents when reflected on their communication during follow - up. a 5-point likert scale from 0 (never) to 4 (always) questionnaire for sections b, c, d, e, and f was adopted with permission from xu.. health literacy on diabetes meant the respondents ' understanding of information in relation to diabetes and its management. a malay language version of the questionnaire was adapted from gazmararian. with permission. all items in the questionnaire that were in english were translated to bahasa melayu version using back to back translation. a pilot study was conducted for the items in sections b, c, d, e, and f and the cronbach 's alpha for the questionnaire ranged from 0.537 to 0.873. although deleting one of the items could improve the cronbach 's alpha to 0.563, it was decided to retain that item. as this item assessed the oral medication adherence, deleting it would lead to incomplete assessment of dsm, as taking medication was an important regime in dsm in this study. the data were analysed using statistical package for social sciences (spss) version 20. a total of 400 respondents were recruited with a mean age of 58.77 (sd = 11.46). majority of the respondents were women (68.6%) and 48.6% were sarawak bumiputra (iban and bidayuh). about 84.5% of them approximately half (50.6%) of the respondents had diabetes mellitus for less than five years with a mean of 6.40 years (sd = 4.46). most of the respondents (84.8%) had one or two chronic illnesses with the commonest being hypertension and hyperlipidemia. a total of 19.6% of them reported to have diabetes - related complications such as neuropathy, nephropathy, retinopathy, and heart problems. the mean fasting blood glucose (fbg) was 8.06 mmol / l (sd = 2.94), in which majority (76.1%) had their fbg more than 6.1 mmol / l. of those with reported hba1c results (n = 137), 56 respondents (40.9%) had their hba1c equal to or more than 6.5%. majority of respondents (79.1%) were not treated with insulin therapy. detailed information on demographic characteristic and health profile of the respondents is presented in table 1. table 2 shows the total mean score of the dsm and the five factors that might influence the dsm behaviours : belief in treatment effectiveness, self - efficacy, family support, healthcare provider - patient communication, and diabetes - related knowledge. the mean score for each factor varied from 8.31 (1.82) to 26.79 (4.60). majority of the respondents reported that they took their oral antidiabetic agents according to the doctor 's prescription (84.0%) and controlled their diet (60.8%) every day for the seven days prior to the data collection date, while 86.3% reported that they took their meal at a regular time every day. in terms of physical activity, only 29.1% of the respondents reported that they participated in physical activity for at least five days in the past week. however, lower mean of 0.67 1.44 was shown in relation to the engagement with specific exercise. three hundred and twelve (78.0%) respondents reported that they dried in between their toes after washing their feet, while 136 (34.0%) reported that they performed foot inspection every day. single linear regression analysis showed that none of the sociodemographic characteristics had significant relationship with dsm. multiple linear regression analysis showed significant linear relationship between dsm and belief in treatment effectiveness (p = 0.001), family support (p = 0.007), and self - efficacy (p = 0.027) (table 4). for one unit, increment of the respondent 's belief in treatment effectiveness would cause 0.301-unit (95% ci : 0.13, 0.47, p = 0.001) increase in the level of dsm and dsm increased 0.198 times in a unit of family support increment (95% ci : 0.05, 0.34, p = 0.007). those type 2 diabetes patients with a unit more in self - efficacy level have 0.210 units higher in dsm performance (95% ci : 0.024, 0.395, p = 0.027). from these findings, the relations among all the predictors in this study can be further described based on the generic regression equation : (1)y = a+b1x1+b2x2+in which y indicate the dependent variable, a is the intercept, and x is the independent variables whereas b is the regression coefficient. thus, the regression equation (final model) was dsm = 12.02 + 0.301(bte) + 0.198(fs) + 0.210(se). r for the regression model was significant, f(4, 395) = 14.59, with p = 0.000. r of 0.129 indicated that this model accounts for about 12.9% of the variance in dsm. it involved the practice of diet control, engaging with adequate physical activities, taking medications, and practicing foot care. performing self - monitoring of blood glucose was not assessed in this study compared to the previous study as it was not a common practice among the individuals with t2 dm as majority of them were treated with oral antidiabetic agents in malaysia. performing good dsm is essential for individuals diagnosed with dm, as it is the key in diabetes management to ensure good control of serum glucose, thus, preventing the occurrence of diabetes - related complications. more than 80% (84.2%) of the respondents in this study reported that they took their oral antidiabetic agents everyday according to doctor 's prescription. this high percentage of medication compliance was also found in china (89.1%) ; however studies in the us population showed that only 64% of their patients did so. this high percentage of medication compliance compared to other behaviours in dsm in this study could indicate that most patients with t2 dm preferred taking medications rather than modifying their behaviour, which is always more difficult. individuals who have better adherence to medications are believed to be significantly associated with positive attitudes towards dsm. findings in the current study indicated that lower percentage of respondents practiced diet control (60.8%). personal unwillingness and numbers of social gatherings as well as time and energy needed for food preparations were some of the barriers highlighted in previous studies that hindered compliance to diabetic diet [14, 15 ]. previous study had shown that proper counselling on diet control would lead respondents to have better understanding of its importance, resulting in them having significant reduction of total hba1c level and bmi. in terms of physical activity, only 29.1% of the respondents engaged in 30-minute physical activities for at least five days a week. in a study conducted in the united states, a slightly higher percentage (35%) of exercise prevalence the barriers to perform physical activity among patients with t2 dm could be related to bad weather (hot or rainy day), staying at housing area (lack of available walking area), and busy routine. age could be another possible contributing factor to this poor performance, taking into account that almost half (45.4%) of the respondents were those aged more than 60 years, who might not be able to perform regular exercise as recommended due to poor health. inadequate or low exercise performance by respondents could also be related to culture. unlike chinese or western population, neither malay nor sarawak bumiputra (the iban or bidayuh) population have specific physical activities such as yoga and tai chi which are culturally related. some studies showed that the chinese tended to exercise more and were more health conscious. proper foot care is essential in preventing complications such as foot ulcer and limb amputation. however, this study found that only 34% of the respondents performed daily foot inspection. previous study showed higher proportion (63%) of diabetes patients practicing daily foot care. one of the possible explanations for such difference could be due to lack of knowledge in relation to the foot care. studies had shown that educational intervention on proper foot care had resulted in better self - foot - care behaviour, improved knowledge, and increased level of confidence in terms of dsm. evidence also showed that providing foot care education to diabetes patients increased their awareness, resulting in low incidence of foot amputation. this study also found that those who had spouse were significantly performing better dsm compared to those who were single, widowed, or divorced. involvement of spouse in self - management of the disease may include providing social support to affirm healthy lifestyle behaviour as well as social control to modify their health behaviour. beverly. found that spousal support was associated with long - term adherence to major lifestyle changes, suggesting that patients with t2 dm were able to obtain emotional and physical support that aided in weight loss, proper diabetic diet, and adherence to the follow - up. this in turn would result in better diabetic control and reduce the risk of complications. individuals who had chronic illness (diabetes, hypertension, and arthritis) were able to demonstrate better levels of adherence to their self - care if they possessed higher level of self - efficacy. other studies revealed that individuals with higher level of self - efficacy were more likely to present better metabolic control as it was a predictor for diet management, self - care, and engagement in physical activities [17, 25 ]. as such, health care professionals should design strategy to enhance patients ' self - efficacy. anderson and funnell suggested empowerment approach to promote patients ' level of confidence which enhances their ability to think critically and act autonomously and improves the level of efficacy. tang. showed that for those patients who had undergone two - year empowerment - based dsm interventions had significant improvements in adherence to the treatment regimens. this study also found belief in treatment effectiveness predictive of better dsm which is consistent with previous studies. as more than 80% of the respondents in this study reported that they took their oral antidiabetic agents everyday according to doctor 's prescription, it is congruent to find belief in treatment effectiveness as one of the predictors for dsm. consistent with previous studies, family support was found to be a predictor for adherence to dsm. strong support from family builds patients ' confidence level, resulting in effective self - management and better disease control. social support can have the appraising and informative effects and provide coping strategies to assist patients to manage diabetes - associated stress and altered daily routines. mayberry and osborn found that family members who showed better supportive behaviours were those who were more knowledgeable about diabetes. thus, in clinical intervention it is important to enhance better understanding of disease and interactions between patients and their family members so as to promote positive and supportive behaviours. this study was conducted in two divisions of sarawak and majority of the respondents were malays and sarawak bumputra ; thus, the results may not be generalizable to all the populations of malaysia. this study added knowledge to the list of predictors for dsm especially for patients with t2 dm in malaysia. as the predictors found are belief in treatment effectiveness, family support, and self - efficacy, health care personnel should convince patients with t2 dm of the effectiveness of the treatment. they also need to empower the patients to enhance their self - efficacy and enlist the support from their family to ensure that patients could sustain their self - management efforts.	diabetes mellitus is a public health concern in malaysia. treatment of diabetes is costly and can lead to complications if disease is poorly controlled. diabetes self - management (dsm) is found to be essential for optimal glycemic control. this cross - sectional study was conducted among samples from four randomly selected diabetes clinics in sarawak, malaysia. the aim was to determine the predictors for dsm. face - to - face interview using questionnaire was used to collect data. four hundred respondents with type 2 diabetes mellitus (t2 dm) were recruited. majority of the respondents were sarawak bumiputra (iban and bidayuh, 48.6%) and female (68.6%). the mean age was 58.77 years (sd = 11.46) and approximately half of the respondents (50.6%) had t2 dm for six years (sd = 4.46). the mean fasting blood glucose (fbg) was 8.06 mmol / l (sd = 2.94), with majority (76.1%) having the level higher than 6.1 mmol / l. multiple logistic regression tests showed significant linear relationship between dsm and belief in treatment effectiveness (p = 0.001), family support (p = 0.007), and self - efficacy (p = 0.027). health care personnel must convince patients with t2 dm of the effectiveness of the treatment, empower and enhance their self - efficacy, and enlist the family support so as to ensure patients sustain their dsm efforts.
regional odontodysplasia is an uncommon, nonhereditary developmental anomaly affecting dental tissues, derived from both the mesoderm and ectoderm. the condition can be differentiated from other odontogenic disturbances, as all the histological elements of the dental organ are abnormal in the affected teeth, while other teeth in the same individual are normal. mccall and wald were credited for publishing the first report of odontodysplasia in 1947, in which they termed the condition arrested tooth development. in 1954, the term shell teeth was introduced by rushton, which the author used to describe the radiographic findings. in 1963, zegarelli. odontodysplasia. as the condition affects a group of several adjacent teeth in a particular segment of the jaw, pindborg added the prefix regional in 1970. the condition can affect both primary and permanent dentition and can occur in the maxilla, the mandible, or both. the etiology of odontodysplasia is uncertain, though many causative factors have been suggested, for example, vascular defects involving ischemia, local infections, trauma, rhesus incompatibility, irradiation, neural damage, activation of latent viruses residing in odontogenic epithelium, hyperpyrexia, metabolic and nutritional disturbances, hereditary and somatic mutation, and also neural crest migration disorders associated with hemangiomas.[57 ] the management of regional odontodysplasia is somewhat controversial and revolves around the question of whether or not to remove the affected teeth. although many clinicians prefer to extract the anomalous teeth as soon the diagnosis is made, some would prefer to retain them as long as they are free of infection until the skeletal growth is complete. this case is interesting and rare because it presents with the condition affecting the right side and the left central incisor is also missing. we present a case report of this rare anomaly with certain unique findings and a multidisciplinary treatment protocol. a 14-year - old girl came to the department of pedodontics and preventive dentistry, bapuji dental college and hospital, davangere, karnataka, india, with a chief complaint of missing or unerupted maxillary anterior teeth. a previous history of trauma to her maxillary anterior region about eight years back was recorded, due to which there was avulsion of few deciduous teeth and few fractured teeth, which were subsequently removed. both parents reported no previous history of tooth or genetic anomalies on either side of the family. on examination, the patient had a deficient maxilla on the right side leading to facial asymmetry. in the maxillary arch on the right side, the central incisor, lateral incisor, and canine were missing or unerupted ; the associated alveolar mucosa was enlarged and covered by fibrous tissue [figure 1 ]. also there was a rotated first premolar, erupting second premolar, and unerupted first and second molar on the right side. however, on the left side of the maxillary arch only a central incisor was missing and the rest of the teeth were present. in the mandibular arch, on both sides, all the teeth were present except the third molars. the oral hygiene was poor with an active carious lesion on the mandibular left first molar. there was deficient maxillary growth on the right side, showing signs of arch collapse [figure 2 ]. maxillary arch with right central incisor, lateral incisor and canine, and left central incisor missing mandibular anterior teeth was in occlusion with maxillary alveolar ridge, and there is no interarch space anteriorly panoramic, occlusal, and periapical radiographs were taken. the mandibular dentition was normal, as was the left maxillary dentition, except the central incisor. in the maxillary anterior region, the right central incisor, lateral incisor, and canine showed malformed and retarded development as compared to the age of the child. the second molar was also malformed, but it was not as severe as the other malformed teeth. the malformed teeth had thin radio - opaque contours with no distinction between the enamel and dentin, and wide pulp chambers, giving a ghost - like appearance. the crowns of the affected unerupted teeth were surrounded by radiolucent areas, probably representing enlarged dental follicles. only an insignificant amount of root formation, was visible radiographically [figures 3 and 4 ]. as the upper right second molar did not look as affected when compared to the other affected teeth, we could wait for its eruption. on the basis of the clinical and the radiographic findings, a provisional diagnosis of regional odontodysplasia was made. considering the situation, the eruption of the affected teeth was not favorable except for the second molar. therefore, it was suggested to surgically remove the affected teeth and go in for a prosthetic replacement to improve the function and for an esthetical purpose. the patient 's family was properly educated about the situation and the need for surgical removal and prosthetic rehabilitation and a complete consent and approval was obtained. panoramic radiographic showing unerupted maxillary right central incisor, lateral incisor and canine occlusal view showing ghost like appearnace of maxillary right central incisor, lateral incisor and canine under local anesthesia, the affected teeth were surgically removed [figure 5 ]. the teeth were of altered morphology, yellowish in color, soft or rubbery in consistency, and had very short or unformed roots, and wide open apices [figure 6 ]. the treatment plan included dietary counseling, instructions on oral hygiene, and prescription of mouth washes. indirect pulp capping and subsequent restoration of the carious mandibular left first permanent molar was also performed. surgical removal of affected maxillary teeth surgically removed teeth with altered morphology, yellowish in colour, very short or unformed roots surgically removed teeth were histologically examined under an optical microscope in the ground and decalcified section. histologically, all structures of the tooth germ were affected. in the ground section, longitudinal ground sections revealed the presence of enamel tissue over dentin and a central enlarged pulp cavity. the dentin layer was abnormally thin with the mantle dentin showing a normal dentinal tubular pattern, while the deeper dentin contained a mixture of predominant interglobular dentin and poorly organized tubular dentin [figure 7 ]. ground sections showing presence of enamel tissue over dentin and central enlarged pulp cavity hematoxylin and eosin staining of the decalcified sections revealed the presence of teeth - like structures, with abnormally formed slight enamel and dentin with wide pulp space [figure 8 ]. few areas of enamel spaces were seen. in most of the areas the dentin was atubular and contained clefts scattered through a mixture of globular areas, with numerous areas of amorphous (basophilic) material [figure 9 ]. decalcified section showing teeth like structure with abnormally formed slight enamel and dentin with wide pulp space decalcified section showing atubular dentin containing clefts scattered through a mixture of globular areas with numerous areas of amorphous (basophilic) material two months postoperatively, a temporary acrylic maxillary partial denture, with a bite plane, was made, to preserve the alveolar ridge during the period of skeletal growth [figure 10 ]. the patient was placed on periodic recall to review and monitor the development of the maxillary arch. clinical view after placing acrylic maxillary partial denture with bite plane the patient will be kept under review until she reaches adulthood, when a final rehabilitation for the loss of teeth might be accomplished after the facial bones had attained complete growth. regional odontodysplasia is a relatively rare, nonhereditary, localized developmental anomaly of the dental hard tissues of a group of contiguous teeth. it occurs in both deciduous and permanent dentitions, and it has a marked preference for the maxilla. other conditions, such as, dentinal dysplasia, shell teeth, hypophosphotasia, dentinogenesis imperfecta, or amelogenesis imperfecta can mimic some features of regional odontodysplasia. however, these disorders tend to affect the entire dentition. although many theories have been proposed, in this particular case it has not been possible to determine a precise etiological factor. the relationship of trauma to the upper front teeth region about eight years back could not be directly co - related to odontodysplasia. it was clear that the underdeveloped maxilla on the right side was due to the teeth being affected by odontodysplasia. in the present case, along with the affected right teeth, the left central incisor and the right first permanent molar were missing. also the second molar was affected to a lesser extent, although one could wait for its eruption. on account of these factors, the present report was considered interesting and rare. the case showed features that were in accordance with the literature, in that the condition seemed to affect females more than males, and most patients came to the dentist complaining of delayed eruption or gingival swelling. the clinical presentation of in this case was failure in eruption of teeth, which could be related to odontodysplasia. in this case, radiographs showed that the unerupted teeth had not achieved complete rhizogenesis. the affected teeth had an abnormal morphology with an irregular surface contour, with pitting and grooves, and a rough surface with defective mineralization.[79 ] the teeth appear to be hypoplastic, hypocalcified, and show yellowish or brownish discoloration. radiographically, the affected teeth have been described as having a ghostly appearance, showing a marked reduction in radiodensity. both the enamel and dentin appear very thin, and the pulp chamber is exceedingly large ; the roots are short with open apices ; and pulp stones or denticles are present in the affected as well as the normal adjacent teeth. histologically, all structures of the tooth germ are affected. in the ground section, the enamel prisms are irregular and show hypoplastic and hypocalcified areas, whereas, the enamel closer to the dentinoenamel junction appears more normal. the dentin is thin, and the tubules are reduced in number and tortuous in shape. the most significant feature is the presence of large areas of amorphous dentin containing irregularly shaped foci of a poorly formed dentinal matrix, within which, capillaries are sometimes present. since the clinical, radiographic, and histological features of regional odontodysplasia are so characteristic, dentists should face no difficulty in the diagnosis of this abnormality. the greater difficulty arises in treatment planning, as no consensus as to the best option has yet been reached. the main question is whether to remove the affected teeth or not, but we believe that this decision should be taken after the assessment of each individual case of regional odontodysplasia. some authors prefer extraction and replacement with removable, and later, fixed prostheses, while others have suggested keeping the unerupted teeth, and waiting for their calcification and thus hope for its eruption. from the evaluation it was decided that the eruption of the affected teeth was questionable, so it would be better to surgically remove the affected teeth and provide prosthetic rehabilitation for the patient. with the consent of the patient and parents, the unerupted teeth were removed and temporary rehabilitation was made with a temporary acrylic prosthesis. the patient will be kept under review until she reaches adulthood, when a final rehabilitation for the loss of teeth may be accomplished, after the facial bones had attained complete growth. consultations between pediatric, prosthodontic, and orthodontic specialties are necessary in each case of odontodysplasia. treatment planning should be designed for each individual case, taking into account factors such as the age of the patient, the medical history, the extent of involvement, the eruption of the teeth, esthetics, the development of pathology, and the wishes of the patient and parents. the treatment of regional odontodysplasia requires a multidisciplinary approach and an organized team effort. in the present case however, further treatment in the form of orthognathic surgery, bone grafting, and implant therapy needs to be anticipated in the future.	regional odontodysplasia is an uncommon developmental anomaly affecting a localized area of dentition, with distinctive clinical, radiographic, and histological findings. this article reviews a case of a 14-year - old female who reported with unerupted maxillary anterior teeth. this case was rare in that it involved maxillary dentition with unerupted maxillary anterior teeth on the right side, but the left central incisor was missing. the molars on the right side were showing some amount of abnormality. radiographically, the affected teeth had a ghostly appearance, showing a marked reduction in radiodensity. both enamel and dentin appeared to be very thin, the pulp chamber was exceedingly large, and the roots were short with wide open apices. all the characteristics were consistent with the diagnosis of regional odontodysplasia. the care and treatment of this patient required a multidisciplinary approach. the unerupted maxillary anterior teeth were surgically removed, following which temporary prosthetic restoration was provided to improve esthetics and to restore the function.
feldspar minerals belong to the group of tectosilicates which make up nearly 75% of the crust of the earth. their abundance makes them relevant to various fields such as water contamination, soil, geochemistry, and atmospheric sciences. despite their ubiquity, how water adsorbs on their surfaces, how ice might form, and aspects of feldspar dissolution behavior are prominent examples. one particular mineral, microcline (a k - feldspar), was put into the spotlight recently. using a droplet freezing technique, atkinson. showed that k - feldspar dominates ice nucleation in earth s atmosphere by mineral dust. while these experiments are well suited to establishing ice nucleating ability, they did not provide the atomistic insight necessary to understand why k - feldspar is such an excellent ice nucleator. this work has prompted a number of follow up studies, and of course water mineral surfaces, although not specifically about microcline, have previously been widely studied. are also to some extent surprising because feldspar surfaces bear no obvious resemblance to the structure of ice. it is commonly thought that good ice nucleators should present surfaces that template ice - like structures. lattice match as in the case of agi or hexagonal arrangements of hydroxyl groups in the case of kaolinite are famous examples. some experiments suggest, however, that other materials without an apparent templating effect can still be good ice nucleators. these cases not only demonstrate that lattice match is probably not a good predictor for ice nucleating ability, but also that we still lack an understanding of what it is that makes a good ice nucleator. recent experimental studies have focused on establishing the ice nucleating ability of various materials, and recent simulations have aimed at characterizing specific water solid interfaces and at understanding general trends in ice nucleating on more idealized model systems. they were able to show that it is a subtle balance between lattice match, surface topology, and adsorption energy that impacts ice nucleation. this means that ice nucleating ability is strongly system dependent and requires investigations on a case by case basis. therefore, by looking at a variety of examples of good ice nucleators, one might be able to deduce a general theoretical framework for what makes materials good at nucleating ice. understanding what it is that makes particularly interesting substrates such as feldspar so good at nucleating ice is therefore desirable. nucleation on real feldspar particles, however, is a complicated process, not least because many different surfaces will be exposed on a real feldspar particle. furthermore, defects, trenches, and wedges might play a role in the nucleation process. no single study can address all of these issues, but here we lay the groundwork by establishing the structure of the clean and water covered microcline (001) surface, which is the surface that is cleaved most easily for feldspars. the rest of the paper is structured as follows : in section 2 we elaborate on the computational method. we then discuss the bulk structure of feldspar in section 3.1 for two reasons. first, we show that our computational setup for our dft calculations is able to reproduce experimental findings very well, which gives us confidence in our methods. second, understanding the bulk is a prerequisite to the surface and the surface / water interface. after covering bulk structures we focus on the surface, for which much less experimental data are available, in section 3.2. we show that the thermodynamics of the al / si distribution on the surface differ significantly from that in bulk and connect this with the dissolution behavior of feldspar minerals. in section 3.3 we move on to the mineral water interface, where we discuss the interaction of water with the microcline (001) surface. at first, we focus on monolayer adsorption and then follow this by a discussion of multilayer ice growth. we demonstrate that the ice - unlike structure of feldspar does not lead to ice - like structures in the contact layer. however, the particular structure of the contact layer has the ability to induce ice - like structures in the second water layer. it is these ice structures that could make feldspar the excellent ice nucleator it is. as well as providing initial understanding of clean and water covered microcline we hope this study will stimulate the general discussion of what makes a material a good ice nucleator. density functional theory (dft) calculations were performed with the periodic plane wave code vasp. core electrons were replaced by projector augmented wave (paw) potentials, and valence electrons were expanded in plane waves. all calculations were done on microcline, a triclinic potassium feldspar with chemical composition k4al4si12o32 (space group c1). its structure is shown in figure 1a and b. (a) one possible arrangement of alo4 (light blue) and sio4 (dark blue) tetrahedra in microcline. a primitive unit cell contains 4 alo4 tetrahedra, 4 k ions (to balance the charge), and 12 sio4 tetrahedra. t1 sites expose bridging oxygen atoms to the (001) surface (these oxygens are highlighted with green circles), and t2 expose bridging oxygens to the (010) surface (oxygens highlighted with yellow circles). al atoms are depicted in blue, si atoms in yellow, k ions in pink, and oxygen atoms in red. all eight t1 and eight t2 sites are labeled. on the bottom of (b) we show the orientation of the unit cell, as well as the location of the two most stable cleavage planes in (a). water fragments (oh and h) used to saturate undercoordinated bonds are shown in green. starting from the experimental unit cell and structure we did a full geometry relaxation with a 600 ev cutoff for the plane wave basis set, which is 50% larger than the recommended 400 ev cutoff for ionic relaxation so as to avoid problems with pulay stresses (see the supporting information (si) for more information). this optimization was followed by an ionic relaxation with a 400 ev cutoff to obtain the equilibrium geometry for any bulk structure. electronic structures were converged to within 1.0 10 ev, and ions were relaxed until the forces acting on them were below 0.005 ev /. pack k - point mesh for bulk calculations and a 3 2 1 monkhorst the (001) slab was generated by cleaving a 1 1 2 supercell and saturating all undercoordinated atoms (si, al, or o) by dissociating water molecules to form al oh and a vacuum parallel to the surface normal of at least 12 was used to separate slabs in adjacent cells, and a dipole correction along the surface normal was applied.figure 1c shows the slab model resulting from this procedure. ernzerhof (pbe) exchange - correlation functional for all calculations, but we also used the optb88-vdw functional to test the role of van der waals forces. both functionals lead to the same qualitative conclusions, as shown in the si. the stability of bulk structures with different al / si order (for a precise definition see sec. 3.1.1) was investigated by comparing their total energy ebulk relative to the most stable bulk structure with energy ebulkmin per al atom. in the primitive unit cell, four al atoms can be distributed among 16 sites, which gives rise to a total of 1820 possible structures. using the experimental crystal structure of microcline, all 1820 structures were generated and minimized with the classical force field clayff using gromacs. the four most stable bulk structures found for every distinct al order were then optimized with dft as described above. additionally, several structures were chosen by hand (some of them at random, some based on chemical intuition) and optimized to guarantee a good sampling of configurational space. in the si we show that energies obtained with pbe and clayff do indeed correlate well with each other, which demonstrates that this a reasonable procedure. the stability of different al / si orders on the surface was evaluated in a similar manner. for this we compared the total energy of surfaces esurf with different al order relative to the most stable surface structure with energy esurfmin per al. water adsorption energies were calculated as follows1where eh2o / surf denotes the total energy of the slab with water adsorbed on it, n the number of water molecules adsorbed, eh2o the total energy of a water molecule in vacuo, and esurf the total energy of the slab in vacuo. with this definition the dimensionality of the configurational space is too large to perform a comprehensive search of all adsorption minima, especially for multilayer water structures which contain up to 24 water molecules. therefore, a guided structure search approach based on the potential energy surface (pes) of water on microcline, chemical intuition based on maximizing the number of hydrogen bonds formed, and structural motifs found in ice ih was chosen to address this problem. the key idea is to guide the structure search in such a way that mainly structures which either have a strong interaction with the surface or strong intermolecular bonds are searched for, leaving out all the structures that are of unlikely relevance. we prescreened the configuration space with clayff combined with the spc water model, and low - energy candidates were then further relaxed using dft. detailed information about our procedure is provided in the si. in total, more than 100 000 structures were screened with clayff, and more than 150 structures were minimized with dft. before investigating the water / feldspar interface one needs to have an atomistic understanding of the bulk. microcline s bulk structure is fairly well established, but there are issues over the al order at an atomistic level that remain unclear. we start with an introduction of the general structural features of microcline, which is followed by a discussion of al order from two perspectives. first, the effect of al order on the unit cell dimensions is investigated, and second, the energetics of different al order states are discussed. our results show that dft is suitable to describe the structure and energetics of feldspar well by comparing our results to experiment. they also reveal, however, that a microscopic structural feature, the appearance of two distinct al o bonds, is not accounted for in macroscopic models. the structure of feldspars in general can be described as mirrored crankshaft - chain frameworks of polymerized al / sio4 tetrahedra. both al and si atoms are 4-fold coordinated by oxygen, forming alo4 and sio4 tetrahedra. figure 1a shows the unit cell of microcline with alo4 and sio4 tetrahedra highlighted. the primitive unit cell contains a total of 16 tetrahedra, of which 4 have al and 12 have si in the center, as well as 4 k ions. aluminum can potentially be located in the center of any of the 16 tetrahedra making up the unit cell. the possible arrangements of al among these sites is referred to as al / si disorder and has been the subject of numerous studies ; see, e.g., ref (54). t1 = 0.00 means that all al are in t2 sites, t1 = 1.00 that all al are in t1 sites. we estimate the al disorder based on dft lattice relaxations combined with the model proposed by kroll and ribbe. the y - axis shows the t1 occupation estimated using this model (eq 2). each black circle represents a different structure which was fully optimized with dft. because we optimized several structures with the same actual t1 order that differed in their atomistic structures, multiple data points for each al / the model and dft agree very well on the trend. also shown on the top are the most stable bulk structures found for t1 = 0.00 and t1 = 1.00. one can distinguish two different tetrahedral sites in monoclinic feldspars, namely t1 and t2 sites. t1 sites expose oxygen atoms to the (001) surface (figure 1a, green circles), while t2 sites expose oxygen atoms to the (010) surface (figure 1a, yellow circles). to clarify this nomenclature, the location of t1 and t2 sites is also highlighted in figure 1b. in a unit cell there are eight t1 and eight t2 sites. if all al occupy the same type of site, the structure is said to be fully ordered, if they are equally distributed between t1 and t2 sites, the structure is said to be fully disordered. we stress that with order we only refer to the distribution of al between t1 and t2 sites throughout the paper. besides being relevant to the physicochemical properties of feldspar minerals, al / si order also plays a crucial role in, for example, zeolites.. a connection between al order and feldspar unit cell dimension has long been known. for a detailed explanation of the origin of this connection, it is sufficient to state that kroll and ribbe proposed the following widely used connection between al order / disorder and unit cell geometry2 in this equation, t1 takes a value between 0 (all al are in t2 sites) and 1 (all al are in t1 sites). b and c are unit cell and reciprocal unit cell vectors, respectively (measured in and). this or similar methods have been useful in understanding al disorder in numerous minerals. however, they all rely on measurements performed on macroscopic samples in which the data are averaged over multiple polycrystalline domains. computational studies have addressed al order issues using classical force fields and monte carlo methods (see for example refs (63 and 64) and references therein). using dft this relationship can be tested at the atomistic level, which, to the best of our knowledge, has not been done before. for every bulk structure computed we estimated the predicted al disorder using eq 2 from the unit cell parameters obtained from the lattice relaxation. the x - axis represents the actual al order in a structure and the y - axis the order estimated with eq 2. the solid red line shows the predictions based on the model, and the black data points are estimates based on our dft results. overall we find that the model and our dft estimates agree very well with each other. the linear relationship between cell dimension and t1 order is clearly well reproduced. what is interesting to note, however, is that different structures at any given t1 occupation result in a number of different estimated disorder states, as can be seen from figure 2. in other words, the suggested 1:1 correspondence between estimated t1 order and actual order breaks down at the atomistic level. this might not be too surprising considering that the model itself is based on data averaged over large samples. o bond length and one characteristic al o bond. however, two different si o bond lengths can be identified, depending on whether al atoms are next nearest neighbors to si or not. we provide a more detailed explanation of why this breakdown happens at the microscopic level in the si. relation between al order and energetics of microcline (bulk and surface). t1 = 0.00 means that all al are in t2 sites and t1 = 1.00 that all al are in t1 sites. the energy per al atom of different bulk (left y - axis, black dots) and surface structures (right y - axis, red squares) relative to the most stable structure is plotted. for clarity, only the most stable structure identified for each t1 occupation is shown. we find that the most stable structures are the ones with t1 = 1.00 for bulk microcline and t1 = 0.00 for the surface. the topmost parts of a t1 = 0.00 and a t1 = 1.00 surface are also shown. while the surface corresponding to t1 = oh groups, the surface at t1 = 1.00 has 50% si oh and 50% al oh groups. after having considered the effect of al order on the unit cell geometry, we now discuss its impact on the energy. figure 3 plots al occupation of t1 sites as a function of energy relative to the most stable bulk structure. on the x - axis the al distribution in t1 sites is plotted, 0.0 meaning that all al are located in t2 sites and 1.0 meaning that all al are located in t1 sites. this again is in very good agreement with experiment, which shows that microcline is highly ordered, with al occupying almost exclusively t1 sites. the second most stable order state is for all al to occupy t2 sites, which is 30 mev per al atom higher in energy. this is considerably more than the thermal energy available at room temperature, which agrees well with the fact that microcline is found experimentally to be highly ordered, with al concentrated in t1 sites. besides energetics many other factors, such as conditions under which the mineral formed (temperature, pressure, ph), will contribute to the final composition of a feldspar mineral. none of these are taken into account here ; however, our results show that it is thermodynamically favorable to highly order al into t1 sites. each of the most stable bulk structures at a given coverage obeys lwenstein s rule (al o al linkages are unfavorable) ; no such al o al linkages appear in any of the most stable structures. furthermore, dempsey s rule, which states that the number of al o si o al linkages shall be minimized at a given al / si ratio, also holds, with the number of such linkages being four for each of the most stable structures. to summarize our bulk results we can reproduce both the empirical relation between al order and lattice dimensions as well as the preferential ordering of al into t1 sites. o bond lengths instead of only one, depending on local environment around that si o bond. having an atomistic understanding of the bulk, one can now move on to the surface. here we focus on the (001) surface because this is the most easily cleaved surface and therefore most likely to play a significant role in dissolution and ice formation. we will show that the energetics of al order on the surface differ significantly from the bulk. while al in bulk prefer t1 sites, they appear to be more stable in t2 sites on the surface. to investigate the surface structure of microcline (001), slab models from the fully relaxed bulk structures with different al / si order were built, as discussed in section 3.1. the (001) cleavage plane is shown in figure 1 a. cleaving the bulk structure along this plane leads to the breaking of si o and al the resulting undercoordinated atoms were saturated with dissociated water molecules, and the resulting slab model is shown in figure 1 c. upon relaxation, none of the surfaces underwent significant reconstructions. our main goal here is to understand how surface stability depends on al order. we show the results in figure 3, where the x - axis corresponds to al t1 site occupation and the y - axis on the right of the graph is the stability of a given surface relative to the most stable surface, esurfmin. oh groups exposed on the surface, whereas structures with t1 = 1.00 have 50% al oh and 50% si our main finding is that, in contrast to the bulk, surfaces with all al in t2 sites (t1 = 0.00, no al the fact that surface composition and bulk composition differ suggests that the chemical difference between them, the presence or absence of hydroxyl groups, is the driving force for this. the likely reason behind this is the stronger electrostatic attraction between a si and an oh compared to an al and an oh. the result agrees well with experimental evidence showing that al groups dissolve prior to si groups. previous computational studies consistently found that, based on kinetic arguments (reaction barriers), al should dissolve faster than si. our results add to this in that they show that it is also thermodynamically more favorable to enrich the surface in si oh groups. having established the structure of the (001) surface of microcline, we now look at its interaction with water. in this section we will look at the water / feldspar interface ranging from water monomers to multilayer ice structures on top of microcline. even though monolayers identified do not resemble the structure of ice, we find second overlayers that are indeed ice - like. this shows how even substrates with a non - ice - like surface morphology can stabilize ice - like multilayers. the feldspar surface is rather complex, and it exhibits a variety of chemical motifs with which water can interact. specifically, there are hydroxyl groups attached to either si or al, which can form hydrogen bonds with water, either as hydrogen bond donors or acceptors. lastly and of less importance, bridging oxygen atoms (al o si or si o si) can act as hydrogen bond acceptors. we define 1.0 monolayer to consist of 8 water molecules because there are 8 strong interaction sites for water available in a primitive unit cell (6 hydroxyl groups and 2 potassium ions). the dashed line represents the cohesive energy of bulk ice ih from pbe (0.66 ev / water). blue data points correspond to adsorption energies on a t1 = 1.0 surface and red data points to energies on a t1 = 0.0 surface. energies of multilayers (coverage of 2.5 and 3.0 ml) are also shown. overlayer structures 1.0 ml + basal and 1.0 ml + prism both have a coverage of 2.5 ml, and overlayer structures basal + basal and prism + prism both have a coverage of 3.0 ml ; they are therefore difficult to distinguish on the basis of coverage. the inset shows the energetics of different multilayer structures more clearly, with (i), (ii), (iii), and (iv) being 1.0 ml + basal, 1.0 ml + prism, prism + prism, and basal + basal, respectively. (b) to (e) show the most stable adsorption structure found for a water monomer (0.125 ml) and monolayers with coverage 0.5, 1.0 and 1.5 ml, respectively. for clarity, we only show the unit cell in (b), and (c)(e) have the same unit cell. we also add red lines connecting neighboring water molecules to highlight the network h2o forms. to understand how water interacts with microcline (001), the adsorption of water in a broad range of adsorption structures over a wide range of coverages was examined. two particular surfaces were considered, the most stable surface (t1 = 0.0) and a surface with al highly ordered in t1 sites, t1 = 1.0. the former will be exposed in weathered feldspar samples, whereas the latter will be present in freshly cleaved samples. we discuss the differences between al- and si - rich surfaces whenever necessary, but our main focus will be on the freshly cleaved surface. one reason for focusing on this (t1 = 1.0) surface rather than the most stable (t1 = 0.0) surface is that there are major chemical processes happening during the weathering process. it is for example well established that k ions will partially be replaced with h3o ions on the surface. furthermore, because weathering happens in non - neutral conditions, hydroxyl groups and bridging oxygens will be partially protonated / deprotonated depending on the ph. having a realistic representation of a weathered microcline surface is therefore beyond the reach of this work. looking at the surface that will be exposed after fresh cleavage on the other hand involves considerably fewer approximations concerning surface structure. in this study all issues arising from the fact that real feldspar surfaces in nature will be imperfect are beyond the scope of this work. the particular t1 = 1.0 surface chosen was one of the most stable t1 = 1.0 surfaces with al distributed between different t1 sites so as to maximize the number of different chemical motifs on the surface. figure 4a shows results for water adsorption on this surface and a t1 = 0.0 surface as a function of coverage. the most stable monomer adsorption site is in between one aloh and one sioh group, as shown in figure 4b. in this configuration, water interacts with two hydroxyl groups, receiving one hydrogen bond (from si the remaining hydrogen atom of the water molecules forms an additional hydrogen bond with a bridging oxygen atom meaning that the water monomer is involved in three hydrogen bonds. the adsorption energy of a water molecule in this position is 0.75 ev. on the most stable t1 = 0.0 surface, the adsorption energy is significantly weaker, only 0.59 ev. because al oh groups (hence the difference in surface energies) they bind water more strongly. even though the energetics of water is quite different on both surfaces, structural features of an adsorbed water molecule are quite similar. the most stable adsorption site for a water monomer on the t1 = 0.0 surface is the same as the one shown in figure 4b. on the t1 = 1.0 surface the o o hydrogen bond lengths involving the water molecule and the surface are 2.65 (hydrogen bond accepted from si oh), and 3.13 (hydrogen bond donated to bridging o) for the water monomer on the t1 = 1.0 surface. on the t1 = 0.0 surface, these hydrogen bond lengths are 2.71 (hydrogen bond accepted from si oh), and 3.26 (hydrogen bond donated to bridging o). the general elongation of hydrogen bonds on the t1 = 0.0 surface agrees with the weaker adsorption energy. in this adsorption site we checked for stable dissociated states but did not find any that came within 1.5 ev of the intact monomer. the fact that water monomers do not dissociate on this surface is not surprising since our surface model is fully hydroxylated. upon increasing the coverage water dimer adsorption metastable hydrogen bonded dimers were identified. however, their adsorption energy was at least 0.05 ev / h2o less than the adsorption energy of two separate adsorbed monomers. similar behavior was seen for higher coverages : isolated water monomers were more stable than hydrogen bonded clusters. this means that water clustering on a freshly cleaved microcline (001) surface is not favored. however, on the most stable surface (t1 = 0.0) this behavior at low coverages is qualitatively different. because of the weaker adsorption energy, clustering of water is favorable on the t1 = 0.0 surface. upon increasing coverage, the water adsorption energies become more similar on the two surfaces (see figure 4a). this is not surprising because at higher coverages the relative importance of surface water interactions versus water the structural features of the adsorbed water cluster do not differ much, and changing the surface mainly impacts the adsorption energies. to understand how ice can grow on surfaces, it is crucial to look at structures that might form on the substrate at high coverages. at these high coverages, the differences in terms of water adsorption between the two different surfaces considered become very small, and so our focus in the discussion will be on the freshly cleaved (001) surface. we looked at a wide range of ice structures including traditional ones (basal and prismatic faces) and nontraditional ones adapting to the potential energy surface of the substrate. figure 4d shows the most stable contact layer structure with an adsorption energy of 0.56 ev / h2o found at a coverage of 1.0 ml. three out of four water molecules in a square form hydrogen bonds with surface hydroxyl groups, and the fourth water molecule is coordinated to a k ion. the average intermolecular water o o bond length in this structure is 2.87, and the o o o angles range from 77 to 103 (compared to 109 in ice ih). this contact layer is rather flat, the average height difference between nearest neighbor water molecules being 0.3 (as opposed to 0.9 in a basal bilayer), and does not resemble any traditional ice - like structure. overlayers that would resemble the structure of an ice ih basal or prism layer have a coverage of 1.5 ml. in terms of energetics, overlayers at 1.0 and 1.5 ml are very similar, the most stable structure having an adsorption energy of 0.60 ev / h2o. the most stable contact layer at such a coverage is shown in figure 4e. irrespective of our starting point (basal or prism face) for the structure search, low energy structures converge to structures similar to the one shown in figure 4e. in these structures the hexagonal arrangement of water molecules persists (as can be seen from the top view). while ice bilayer structures have water molecules located at two well - defined heights, water molecules in these monolayers are at a broad distribution of heights, as exemplified by the side view in figure 4e. in the most stable 1.5 ml ice structure on microcline, intermolecular o o bond lengths range from 2.59 to 2.91 and intermolecular o o o angles from 95 to 128. low energy monolayer coverage structures at 1.5 ml therefore do not resemble either basal or prism structures. the broad range of bond lengths and angles is a result of the water overlayer optimizing its interaction with the surface, which comes at the cost of distorting hydrogen bonds. previous work has shown that, for example, on metal surfaces ice bilayers do not form in general. even surfaces with a near perfect lattice match to ice do not necessarily tend to form ice bilayers. interestingly on other surfaces, such as for example kaolinite, more regular ice - like structures can form in the contact layer. in this case this is made possible by the particular arrangement of hydroxyl groups on the kaolinite surface. they are arranged in an almost uniform hexagonal pattern and match the ice bilayer structure rather well. a hexagonal layer of ice can therefore bind very strongly to the kaolinite surface. it should be noted that the hexagonal structure on kaolinite has no dangling hydrogen bonds and is therefore not amenable to further water adsorption. on microcline, the situation is very different because the surface does not match the bilayer structure at all. not only are different interaction sites exposed at different heights (the height difference between k ions and the oxygen atom of a oh group is 0.6 at the surface) but also the spacing between different hydroxyl groups itself varies significantly (o o distances range from 4.7 to 6.1). as a result of this very complex surface, we do not observe contact layers resembling a regular ice ih bilayer structure. the ability of a substrate to stabilize multiple ice layers appears to be a trivial prerequisite for any substrate to be a good ice nucleator. based solely on this, kaolinite should not drive multilayer ice growth, which would make it a bad ice nucleator. classical md simulations addressing ice nucleation on kaolinite revealed however that it nevertheless promotes ice growth. at least of equal importance is to understand the full configurational space of water on a substrate and any low energy metastable structures that might form. in the case of kaolinite it was for example a prismatic face of ice forming on the surface that drove ice nucleation and not the 0.04 ev / h2o more stable bilayer - like structure obtained from first - principles calculations. to explore this system we performed an extensive search of possible ice structures using our guided structure search method. we specifically looked at four different ice multilayer structures on microcline : (i) 1.0 ml in the contact layer plus an ice ih basal face in the second layer (1.0 ml + basal) (coverage of 2.5 ml) ; (ii) 1.0 ml in the contact layer plus an ice ih prism face in the second layer (1.0 ml + prism) (coverage of 2.5 ml) ; (iii) two layers of the ice ih prism face (prism+prism) (coverage of 3.0 ml) ; (iv) two layers of the ice ih basal face (basal+basal) (coverage of 3.0 ml). the energies of the different multilayer structures are plotted in figure 4a as a function of coverage and, in the inset, for the four structures just described. figure 5 shows the most stable structure for each of these cases from a side and top view. before discussing the details of different multilayers, we compare monolayer and multilayer ice structures. by adding an additional water layer on top of the contact layer, the adsorption energy (eads = 0.60 ev / h2o) either increases slightly (compared to the 1.0 ml coverage) or stays the same (compared to the 1.5 ml coverage). we show the most stable structure found for a double basal layer (a), a double prism layer (b), a combination of a contact layer with 1.0 ml coverage and a basal layer (c), and a combination of a contact layer with 1.0 ml coverage and a prism layer (d). the contact layer in each case is shown in dark blue and the second layer in light blue. (b) and (c) are the most stable structures of all multilayer structures and very closely mimic the prism face (b) or basal face (c) of ice ih. the inset shows that the energetics of the different types of overlayer are very similar and range from eads = 0.60 ev / h2o (for structures (ii) and (iii)) to eads = 0.58 ev / h2o (for structure (iv)), (i) being in the middle with an adsorption energy of eads = 0.59 ev / h2o. these tiny energy differences of a few mev / water are beyond the accuracy of dft (at the generalized gradient approximation level) for hydrogen bonded systems, and we therefore do not attempt to say which structure is most stable. even though the various multilayer ice structures have similar energies, their structures differ substantially. first, the second ice layer in the prism+prism structure very closely resembles the prism face of ice ih, as can be seen from the red guidelines in figure 5b. the average o o distance of the second ice overlayer in this structure is 2.82, and the average o o o angle is 113. note that the first overlayer in this case does not adopt a prism face - like structure but instead acts as a mediator between the substrate structure of microcline and the prism face formed in the second overlayer. the ice configuration shown in figure 5c also quite closely resembles an ice - like structure, in this case the basal face of ice ih (average o o distance : 2.72, average o o o angle : 107). recent unbiased molecular dynamics simulations on model systems revealed that the formation of an ice - like overlayer seems to enhance ice nucleating ability. both of these structures are therefore viable candidates for how ice might form on a clean and defect - free microcline (001) surface. in the other two scenarios, shown in figure 5a and 5d, the second layer gets significantly more distorted upon relaxation. in both structures, the in - plane symmetry resembles the hexagonal structure of ice, and the side view reveals however that the buckling is very different compared to a basal and prism face. this makes both of these structures unlikely candidates for explaining what makes feldspar a good ice nucleator. we now try to understand the structures obtained. from the calculations at 1.5 ml we know that neither basal - like nor prism - like contact layers can form (see figure 4e for a reminder). it makes sense that this irregular first layer induces strong distortions in a basal - like overlayer on top of it, simply to make both layers fit onto each other. not surprisingly, therefore, a multilayer consisting of two basal - like ice layers, as in figure 5a, will distort significantly upon relaxation. in contrast, the contact layers found for a coverage of 1.0 ml were much more regular (see figure 4d for one particular example). as a consequence, forming a regular basal - like overlayer is much more feasible in this case. this is why the second water layer is able to maintain its basal - like structure in the scenario shown in figure 5c. on the other hand, forming a prism - face - like structure on this regular contact layer with 1.0 ml coverage is not favorable. thus, the second layer in figure 5d, which started off as prism - like face, deforms significantly. to summarize, our results show that 1.0 and 1.5 ml contact layers on microcline can template the formation of an ice - like structure in the second layer. this is interesting because neither the substrate nor the contact layer adopts an ice - like structure. nevertheless, basal and prismatic ice - like configurations can be formed on top of them. these ice - like arrangements of water molecules become possible in the second overlayer because the first contact layer, even though overall not being very ice - like, still exhibits a local structure akin to that of ice (o o bond lengths for example are comparable to ice in the first contact layer). in contrast, the underlying substrate, as in the case of feldspar (001), can be very different from an ice - like structure. in other words, the non - ice - like contact layer can be thought of as having a mediating function between the ice - unlike substrate and an ice structure. this finding might help to stimulate thinking in relation to ice nucleation because it greatly enlarges the compound space of potentially efficient ice nucleators. our finding, based on ab initio calculations, shows that the surface structure of a good ice nucleator need not necessarily to match the structure of ice. density functional theory combined with a guided structure search approach was used to investigate the feldspar / water interface. at first, we looked at bulk microcline and focused in particular on al order. we reproduced not only the empirical relation between al order and lattice dimension but also the fact that microcline has al highly ordered in its t1 site. furthermore, our microscopic understanding allowed us to address an issue that was previously overlooked, namely, that there are two rather than one characteristic si the 1:1 relation suggested by the empirical model breaks down at the atomistic level. after establishing a clear picture of the bulk structure the thermodynamically most favorable order state on the surface was different from the bulk. instead of al preferring t1 sites as in the bulk, this was explained by the greater stability of sioh groups compared to aloh groups. our numbers provide clear support for the well - known experimental fact that al dissolves prior to si. when it came to water adsorption we focused on investigating the configurational space of water on microcline. we found that at low coverages single water molecules are able to bind strongly to the surface. as more water is adsorbed, the adsorption energy per water molecule decreases up to a coverage of 1.0 ml. contact layers with a coverage of 1.0 and 1.5 ml were found to be ideal candidates to stabilize ice - like structures. multilayer ice configurations closely resembling the prism face or the basal face of ice ih were identified. this was somewhat surprising because neither the microcline surface nor the contact layer resembles an ice - like structure. besides feldspar, other substrates such as testosterone or marine biogenic particles are also good ice nucleators without having an apparent match with ice. our findings here could potentially apply to these systems as well ; just because the substrate itself does not resemble the structure of ice it does not automatically follow that ice - like structures can not be stable on them. it is interesting to briefly consider how this work could be taken forward. the accuracy of pbe for hydrogen bonded systems is around 50 mev / h2o or better depending on the system. the most obvious shortcoming of pbe is that it neglects van der waals dispersion forces. however, as reported in the si we have performed a series of tests for feldspar bulk and water covered surfaces which show that the inclusion of dispersion does not alter any of the key conclusions reached in this particular system. the main effect of dispersion is to reduce the energy spacing between stable and metastable structures, in a manner similar to what has been observed for bulk ice structures. given the limitation of pbe, and any exchange - correlation functional for that matter, we have been careful not to attempt to discriminate between low energy structures of similar stability. rather we have focused on obtaining a general understanding of the low energy structures of ice that are accessible on the surface. besides approximations coming from the choice of exchange - correlation functional, our study focused exclusively on a perfect (001) surface of microcline at 0 k. real feldspar particles in our atmosphere will expose a large variety of different surfaces, all of which might contribute to the outstanding ice nucleating ability of this material. furthermore, the effect of temperature, defects, trenches, wedges, and impurities on its surface remains unclear. addressing these issues is important for future studies aiming to explain what makes feldspar an excellent ice nucleator. even though we did not directly look at ice nucleation on microcline, the dft data can be useful for guiding and validating future force field studies which might probe the ice nucleating ability of feldspar explicitly. the fact that we now have a good idea about the microcline (001) surface as well as the finding that water does not appear to dissociate on this surface suggests that classical force field simulations are a reasonable choice to investigate this interface further. this is particularly promising because it is becoming feasible to study ice nucleation with unbiased molecular dynamics simulations. furthermore, recent force field studies suggested that the formation of ice - like water overlayers on top of substrates can promote ice nucleation. we therefore envisage future studies, in which a large number of materials could be screened with the method introduced here to identify water structures at complex substrates, for which the ice nucleating ability could subsequently be probed with molecular dynamics simulations. this could make it feasible in the future to rationally design highly efficient ice nucleating agents.	feldspar minerals are the most common rock formers in earth s crust. as such they play an important role in subjects ranging from geology to climate science. an atomistic understanding of the feldspar structure and its interaction with water is therefore desirable, not least because feldspar has been shown to dominate ice nucleation by mineral dusts in earth s atmosphere. the complexity of the ice / feldspar interface arising from the numerous chemical motifs expressed on the surface makes it a challenging system. here we report a comprehensive study of this challenging system with ab initio density functional theory calculations. we show that the distribution of al atoms, which is crucial for the dissolution kinetics of tectosilicate minerals, differs significantly between the bulk environment and on the surface. furthermore, we demonstrate that water does not form ice - like overlayers in the contact layer on the most easily cleaved (001) surface of k - feldspar. we do, however, identify contact layer structures of water that induce ice - like ordering in the second overlayer. this suggests that even substrates without an apparent match with the ice structure may still act as excellent ice nucleating agents.
historically, patients receiving animal insulin preparations of low purity developed high levels of insulin antibodies, potentially affecting efficacy 1. after the development of recombinant human insulin, and the rapid and longacting analogues, the number of patients developing high levels of insulin antibodies substantially decreased 2, 3, with high levels of insulin antibodies rarely observed and with no apparent effects on efficacy 4, 5, 6. insulin degludec (ideg) is a new basal insulin analogue with an ultralong duration of action (> 42 h) 7, 8, 9. we measured insulin antibody levels in six randomized, controlled, openlabel trials in patients with type 1 (t1d) or type 2 diabetes (t2d) who received ideg (n = 2550) or insulin glargine (iglar) (n = 1184) once daily (table s1, supporting information) 10, 11, 12, 13, 14, 15 to assess the impact of antibody formation on the change in hba1c from baseline to end of trial (eot), on insulin dose at eot and on the incidence of specific adverse events (aes). two trials [the begin basal bolus type 1 long (3583) 10 and the begin flex type 1 (3770) 11 ; treatment periods : 52 and 26 weeks, respectively ] compared the efficacy and safety of ideg with iglar (both once daily at 100 u / ml) in patients with t1d also treated with insulin aspart [iasp (100 u / ml) ] in a basalbolus regimen (initiated 12 months before the trial). three trials [the begin once long (3579) 12, the begin once asia (3586) 13 and the begin flex type 2 (3668) 15 ; treatment periods : 52, 26 and 26 weeks, respectively ] in patients with t2d compared ideg with iglar (both once daily at 100 u / ml) oral antidiabetic drugs. the begin low volume trial (3672) 14 compared ideg (200 u / ml) with iglar (100 u / ml) administered once daily for 26 weeks in combination with metformin a dipeptidyl peptidasefour inhibitor. patients with t2d in trials 3579, 3672 and 3586 were insulinnave before the trial. patients in trial 3668 were either insulinnave or receiving basal insulin oral antidiabetic drugs. data were not collected from patients in the begin basalbolus type 2 trial (3582), as insulin antibody levels were measured for insulintreated patients with t2d in trial 3668. antibody measurements, from fasting serum samples, were carried out at baseline (week 0), weeks 12, 26, 40 and 52 (depending on treatment duration) and at end of followup (eof), after a 1week washout period (week 27 or week 53) while using nph insulin. the washout was used to minimize interference of high eot plasma concentrations of the recombinant insulin analogues (resulting from their longer halflives 7, 16) with the antibody assays. antibody levels were measured using a validated subtraction radioimmunoassay (file s1, supporting information). antibody levels were expressed as % b / t, the percentage of bound radioactivity (b) relative to total radioactivity (t) added to the samples. spearman 's correlation coefficient was calculated to investigate the association between crossreacting antibodies at eof and change in hba1c from baseline to eot, as well as total daily insulin dose at eot. standardized medical dictionary for regulatory activities (meddra) queries were used to identify patients experiencing immunogenic or hypersensitivity reactions. for evaluation of aes, patients who exhibited > 10% b / t absolute increase in crossreacting antibody level or > 5% b / t absolute increase in ideg or iglarspecific antibody level were considered to have increased levels of antibodies. mean levels of ideg and iglarspecific antibodies remained low for both treatment groups (table s2, supporting information) at baseline and eof, with little variation in idegspecific (0.00.1% b / t at baseline and 0.00.4% b / t at eof) and iglarspecific antibodies (1.3 to 0.9% b / t at baseline and 1.1 to 1.1% b / t at eof) across t1d and t2d studies. mean levels of antibodies crossreacting with human insulin remained low for both ideg and iglar treatment groups (table 1). in t1d the % b / t ranged from 11.2% b / t at baseline to 19.3% b / t at eof (ideg) and from 11.5% b / t to 14.3% b / t (iglar). in t2d, crossreacting antibody levels ranged from 0.2% bt at baseline to 5.1% b / t at eof (ideg) and from 0.2% b / t at baseline to 6.0% b / t at eof (iglar). figure 1 shows levels of crossreacting antibodies over time during the two 52week trials in patients with t1d (panel a : trial 3583, n = 629) and t2d (panel b : trial 3579, n = 1030). the mean daily basal insulin (table s1, supporting information) and bolus insulin doses (data not shown) were similar at baseline and eot in the t1d trials of ideg and iglartreated patients ; however, in the t2d trials, the mean daily basal insulin dose increased from baseline to eot in both groups. in several trials, the basal insulin doses at eot were lower in the ideg group compared with the iglar group 10, 13, 14. scatter plots showing levels of crossreacting antibodies versus total daily insulin dose at eot for patients in the 52week trials 10, 12 did not suggest that the level of crossreacting antibodies had an influence on insulin dose or that dose influenced the level of crossreacting antibody formation (figure s1, supporting information). antibodies crossreacting with human insulin (% b / t) at baseline and end of followup in phase iiia trials comparing the safety and efficacy of insulin degludec with insulin glargine in patients with type 1 and type 2 diabetes. % b / t, percentage bound of total radioactivity ; bb, basalbolus ; eof, end of followup ; ff, forced flexible dosing ; ideg, insulin degludec ; iglar, insulin glargine ; s.d., standard deviation ; t1, type 1 diabetes ; t2, type 2 diabetes. crossreacting antibody levels to human insulin (hi) over time in patients with (a) type 1 diabetes (begin basalbolus type 1 long 10) and (b) type 2 diabetes (begin once long 12). antibody levels were measured at week 0 (baseline), week 12, week 26, week 40, week 52 (end of trial, eot) and week 53 (end of followup, eof) after a 1week washout period. patients were treated with nph insulin during the washout period to minimize interference of trial drugs with the antibody assay. dark blue circles = insulin degludec (ideg)treated patients ; light blue diamonds = insulin glargine (iglar)treated patients. data are presented as mean standard error of the mean. to evaluate associations between insulin antibodies and efficacy, levels of crossreacting antibodies at eof versus change in hba1c at eot from all patients in the two 52week trials 10, no patterns were observed in the change in hba1c with respect to the level of crossreacting antibodies for patients with t1d or t2d treated with either ideg or iglar. spearman 's correlation coefficients evaluating the correlation between the levels of insulin antibodies at eof and change in hba1c from baseline to eot, and between insulin antibody levels at eof and basal insulin dose at eot are shown in table s3, supporting information. all correlation coefficients were low, suggesting there was no clinically relevant association between insulin antibodies and change in hba1c or insulin dose. rates of aes associated with immunogenic reaction are shown in table s4, supporting information. the ae rates (per 100 patientyears of exposure) in idegtreated patients with increased levels of crossreacting antibodies (> 10% b / t) were 15.4, 14.6 and 1.7 for injection site reactions, skin and subcutaneous tissue disorders and immune system disorders, respectively. the higher number of immune system disorders in iglartreated patients was driven by a higher rate of seasonal allergy and multiple allergies, thus events unrelated to treatment with iglar. in patients with no or 10% b / t absolute increase in crossreacting antibodies, event rates were generally lower than those with > 10% b / t. in idegtreated patients with 10% b / t absolute increase in crossreacting antibodies, the ae rates (per 100 patientyears of exposure) were 13.0, 8.4 and 2.1 for skin and subcutaneous tissue disorders, injection site reactions and immune system disorders, respectively.. two hypersensitivity reactions, both considered unlikely to be related to trial drug, were experienced by two patients in the ideg group with > 10% b / t absolute increase in crossreacting antibodies. no reactions were reported in the iglar group. in the population with no or 10% b / t absolute increase in crossreacting antibodies, 19 patients experienced hypersensitivity reactions ; the event rates in the ideg and iglar groups were 1.1 and 0.7 events per 100 patientyears of exposure, respectively. none of the patients with > 5% b / t absolute increase in insulinspecific antibody levels (ideg or iglarspecific antibodies) experienced hypersensitivity reactions. there was no indication of a relationship between crossreacting antibodies at eof and the rate of confirmed hypoglycaemic episodes in either group (data not shown). patients treated with ideg for 2652 weeks had low levels of both idegspecific antibodies and antibodies crossreacting with human insulin at eot. crossreacting antibody levels were similar in the idegtreated and iglartreated groups at eof and, in general, were higher at eof in trials with patients who were previously exposed to insulin. no association between change in hba1c and insulin antibody levels was noted from scatter plots. furthermore, spearman 's correlation coefficients evaluating the degree of association between insulin antibody levels and change in hba1c from baseline were low. these results suggest that insulin antibody levels in idegtreated patients were not associated with the change in hba1c in these trials. the higher levels of crossreacting antibodies reported from the begin t1d trial (3583), compared with the t2d trial (3579), may be explained by differences in pretrial insulin exposure, i.e. those with t1d have previously been treated with insulin for many years and therefore have a higher antibody level at baseline when compared with those with t2d, who were insulinnave at randomization. the correlation coefficients between total daily insulin dose and insulin antibody levels were low, suggesting that neither the level of idegspecific antibodies, nor the level of antibodies crossreacting with human insulin, were associated with the insulin dose. no clinically meaningful differences were observed in ae types or rates between patients with or without an absolute increase of > 10% b / t in crossreacting antibody levels from baseline to eof. in conclusion, the immunogenic response to longterm treatment with ideg was low in patients with t1d and t2d. the development of insulin antibodies to ideg was not associated with change from baseline hba1c or total daily insulin dose at eot, nor was it associated with higher rates of immunogenic reactions compared with iglar. j. v. has served on advisory boards for novo nordisk, eli lilly, sanofi aventis, msd, boehringer ingelheim, bristolmyers squibb, novartis and abbott, has received research support from novo nordisk, msd, eli lilly and sanofi aventis, and served on speakers ' bureaux for novo nordisk, eli lilly, sanofi aventis, msd, novartis, abbott and boehringer ingelheim. j. s. has attended advisory boards for takeda, bayer, novartis, merck sharp dohme, astra zeneca, bristolmyers squibb, novo nordisk, sanofi aventis, berlin chemie, lilly, boehringer ingelheim, merck, roche, ipsen, pfizer, janssen and lifescan, and has attended speakers bureaux for takeda, bayer, novartis, merck sharp dohme (msd), astra zeneca, bristolmyers squibb, novo nordisk, sanofi aventis, berlin chemie, lilly, boehringer ingelheim, roche, ipsen, pfizer, janssen and lifescan. h. s., o. k. and t. j. are employees of novo nordisk and have shares in the company. some data have been previously presented in abstract form at the following meetings : 48th annual meeting of the european association for the study of diabetes in 2012, 94th annual meeting of the endocrine society in 2012, 9th western pacific region congress of the international diabetes federation in 2012 and 56th annual meeting of the japan diabetes society in 2013. the title of all the abstracts was insulin degludec does not increase antibody formation compared to insulin glargine : an evaluation of phase 3a trials. j. v. acts as the guarantor for this manuscript and takes full responsibility for the work as a whole, including the study design, access to data, and the decision to submit and publish the manuscript. all authors (j. v., j. s., h. s., o. k., t. j. and p. h.) were involved in critical analysis and interpretation of the data, drafting / critically revising the article and shared in the final responsibility for the content of the manuscript and the decision to submit it for publication. description of six phase iiia clinical trials comparing the safety and efficacy of insulin degludec with insulin glargine. insulin degludec (ideg) and insulin glargine (iglar)specific antibodies (% b / t) at baseline and eof in phase iiia trials comparing the safety and efficacy of ideg with iglar in patients with type 1 and type 2 diabetes. spearman 's correlation coefficients for antibodies versus change in glycated haemoglobin level and antibodies versus total daily insulin dose. rates of adverse events associated with immunogenic reaction by the absolute increase in crossreacting antibody levels from baseline to end of followup. total daily insulin dose at end of trial versus crossreacting antibody level at end of followup for patients in trials with a treatment duration of 52 weeks. (a) data from patients with type 1 diabetes (begin basalbolus type 1 long 5). (b) data from patients with type 2 diabetes (begin once long 7. change in glycated haemoglobin level from baseline to end of trial versus crossreacting antibody level at end of follow up for patients in trials with a treatment duration of 52 weeks. (a) data from patients with type 1 diabetes (begin basalbolus type 1 long 5). (b) data from patients with type 2 diabetes (begin once long 7.	we examined insulin antibody formation in patients with type 1 (t1d) or type 2 diabetes (t2d) treated with oncedaily insulin degludec (ideg) or insulin glargine (iglar) to evaluate the impact of antibody formation on efficacy and safety. insulin antibodies were measured using subtraction radioimmunoassays in six phase iiia clinical trials using ideg (n = 2250) and iglar (n = 1184). spearman 's correlation coefficient was used to evaluate associations between crossreacting antibodies and change from baseline glycated haemoglobin (hba1c) and insulin dose. ideg and iglarspecific antibodies remained low [10% b / t or without an absolute increase in antibodies crossreacting with human insulin. ideg treatment resulted in few immunogenic responses in patients with t1d and t2d ; antibody formation was not associated with change in hba1c, insulin dose or rates of aes.
two years ago, fujita and colleagues published a landmark study that sparked the current advances in our understanding of antiviral immune responses (5). the group screened for cdnas that could enhance the activation of an ifn regulatory factor 3 (irf3) reporter after transfection with polyinosinic polycytidylic acid (poly i : c)a synthetic double - stranded (ds)rna polymer that potently induces the production of type i ifns. their screen turned up the caspase - recruitment domain (card)containing cytosolic rna helicase rig - i and the closely related mda5 (5). shortly afterward, balachandran. demonstrated that the signaling proteins fas - associated via death domain (fadd) and receptor - interacting protein 1 (rip1) were essential for the production of type i ifns in response to infection with vesicular stomatitis virus (vsv) and transfection with poly i : c (6). another signaling protein, tnf receptor associated factor 3 (traf3), was also shown to be involved (7). less than a year ago, four groups independently identified this missing link as a card - containing adaptor protein, naming it ifn- promoter stimulator-1 (ips-1) (8), mitochondrial antiviral signaling protein (mavs) (9), virus - induced signaling adaptor (visa) (10), and card adaptor inducing ifn- (cardif) (11). here overexpression of ips-1 activated irf3 and nf-b, and knockdown of ips-1 expression blocked signaling in response to both rna viruses and poly i : c. interestingly, one group found that ips-1 is localized to the outer membrane of mitochondria (9), and another group showed that it is cleaved and inactivated by the hepatitis c virus (hcv) protease ns3/4a (11), which had previously been shown to intercept dsrna - activated signaling to irf3 and ifn- (12, 13). studies of mice lacking either rig - i or mda5 showed that the two helicases are essential for type i ifn production in response to distinct classes of rna virus. transcribed dsrna and is required for the in vivo response to vsv, newcastle disease virus (ndv), sendai virus, and influenza virus (14, 15). mda5, by contrast, is the principal receptor for poly i : c and is essential for the antiviral response to the picornavirus encephalomyocarditis virus (emcv) (15, 16). two recent studies, one in the july issue of the jem and another in a recent issue of immunity, reported that the phenotype of ips-1deficient mice is essentially the sum of the phenotypes of rig - i and mda5 single knockout mice (17, 18). in cells from ips-1deficient mice, rna virus and dsrna - induced signaling and type i ifn production were profoundly impaired. the only exception to this rule was in plasmacytoid (p)dcs, which exclusively use tlrs to link rna recognition to type i ifn production (14, 18). ips-1deficient cells were unable to activate irf3 and nf-b, failed to produce type i ifns and did not activate ifn - inducible genes downstream of rig - i and mda5 (17, 18). in vivo, ips-1 mice were highly susceptible to infection with vsv (17, 18), despite normal serum levels of type i ifns (probably induced by pdcs through tlr - dependent mechanisms) (18). interestingly, ips-1 was highly haploinsufficient in vivo : heterozygous (ips-1) and homozygous (ips-1) mice were equally susceptible to infection with vsv (18). furthermore, as with mda5 deficiency (15, 16), ips-1 mice were more susceptible to lethal infection with emcv (17). unlike vsv infection, however, emcv infection failed to induce the early production of type i ifns in the serum of ips-1 deficient mice, suggesting that this nonenveloped picornavirus might evade tlr - dependent detection by pdcs. these studies show that ips-1 is essential for antiviral responses to rna viruses and synthetic dsrna. in contrast, ips-1 was not required for type i ifn production in response to transfected dna (17, 18), or to infection with the intracellular bacterium listeria monocytogenes (18) or the vaccinia poxvirus (17). these data, together with recent reports demonstrating a tlr - independent response to intracellular dna (1921), suggest that cytosolic rna and dna recognition activate the production of type i ifns via distinct signaling pathways that converge on irf3. the characterization of ips-1deficient cells and mice is an important advance in our understanding of how cells respond to infection with rna viruses. but these studies also raise interesting questions regarding the nature of nucleic acid recognition and the different ways in which infected cells coordinate antiviral immunity. the phenotype of ips-1deficient cells formally demonstrates that rig - i and mda5 are the primary, nonredundant sensors that link cytosolic dsrna detection to the type i ifn response. before the characterization of rig - i and mda5, the ifn - inducible protein kinase r (pkr) had been thought to be important for the type i ifn response to dsrna, because pkr - deficient cells and animals show reduced type i ifn production and are susceptible to infection with vsv (22). however, in light of the identification of the rna helicase pathway of viral recognition, it now appears that pkr does not induce the type i ifn response to cytosolic dsrna. instead, pkr appears to have a separate, but still important, role in cell - autonomous control of viral infection. the function of pkr most likely involves its ability to inhibit translation of host cell mrna by phosphorylating the translation initiation factor eif2the only known substrate of pkr (22). perhaps the reduced type i ifn production by pkr - deficient cells reflects the possibility that type i ifns are more efficiently translated under conditions where eif2 is limiting, as would occur during viral infection. not only do these findings clarify the relative contribution of pkr to the antiviral response, they also imply a more general principle with fundamental implications for antiviral responses and autoimmunity : nucleic acid recognition might be the only way for most cells to induce type i ifn production. in models of infection with various classes of rna virus, this suggests that no component of these viruses other than the rna, including envelope glycoproteins and capsid proteins, can induce the production of type i ifns. moreover, it indicates that stress responses, which are activated in cells supporting rapid viral replication, are also insufficient to induce the production of type i ifns. in this regard, it would be interesting to examine mitogen - activated protein kinase activity during viral infection in ips-1deficient cells, as it is likely that intact stress response pathways are activating the expression of genes other than type i ifns. if nucleic acid recognition is the only way for most cells to induce the production of type i ifns in response to infection with rna viruses, does a similar principle apply to recognition of dna viruses ? until recently, dsrna was thought to be the main signature of viral replication inside cells, produced by replicating rna viruses and bidirectional transcription of dna viruses. however, the finding that ips-1deficient cells mount a normal antiviral response to transfection with dna, and to infection with l. monocytogenes or a poxvirus (17, 18), indicates that cytosolic dsrna is not the only trigger of the type i ifn response. this raises the question of why two signaling pathways are needed to link nucleic acid detection to the antiviral response. the simplest explanation is that rna and dna viruses activate different cell - intrinsic effector responses that are tailored to combating the particular type of virus. importantly, neither type i ifns nor generic ifn - inducible genes account for the specificity of this response, as transfection with dna and poly i : c activates a similar profile of ifn - inducible genes (21). instead, the two signaling pathways might drive the transcription of distinct ifn - independent genes and might also activate different transcription - independent antiviral mechanisms. if so, this would have important implications for the design of antiviral therapeutics, which would need to target the ifn - independent response appropriate for the type of virus infection. another implication of these studies relates to autoimmune disorders, which are often associated with the production of type i ifns (23). most cells tested respond to rna viruses and dsrna in an ips-1dependent manner, and a similarly ubiquitous response exists to cytosolic dna (20, 21). thus, an inappropriate response of these sensor proteins to self - derived, intracellular nucleic acids might be a critical contributor to autoimmunity. this scenario implies a cell - autonomous initiation of autoimmunity, which is distinct from several current models in which defective clearance of apoptotic cells results in the accumulation of extracellular nucleic acids (24). tlrs might have a key role in the detection of these extracellular self - nucleic acids (25), particularly chromatin and rna protein complexes (2630), whereas the cytosolic rna and dna sensors might be responsible for a distinct set of autoimmune disorders that may only somtimes be associated with the failed clearance of apoptotic cells (19). breeding ips-1deficient mice with autoimmune - prone mouse strains would be a simple way to begin to address this hypothesis. the observation that ips-1deficient mice are highly susceptible to infection with vsv despite generating a normal systemic type i ifn response (18) is reminiscent of the phenotype of fadd - deficient fibroblasts, which are unable to control vsv replication even after pretreatment with type i ifns (6). it is possible that local concentrations of type i ifns are too low to control viral replication in peripheral tissues in the absence of ips-1. but there is another possible explanation that would suggest a role for ips-1 signaling beyond the induction of type i ifns : detection of cytosolic nucleic acids might trigger cell - intrinsic apoptosis and/or flag the infected cells but not their uninfected neighbors for elimination by nk cells and ctls. one of the key functions of type i ifns is to provide a paracrine alarm signal and induce an antiviral state in uninfected neighboring cells. for example, rig - i and mda5 are induced by type i ifns, which then increase the sensitivity of nucleic acid detection in neighboring cells. another key role of type i ifns is to enhance the cytotoxic functions of nk cells and cd8 t cells, which are important for inducing non cell - autonomous apoptosis in infected cells. the recognition and elimination of infected cells must be carefully regulated such that only the infected cells and not their immediate neighbors are targeted. the ips-1dependent production of type i ifns can not supply the dominant kill signal for cytotoxic cells ; this signal must be cell bound and it must be unique to infected cells (fig. 1). because rig - i and mda5 (and the currently unidentified dna sensor(s)) detect viral nucleic acids within infected cells, they are perfectly suited to induce surface expression of the ligands that uniquely flag infected cells for elimination. (a) in normal cells infected with an rna virus, ips-1dependent signaling activates type i ifns, which turn on ifn - inducible genes in both infected cells and neighboring, uninfected cells. ips-1 might also control cell - intrinsic apoptosis and the induction of ligands for nk cells and ctls, but only in infected cells. (b) in ips-1deficient cells, all four functions depicted in part a are lost and antiviral defenses are fully compromised. (c) treatment of ips-1deficient cells with type i ifns (either tlr - activated systemic ifns or exogenous type i ifns) only restores the ips-1independent response mediated by ifn - inducible genes. what might these ligands be ? promising candidates are ligands for activating and inhibitory receptors that are expressed by nk cells and cytotoxic cd8 t cells (31). coordinated down - regulation of inhibitory ligands and up - regulation of activating ligands can determine the susceptibility of target cells to nk cell mediated killing (31), but the mechanisms and signaling pathways responsible for altering the expression of these ligands remain largely unknown. these mechanisms must be cell autonomous, however, and could conceivably be controlled by ips-1dependent signals in infected cells. one activating receptor expressed by nk cells and some effector cd8 t cells is nkg2d, which recognizes cell surface ligands induced by various stresses, including viral infection (32). one prediction of this model is that ips-1deficient mice would initiate adaptive immune responses normally after infection with rna viruses but that effector cd8 t cells and nk cells would be unable to kill infected cells. another clinically relevant prediction is that treatment of virus - infected individuals with recombinant type i ifns would only restore paracrine ips-1dependent immune responses and not the cell - autonomous effector functions downstream of ips-1 (fig. 1). for viruses such as hcv, which encodes the ips-1cleaving protease ns3/4a, pharmacological inhibitors that block ns3/4a would restore all functions of ips-1 and would thus be far more effective than treatment with type i ifns alone (33). ips-1 might also control induction of cell - intrinsic apoptosis in an ifn - independent manner (fig. because ips-1 is a transmembrane protein localized to mitochondria, it would be interesting to see if it interacts with or alters the function of the bcl2 family of proteins, which control mitochondria - dependent apoptosis. however, infection with viruses other than vsv would be required to firmly establish this, as vsv - infected cells can be killed by the strong cytopathic effect of this virus, especially in the absence of type i ifn - induced signaling. importantly, anchoring ips-1 to other membranes in the cell impairs signaling, suggesting that mitochondrial localization, and not membrane attachment itself, is essential for its function (9). this observation, coupled with the fact that ips-1 is not ifn inducible and is strongly haploinsufficient in vivo, implies that ips-1 is coordinately regulated with the numbers of mitochondria and might be most abundant (and most sensitive to nucleic acid detection) in cells with the highest metabolic activity. the description of ips-1deficient mice marks an important milestone in the rapidly advancing field of nucleic acid recognition. in just two years, the nonredundant cytosolic rna sensors and several of the key signaling molecules that link rna recognition to the antiviral response have been identified and characterized in vitro and in knockout mice. along the way, we have realized that an entirely separate system exists for the detection of intracellular dna that is likely to be important for host responses to dna viruses and intracellular bacteria. together with the current knowledge of tlr - mediated nucleic acid recognition, we are now in a position to dissect the relative contribution of all of these systems to immunity and immunopathology.	mice lacking the adaptor protein that initiates an antiviral response downstream of the rna helicases retinoic acid inducible gene i (rig - i) and melanoma differentiation - associated gene 5 (mda5) have recently been described. these studies highlight the essential and nonredundant role of nucleic acid recognition in the induction of type i interferon production and raise important questions regarding the nature of cell - autonomous virus detection in coordinating the antiviral response.
obesity is one of the major predictors for heart disease and other chronic diseases including hyperlipidemia, hypertension, atherosclerosis and hyperinsulinemia (1). the principal causes of the increasing obesity problem have known to be sedentary lifestyles and a high - fat and energy rich diet. body mass index (bmi) is widely used in the adult population to define overweight and obesity although it is a less ideal method for measuring body fat. the cut off points of 25 kg / m and 30 kg / m have been recognized internationally as definitions of adult overweight and obesity, respectively (3). however, recent studies have shown that asians have a lower bmi but a higher percentage of body fat than caucasians at the same bmi (4, 5). deurenberg. also suggested that the relationship between bmi and body fat percentage differs between ethnic groups (6). therefore, increased health risks associated with obesity appear to occur at a lower bmi in asians. several asian countries and the world health organization (who) expert panel proposed different cut - off values as overweight (bmi 23 - 24.9 kg / m) and obesity (bmi 25 kg / m) based on the morbidity of their own population (7 - 10). cross - sectional and longitudinal studies suggest an increase in body mass and adiposity with aging despite decreases in energy intake or considerable physical activity especially until the ages of 65 - 70 yr although the patterns seem to differ by gender (11 - 13). those studies reported that men had a progressive body mass gain from early adulthood to late middle age while women had a continuous increase from early adulthood to postmenopause. nationally representative cross - sectional data showed that the prevalence of obesity by bmi in men increased with aging until 35 - 45 yr of age, followed by plateau in japan, but a continuous increase in united states until 65 - 75 yr of age (14, 15). on the contrary, the bmi started to decrease after 65 - 75 yr of age in both men and women in those countries. the economic growth in the republic of korea increased markedly over the past several decades. at the same time, western lifestyles including their diet has been introduced rapidly into the country. in addition, industrialization and growth of the technology sector have led to a decrease in physical activity. as those lifestyles change, obesity has also increasingly become a social problem in korea (16, 17). a study from the indian population reported the prevalence of obesity by residential difference, which may reflect the degree of industrialization in a country, and suggested that those urban - rural differences would be due to the differences in lifestyle factors including physical activity and nutrition by residents (18). however, few studies focused on the distribution of bmi and trends of related health behaviors, especially based on the effect of gender, aging and residential areas in current representative korean population although certain transition must exist cross - sectionally. the purpose of this study was to discuss the distribution of bmi and obesity related health behaviors by gender, age and residence in korea using the data obtained from the 1998 national health and nutrition survey gathered by ministry of health and welfare of republic of korea and korea institute for health and social affairs in november 1998. data from the 1998 korean national health and nutrition survey were used in the study. the survey employed a stratified multistage probability sampling design. a total number of 12,283 korean households with 39,060 household members participated in the health interview survey. one out of three samples from the health interview survey were selected to conduct a health examination and nutrition survey (19). anthropometric data were available for 10, 880 participants aged 10 - 94 yr. among those, adults aged 20 - 64 yr were selected to examine the differences in the trends of obesity and related health behaviors. subjects over the age of 65 yr were excluded from this analysis because bmi started decreasing after 65 yr of age in korean women. height and weight were measured based on standard procedures and bmi was calculated by dividing the weight in kilograms by the square of the height in meters. dietary intake was assessed by the single 24-hr dietary recall method. experienced interviewers instructed respondents to recall and describe the foods and beverages consumed over the previous 24 hr. energy, protein, fat and carbohydrate intake were calculated and reported, and they were also expressed as a percentage of total energy. adults aged 20 - 64 yr were further divided into three groups using a 15-yr age interval, 20 - 34, 35 - 49 and 50 - 64 yr. residential areas were divided into three categories as big cities, small cities and rural areas. the average bmi and energy, protein, fat and carbohydrate intakes were calculated and compared by age and residential areas in each gender. after the distribution of bmi was analyzed by original who standards for western countries and recent suggestions for asians by who expert panel (bmi30 as obesity for westerners, 30>bmi25 as overweight for westerners or obesity for asians, 25>bmi23 as overweight for asians, 23>bmi18.5 as normal weight for asians, bmi 20% of energy from fat and 20% of energy from fat. self - reported alcohol consumption, smoking, exercise, the degree of usual daily activity and weight reduction information were obtained from the questionnaire and the proportions were compared by age and region as well. alcohol consumption was classified as heavy (heavy drinking once or more than once per month) and non - heavy drinkers (drinking less than once per month or none) from the question " how often do you drink heavily ? ". smoking status was classified by the participants ' self - report as current versus former or nonsmokers. exercise was classified as regular (at least 20 min at a time, 3 or more times per week) and non - regular (the rest of them). the degree of daily activity was classified as intense versus nonintense from the question " how intensely can you rate your daily activity ? " information on weight reduction status was classified into the current weight reduction group when the answer was weight reduction from a self - reported question " which weight control do you try ? " all statistics were calculated using sudaan (version 9.0) to consider the sample design (20). sudaan was used to increase the accuracy and validity of results through computing variance estimates and test statistics for a stratified, multistage probability survey design. sample weights were applied to all analyses to account for the unequal probability of selection, non - coverage and nonresponse bias from sampling. mean and standard errors, which represent variability of the estimates, were reported for bmi, energy, protein, fat and carbohydrate intakes. two way anovas were performed on bmi and health related behaviors to investigate the main effect of age and residential areas and their interaction, separating men and women. contrasts by bonfferoni were used to examine the mean differences by age in each residential area and residential area in each age group. general linear model and its contrast by bonfferoni were used to test the significant difference in the prevalence of bmi and proportion of health behaviors by age and residential areas in each gender. all statistics were calculated using sudaan (version 9.0) to consider the sample design (20). sudaan was used to increase the accuracy and validity of results through computing variance estimates and test statistics for a stratified, multistage probability survey design. sample weights were applied to all analyses to account for the unequal probability of selection, non - coverage and nonresponse bias from sampling. mean and standard errors, which represent variability of the estimates, were reported for bmi, energy, protein, fat and carbohydrate intakes. two way anovas were performed on bmi and health related behaviors to investigate the main effect of age and residential areas and their interaction, separating men and women. contrasts by bonfferoni were used to examine the mean differences by age in each residential area and residential area in each age group. general linear model and its contrast by bonfferoni were used to test the significant difference in the prevalence of bmi and proportion of health behaviors by age and residential areas in each gender. a total of 2,583 men and 3,087 women (20 - 64 yr of age) were included in this analysis. the distribution of subjects by age categories and residential areas is presented for korean men and women in table 1. table 2 and 3 show the mean and standard error of bmi and energy, protein, fat and carbohydrate intakes by age and residential areas in korean men and women. the means of bmi (kg / m) by age groups from three residential areas were 22.6 - 23.9 for men and 21.7 - 24.8 for women. the average bmi for men was slightly higher than that for women in most age groups, except for those between the ages of 50 - 64 yr. mean energy intake ranged from 2,079 to 2,554 kcal for men and 1,679 - 1,938 kcal for women. the average percentages of energy from fat intake were 12.5 - 20.3% and 10.9 - 19.8% for men and women, respectively. overall significant age differences were found in bmi, energy and macronutrient intakes in korean men and women (p<0.05) except carbohydrate intake and the percentage of energy from protein for women. residential differences were observed by higher fat intake and higher percentage of energy from fat and lower percentage from carbohydrate in urban areas (both big and small cities) compared with rural areas among the entire men. urban women showed higher bmi and fat intake and higher percentage of energy from protein and fat and lower percentage from carbohydrate. different patterns of age - specific mean bmi by residential areas existed, presenting a significant interaction of age and residential areas for both men and women (p=0.0025 and p<0.0001, respectively). in the comparison of the mean levels of bmi and intakes by residential areas in each age group, the bmi in men aged 20 - 49 yr did not show any difference by residential areas. men aged 50 - 64 yr in urban areas had a higher bmi than men in the same age group in rural areas. urban women in the 20 - 34 and 35 - 49 yr age group showed a lower bmi than that for rural women, on the contrary, urban women aged 50 - 64 yr showed a higher bmi. in big cities, men aged 35 - 49 and 50 - 64 yr showed a higher bmi than men aged 20 - 34 yr. whereas, in small cities and rural areas, bmi of men aged 50 - 64 yr did not show significant differences from the bmi of men aged 20 - 34 yr. in women, bmi was the highest for those aged 50 - 64 yr in urban area, however, bmi in those aged 35 - 49 yr was higher than those aged 50 - 64 yr in rural area. the older population consumed lower energy in each residential area for both men and women. people aged 50 - 64 yr had lower protein intakes than those aged 20 - 49 yr in small cities and rural areas, but those in big cities did not have significantly different protein intakes by age. carbohydrate intakes were not significantly different according to different age and residential areas except for men living in rural areas. the percentage of energy from fat tended to be higher in younger people and those living in big cities. younger people aged 20 - 34 yr, did not show any difference in the energy percentage from fat by residential areas in the current korean population. the age - specific prevalence of overweight and obesity across various age categories is presented in fig. 1. according to the standard for asians suggested by who expert panel (obesity25), 22.5% and 15.1% were obese in korean men and women aged 20 - 34 yr, respectively. the highest prevalence was found in the 30 - 49 yr for men and 50 - 64 yr for women. in the 50 - 64 yr age group, women showed a much higher prevalence than men (28.6% vs. 41.2%). the prevalence of subjects with bmi23 (asian criteria for overweight and obesity) was 61.4% in men aged 35 - 49 yr and 67.8% in women aged 50 - 64 yr, whereas 9.8% (bmi<18.5) of women aged 20 - 34 yr were underweight. 2, the results revealed that obesity is widely prevalent in the older urban population in both men and women except a higher prevalence in younger rural women compared with younger urban women. the prevalence of obesity was significantly different by age (age effect p=0.0001 for men and p<0.0001 for women by general linear model) and the age - specific prevalence trends of obesity were significantly different by residential areas in both men and women (interaction effect, p=0.0023 for men and p=0.0001 for women). among men, the highest prevalence was observed in the 50 - 64 yr of age in big cities, but in the 35 - 49 yr of age in small cities and rural areas. however, women showed opposite patterns, the highest prevalence at 50 - 64 yr in urban areas and at 35 - 49 yr in rural areas. no residential area difference on prevalence was found in the 20 - 34 and 35 - 49 yr of age in men, but a significant difference was observed in the 50 - 64 yr of age (35, 29 and 17% in big city, small city and rural area, respectively). as with women, the 20 - 34 and 35 - 49 yr age group showed a higher prevalence in rural areas than in urban areas. age - specific health behaviors by residential areas were only significantly different in exercising, daily activity and weight reduction for men and women (fig. there was a higher percentage of regular exercising in urban areas than in rural areas in men at the ages of 50 - 64 yr and in women at 35 - 64 yr. the proportion of people who self - rated the degree of their activity as intense was the highest in older rural men and women, but the 20 - 34 yr age group did not show any residential difference. almost half of the women in the 20 - 34 yr of age group tried to lose weight regardless of the residential area. residential difference in weight reduction existed only in the 50 - 64 yr age group in men and women. smoking and alcohol drinking only showed a significant age difference, but not residential difference in any age group. the population rate of eating over 20% energy from fat was significantly lower in the older age group in both men and women. adults aged 20 - 34 yr did not show any significant difference in fat intake by residential area, but the 50 - 64 yr age group showed a lower percentage in rural areas compared to urban areas. a total of 2,583 men and 3,087 women (20 - 64 yr of age) were included in this analysis. the distribution of subjects by age categories and residential areas is presented for korean men and women in table 1. table 2 and 3 show the mean and standard error of bmi and energy, protein, fat and carbohydrate intakes by age and residential areas in korean men and women. the means of bmi (kg / m) by age groups from three residential areas were 22.6 - 23.9 for men and 21.7 - 24.8 for women. the average bmi for men was slightly higher than that for women in most age groups, except for those between the ages of 50 - 64 yr. mean energy intake ranged from 2,079 to 2,554 kcal for men and 1,679 - 1,938 kcal for women. the average percentages of energy from fat intake were 12.5 - 20.3% and 10.9 - 19.8% for men and women, respectively. overall significant age differences were found in bmi, energy and macronutrient intakes in korean men and women (p<0.05) except carbohydrate intake and the percentage of energy from protein for women. residential differences were observed by higher fat intake and higher percentage of energy from fat and lower percentage from carbohydrate in urban areas (both big and small cities) compared with rural areas among the entire men. urban women showed higher bmi and fat intake and higher percentage of energy from protein and fat and lower percentage from carbohydrate. different patterns of age - specific mean bmi by residential areas existed, presenting a significant interaction of age and residential areas for both men and women (p=0.0025 and p<0.0001, respectively). in the comparison of the mean levels of bmi and intakes by residential areas in each age group, the bmi in men aged 20 - 49 yr did not show any difference by residential areas. men aged 50 - 64 yr in urban areas had a higher bmi than men in the same age group in rural areas. urban women in the 20 - 34 and 35 - 49 yr age group showed a lower bmi than that for rural women, on the contrary, urban women aged 50 - 64 yr showed a higher bmi. in big cities, men aged 35 - 49 and 50 - 64 yr showed a higher bmi than men aged 20 - 34 yr. whereas, in small cities and rural areas, bmi of men aged 50 - 64 yr did not show significant differences from the bmi of men aged 20 - 34 yr. in women, bmi was the highest for those aged 50 - 64 yr in urban area, however, bmi in those aged 35 - 49 yr was higher than those aged 50 - 64 yr in rural area. the older population consumed lower energy in each residential area for both men and women. people aged 50 - 64 yr had lower protein intakes than those aged 20 - 49 yr in small cities and rural areas, but those in big cities did not have significantly different protein intakes by age. carbohydrate intakes were not significantly different according to different age and residential areas except for men living in rural areas. the percentage of energy from fat tended to be higher in younger people and those living in big cities. younger people aged 20 - 34 yr, did not show any difference in the energy percentage from fat by residential areas in the current korean population. the age - specific prevalence of overweight and obesity across various age categories is presented in fig. 1. according to the standard for asians suggested by who expert panel (obesity25), 22.5% and 15.1% were obese in korean men and women aged 20 - 34 yr, respectively. the highest prevalence was found in the 30 - 49 yr for men and 50 - 64 yr for women. in the 50 - 64 yr age group, women showed a much higher prevalence than men (28.6% vs. 41.2%). the prevalence of subjects with bmi23 (asian criteria for overweight and obesity) was 61.4% in men aged 35 - 49 yr and 67.8% in women aged 50 - 64 yr, whereas 9.8% (bmi<18.5) of women aged 20 - 34 yr were underweight. 2, the results revealed that obesity is widely prevalent in the older urban population in both men and women except a higher prevalence in younger rural women compared with younger urban women. the prevalence of obesity was significantly different by age (age effect p=0.0001 for men and p<0.0001 for women by general linear model) and the age - specific prevalence trends of obesity were significantly different by residential areas in both men and women (interaction effect, p=0.0023 for men and p=0.0001 for women). among men, the highest prevalence was observed in the 50 - 64 yr of age in big cities, but in the 35 - 49 yr of age in small cities and rural areas. however, women showed opposite patterns, the highest prevalence at 50 - 64 yr in urban areas and at 35 - 49 yr in rural areas. no residential area difference on prevalence was found in the 20 - 34 and 35 - 49 yr of age in men, but a significant difference was observed in the 50 - 64 yr of age (35, 29 and 17% in big city, small city and rural area, respectively). as with women, the 20 - 34 and 35 - 49 yr age group showed a higher prevalence in rural areas than in urban areas. age - specific health behaviors by residential areas were only significantly different in exercising, daily activity and weight reduction for men and women (fig. 3, 4). there was a higher percentage of regular exercising in urban areas than in rural areas in men at the ages of 50 - 64 yr and in women at 35 - 64 yr. the proportion of people who self - rated the degree of their activity as intense was the highest in older rural men and women, but the 20 - 34 yr age group did not show any residential difference. almost half of the women in the 20 - 34 yr of age group tried to lose weight regardless of the residential area. residential difference in weight reduction existed only in the 50 - 64 yr age group in men and women. smoking and alcohol drinking only showed a significant age difference, but not residential difference in any age group. the population rate of eating over 20% energy from fat was significantly lower in the older age group in both men and women. adults aged 20 - 34 yr did not show any significant difference in fat intake by residential area, but the 50 - 64 yr age group showed a lower percentage in rural areas compared to urban areas. the predominant finding of this study was that the prevalence of obesity (bmi 25) was significantly different by age and residential areas in korean men and women. aging seemed to be a significant contributor in the increase of obesity for both korean men and women despite the gender difference pattern. this increasing trend by aging in a cross - sectional study was a common phenomenon in other countries as well (9, 21, 22). the 1999 - 2000 national health and nutrition examination survey of the u.s.a. showed a continuously increasing prevalence of obesity by aging for men. the 1998 nutrition survey in japan showed that the prevalence of obesity in men increased to 35 - 45 yr of age, followed by plateau. on the contrary, when the effects of residential areas were considered for obesity trends, these aging effects were different by residential areas in korea. although younger people aged 20 - 49 yr did not show a prevalence difference of obesity by residential areas, people at the ages of 50 - 64 yr living in urban areas, which are more industrialized, showed the highest prevalence of obesity for men. in rural areas, which usually keep a more traditional lifestyle, men aged 50 - 64 however, in women, these urban - rural effects were applied differently in each age group. in korean women, a higher prevalence was found in rural areas compared to urban areas in the younger age group (20 - 49 yr), but not in the older age group. the urban - rural differences of obesity prevalence in this study demonstrate the various stages of behavioral transition that korea is currently undergoing, which may be explained by lifestyle and certain social factors. urban areas had significantly higher rates of people who ate more than 20% energy from fat than rural areas did, especially in older population for both men and women, although age had a greater effect on fat intake than residential areas did. among lifestyle factors, self - rated usual activity as intense showed the biggest urban - rural differences, which may explain the lower prevalence of obesity in rural older population. the higher exercise rate in urban areas might show that koreans in urban areas did exercise more in their leisure time than those in rural areas, while non - leisure daily activity level were higher in rural areas. we believe that the industrialized lifestyles including high fat intakes and low usual intense activity led to a high prevalence of obesity, whereas, this higher obesity status combined with higher health concerns got older urban people more involved in exercise. higher prevalence of obesity in younger rural korean women was not explained by lifestyle factors that were analyzed in this study. we think this may reflect one industrialized trend, having a strong desire to be " thinner " due to social pressure, hence, young urban women showed a lower prevalence of obesity than rural women did (23). however, since we only had information about the rate of weight reduction, and not on the degree of weight control or desire in this study, further research is needed to be conducted. longitudinal trend analysis on bmi and lifestyle factors in japan by yoshiike. from 1976 - 2000 also reported similar obesity trends by residential areas to those of koreans. they showed a very prominent increasing trend in overweight among males in rural populations and a significant decrease in overweight among females in the metropolitan areas in japan (14). although the overall prevalence of obesity (bmi25) in the korean population is quite low compared with western societies, it reached around one - fourth of korean adults, which is higher than the prevalence in other asian countries such as china, the philippines and japan (2, 14). a higher prevalence in korea than even in japan, a developed country, was a surprising result, which should be taken into consideration for obesity control at national levels. the strength of this study was that it analyzed a national representative sample obtained from the nationwide health examination survey in korea (1998). external validity is therefore sufficient to interpret the findings for the general population of korea. our study provided variable information about how the distribution of obesity would be characterized in korea, connected with other studies using this data mostly providing a significant relationship between obesity and metabolic disorders (24 - 26). a major limitation of this study would be its cross - sectional nature, suggesting that it does not provide sufficient evidence of causality. other limitations include the recall bias of the single 24-hr recall method although we used the information for only estimating population means. the reliance on self - reporting answers with undefined self - perceived degree on questions such as " heavy " for alcohol drink and " intense " for usual activity could be also the limitation to interpret our results. in conclusion, our study revealed that different age - specific trends of obesity coexists depending on residential areas in korea, especially more in older generation. these urban - rural differences on obesity might reflect the difference of other health related behaviors such as lifestyle traditions, in which rural residents necessarily impose a more active and traditional lifestyle.	the objective of this study was to discuss the residential difference in gender and age specific prevalence of obesity by body mass index (bmi) and obesity related health behaviors in the republic of korea. a total of nationally representative 2,583 men and 3,087 women (age : 20 - 64 yr) was used as subjects from 1998 national health and nutrition survey. all statistics were calculated using sudaan to consider a stratified multistage probability sampling design. the prevalence of obesity (bmi25) was significantly different by age, gender and residential areas. although younger men aged 20 - 49 yr did not show a residential difference in the prevalence of obesity, men aged 50 - 64 yr showed differences, highest in big cities and lowest in rural areas. however, in women, a higher prevalence was observed in rural areas compared to urban areas in the younger age group (20 - 49 yr), but not in the older age group. residential differences of obesity related health behaviors existed mostly in the older population, but not in the younger population. the urban - rural differences demonstrate the various stages of behavioral transition that korea is currently undergoing. therefore, different strategies considering those factors are needed to manage obesity problems in korea.
the public - private mix (ppm) approach is an effective way to improve detection rates of the directly observed treatment, short course (dots) approach with comparative treatment success rate in tuberculosis.1,2 however, the ppm approach has a lower rate of success than expected in some situations.3 for the ppm strategy to be successful, it is important to find ways to improve the adherence of patients in clinics, especially those patients of low socioeconomic status (ses). however, few studies have investigated the impact of education on adherence, and existing findings are inconsistent.4 - 7 we evaluated the impact of physician - provided education on adherence to treatment in patients of low ses in a primary health care clinic. the control group consisted of tuberculosis patients treated from march 2005 to april 2006, and the intervention group consisted of patients treated from may 2006 to december 2007 at bangladesh - korea friendship hospital. the facility is a primary health care clinic located in savar, a suburban industrial complex area adjacent to dhaka, the capital of bangladesh. we followed the standard short course treatment regimen recommended by the world health organization and used a fixed - dose formula provided free of charge by the national tuberculosis control program in bangladesh. this study was approved by the institutional review board of daegu fatima hospital (no. dfh10ot088). from may 2006, we changed the irregular visiting schedules of the patients into regular ones as follows : every week during the initial month of treatment, every other week during the next month, and then every month until the end of treatment. we scheduled the next visit 3 days before drug exhaustion and directed each patient to bring the remaining pills at every visit. the contents of our education included the high incidence and mortality rates of tuberculosis in bangladesh, how to take anti - tuberculosis drugs and their major side effects, and the consequences of nonadherence and of early discontinuation of medication because of symptom relief (e.g., treatment failure, emerge of drug resistance, death). to confirm patients ' full understanding, we asked the following questions : what could happen when you stop taking the medicine ? what could happen if you do n't take the medicine regularly ? adherence was defined as patient 's attendance at the scheduled visit and regular medication with over 90% of doses prescribed. categorical and continuous variables were compared by using the pearson chi - square test and student 's t - test, respectively, to test for homogeneity between the intervention and control groups. the kaplan - meier method and cox 's proportional hazards model were used to evaluate the relationship between adherence to medication and education in the two groups. multivariate analyses were performed by using the cox - regression hazard model in the backward stepwise conditional manner. statistical analyses of the data were performed with spss for windows (version 19.0 ; spss inc., chicago, il, usa). a total of 354 patients participated : 198 patients (56%) were treated in the pre - intervention period, and 156 patients (44%) were treated in the post - intervention period. the patients ' mean (sd) age was 35 (15) years in the pre - intervention group and 35 (16) years in the post - intervention group (p=0.90). mean body weight (sd) was 42 (9) kg in the pre - intervention group and 42 (7) kg in the post - intervention group (p=0.46). a total of 126 (64%) patients in the pre - intervention group and 85 (55%) in the post - intervention group were male (p=0.08). of these patients, 145 (73%) in the pre - intervention group and 118 (76%) in the post - intervention group lived within 30 minutes of the clinic by three - wheeler rickshaw (p=0.43). two hundred seventy - two patients (77%) had pulmonary tuberculosis, and 142 (52%) of them were smear - positive for acid - fast bacilli (afb) on sputum examination. extrapulmonary tuberculosis was significantly more common in the control group (56/198, 28%) than in the intervention group (26/156, 17% ; p=0.010). however, other characteristics did not differ significantly between the two groups (p>0.05, table 1). the proportion of patients who remained adherent at day 168 and the cumulative adherence to anti - tuberculosis medication was significantly greater in the intervention group than in the control group (table 1, fig. 1). the multivariate analysis included the following variables : age, male sex, afb - negative, intervention group (p<0.10 in the univariate analysis), and tuberculosis type (which was significantly different between the intervention group and the control group). independent risk factors for nonadherence were pre - intervention group, age, and afb - negative in the cox - regression multivariate analysis (p<0.05, table 2). subjects in the intervention group were more likely than those in the control group to continue taking medication in the multivariate cox 's proportional hazards model (table 2). because the prevalence of pulmonary and extra - pulmonary tuberculosis differed significantly between the groups, we stratified the analyses by tuberculosis type. cumulative adherence in the intervention group was higher than in the control group among both pulmonary and extra - pulmonary tuberculosis patients (kaplan - meier method and log - rank test ; chi - square 8.4 and 11.3, p<0.05 each). bangladesh, which ranks sixth in the world in terms of tuberculosis burden, first implemented a tuberculosis program with a dots strategy in 1984. treatment success rates reached 92% in 2007.8 however, detection rates are still only 66%,8 and many tuberculosis patients are still treated in clinics. we treated suburban tuberculosis patients in a clinic ; however, treatment success rates were not satisfactory. because of poorly available resources, to improve adherence, we provided patients with direct education from physicians. for example, knowledge about the disease, the duration of treatment, and schedule of follow - up are associated with adherence to anti - tuberculosis medication.9 distance and time to health facilities, male gender (male patients are usually the breadwinners of their families), and stigma that tuberculosis is incurable are also major constraints to treatment adherence, especially in developing countries.10,11 consistent with the previous studies, compliance was also affected by multiple factors in our study. thiam.6 reported that a strategy including reinforced counseling between health personnel and patients improved adherence to the anti - tuberculosis medications. however, a study conducted in pakistan showed that intensive counseling had a limited impact on adherence.4 morisky.5 also reported that there was no significant improvement in treatment adherence in the intervention group even though intensive education and a monetary incentive were provided to the patients. in our study, education was effective even though most patients were of low ses, most were poorly educated, half were completely uneducated and illiterate, and defaulter tracing was not applicable. in our opinion, the success of our intervention, in contrast with other previous studies that failed, was possibly due to the qualified health care staff and their desire to improve treatment outcomes. in a recent study, hane.12 found that health care staff can play a key role in promoting adherence to treatment, which justifies the training component of the strategy tested and the need for improved counseling. a major limitation of our study on physician - provided patient education was that defaulter tracing could not be applied. second, we started to educate the patients and changed the irregular visiting schedules of the patients into regular ones in a bundle of patients at the same time. for this reason, it is possible that not only the education but also the change of schedule contributed to the improved adherence of the educated patients. third, this study was not designed as randomized controlled trial but was designed as a before and after study. in conclusion, our data demonstrate that direct education from physicians can improve the adherence of patients to tuberculosis treatment even when the patients are poorly educated and of low ses. physician 's education is also important and can contribute to increasing the adherence of patients in resource - limited settings, as can the education of other health care workers.	successful tuberculosis control depends on good adherence to treatment. yet, limited data are available on the efficacy of methods for improving the adherence of patients of low socioeconomic status. we evaluated the impact of physician - provided patient education on adherence to anti - tuberculosis medication in a low socioeconomic status and resource - limited setting. a pre-/post - intervention study was conducted at a suburban primary health care clinic in bangladesh where an intensive education strategy was established in may 2006. treatment outcomes of tuberculosis patients from march 2005 to april 2006 (pre - intervention) and from may 2006 to december 2007 (post - intervention) were compared. among 354 patients, 198 (56%) were treated before intervention and 156 (44%) were treated after intervention. cumulative adherence to anti - tuberculosis medication was significantly greater in the intervention group than in the control group in univariate and multivariate analyses. physician 's education can contribute to increasing the adherence of patients in resource - limited settings.
they represents about 2% of all soft tissue sarcoma, which can often metastasize through the hematogenous route. the radiological features have been analyzed in 4 patients with metastatic angiosarcoma in the chest. the main radiologic findings included nodules, cysts, nodules with halo sign, and vascular tree - in - bud. morphologic features, as observed in the histologic specimen, have been correlated with radiologic appearance. metastatic angiosarcomas to the lung are characterized by a wide variety of radiologic appearances that can be very characteristic. computed tomographic findings observed include bilateral solid nodules, cystic, and bullous lesions sometimes associated with spontaneous hemopneumothoraces. angiosarcomas are rare, malignant vascular tumors, representing about 2% of all soft tissue sarcomas. the most frequently primarily affected sites include the heart, liver, breast, skin, and scalp, and they have a high rate of metastases to the lungs and, less commonly, liver, regional lymph nodes, and bone. primary extrapulmonary angiosarcomas originating in the chest include those in the heart, aorta, or great vessels and, because of their rarity, are poorly characterized. they have a clinical presentation that can include dyspnea, chest pain, or syncope. their radiological findings are characterized by filling defects occupying the lumen of great vessels, or thickening of one of the heart chambers. pulmonary artery sarcomas may include an angiosarcomatous component, but often they show patterns of other soft tissue sarcomas and must also be distinguished from chronic pulmonary embolism (pe). the extravascular spread / invasion of the lesion and the heterogeneous enhancement of contrast medium in the mass occupying the lumen typically characterize this lesion. pulmonary artery sarcoma can also be suspected in patients undergoing a computed tomography (ct) angiography with a low to intermediate probability of pe. on the contrary, metastatic angiosarcoma to the lung ct findings can include bilateral solid nodules or cystic and bullous lesions sometimes associated with spontaneous hemopneumothoraces. tateishi also described cystic lesions with air fluid level related to recent hemorrhage. in addition, they reported areas of ground glass attenuation related to areas of focal hemorrhage and nodules with miliary distribution. a distinctive feature in some metastatic angiosarcomas is a halo of ground glass change around nodules representing perinodular hemorrhage into alveoli, the so - called halo sign. most published series of metastatic angiosarcoma to the lung have detailed either the radiologic or pathologic changes. the aim of this report is to review the radiological - pathological correlation based on review of the literature and on 4 additional cases of angiosarcoma in the chest that we describe. the clinical findings of the 4 cases are summarized in table 1 and the radiologic features are summarized in table 2. radiological features of pulmonary lesions. an 84-year - old male (patient 1), former smoker, with a prior history of gastric and prostatic cancer, both surgically resected, presented with a recent episode of hemoptysis. ct scan of the chest without contrast showed numerous tiny solid pulmonary nodules, measuring 2 to 5 mm in size, most of them in the periphery of the lung, forming a vascular tree - in - bud pattern. furthermore, some slightly bigger lesions (measuring 58 mm) surrounded by ground glass (halo sign) were also visible, mainly in the left upper lobe. a mild smooth thickening of interlobular septa was also present in both lower lobes (fig. ct scan shows numerous bilateral tiny nodules mainly in the middle and lower zones of hemithoraces. many of these nodules show a vascular tree - in - bud pattern related to neoplastic thrombotic microangiopathy (red circle). moreover vascular branches are focally enlarged. bone window settings showed an osteolytic lesion of the seventh left rib, with a complete erosion of cortical profile. differential diagnosis of these findings included a neoplastic thrombotic microangiopathy associated with one of his prior carcinomas, particularly gastric cancer. the bone lesion was not sclerotic (as would be typical for metastatic prostate carcinoma). the patient underwent pet - ct scan that showed 3 sites of uptake highly suggestive of neoplastic lesions located in the bone (seventh left rib and sacrum) and in the wall of right atrium. however, the numerous pulmonary lesions did not show any uptake, possibly because of their small size. the transoesophageal ultrasound confirmed the presence of a posterolateral thickening of the right atrial wall, with a homogeneous density and irregular margins, consistent with a neoplastic process. the patient underwent broncho - alveolar lavage (bal) that showed evidence of chronic hemorrhage. subsequently, a transbronchial cryobiopsy was performed in the lateral segment of left lower lobe, confirming the presence of multiple alveolar hemosiderin laden macrophages in airspaces and a mild lymphocytic infiltrate. the patient underwent ct - guided biopsy of sacrum that confirmed presence of angiosarcoma showing vasoformative regions and confirmed as endothelial in origin by immunohistochemistry (cd31) (fig. dissecting vascular channels lined by endothelial cells showing various degrees of cytologic pleomorphism and nuclear atypia. a 55-year - old male (patient 2) underwent resection of an enlarging mass of the right foot. the specimen showed an epithelioid neoplasm with positivity for cd31 and a focal positivity for cytokeratin ae1/ae3. after a minor trauma, 1 month later, the patient presented to the emergency department because of an intense chest pain on the right side. chest x - rays documented a large right pleural effusion, and no rib fractures were present (fig. 3). thoracentesis showed a hemorrhagic pleural effusion with reactive mesothelial cells seen cytologically. ct scan with contrast showed 2 adjacent parietal pleural lesions measuring 75 and 15 mm in greatest dimension, respectively (fig. these lesions had smooth margins and did not cause erosion on the ribs, whereas they completely obliterated the extrapleural space. in addition, moderate diffuse thickening of the pleura was visible in the whole right hemithorax. multiple sites of pathological uptake were then confirmed with pet - ct in the right hemithorax suggesting multiple secondary pleural metastases. presence of 2 contiguous opacities in the middle zone of right hemithorax, both subpleural and with an obtuse angle with the pleural margin, suggesting the feature of pleural lesions. ct scan confirms the presence of 2 pleural lesions, the biggest one measures 2.6 3.5 cm and it is in the right middle lobe. (a) atypical, multilyering pleomorphic cells often forming tuft small papillae (h&e, midpower). he also underwent several cycles of chemotherapy first with taxol and, afterwards, ifosfamide and epirubicin. two years later, he developed osteolytic secondary lesions in the cervical spine at the level of c3 and c4 with invasion of the spinal canal for which he underwent radiotherapy and surgical stabilization. one year later, 3 years after initial diagnosis, the patient showed multiple new bilateral pulmonary nodules with halo sign, measuring 8 to 15 mm in size consistent with metastases (fig. 6). two months later, he developed a severe worsening of respiratory symptoms and repeated ct angiography of the chest to exclude pe. multiple areas of diffuse interlobular septal thickening were in association of crazy paving pattern related to diffuse alveolar hemorrhage (fig. ct shows bilateral pulmonary metastatic lesions characterized by nodules with halo sign (the biggest measuring about 1 cm) and ground glass lesions. ct scan 2 months after the evidence of pulmonary progression shows the increase of pulmonary nodules, bilateral pleural effusion and wide ground glass attenuation, mainly in the right upper lobe. a 47-year - old male (patient 3), truck driver, former smoker, presented with recent episodes of hemoptysis, in absence of dyspnea or chest pain. his medical history was noncontributory except for an allergic history and recurrent upper airway symptoms. ct scan of the chest showed several bilateral consolidations, ranging from 20 to 60 mm in size, most of them with a halo sign (fig. laboratory data showed only a mild increase of c reactive protein (6.8 mg / l ; normal value < 5 pr3-anticytoplasmic autoantibodies, myeloperoxidase antibodies anca, anticardiolipin antibody igg and igm, and antibeta 2 glycoprotein 1 antibody igm and igg were negative. ct scan shows several consolidations with halo sign, the biggest in the right middle lobe. some ground glass lesions are also present ; the biggest in size is in the posterior segment of right upper lobe. bal showed red blood cells and numerous hemosiderin laden macrophages ; however, no neoplastic cells were identified. ct - guided needle biopsy showed organizing pneumonia. in absence of serological or clinical data supporting the hypothesis of a cvd or vasculitis, (a) a small pulmonary artery has the lumen completely obliterated by neoplastic cells. the surrounding airspaces are filled by red cells and hemosiderin laden macrophages (h&e, midpower). this showed small pulmonary arteries with the lumen completely obliterated by cd31 + neoplastic cells that showed cytoplasmic vacuoles. the surrounding alveolar spaces were full of red cells and contained hemosiderin - laden macrophages. patient underwent pet - ct scan that confirmed the uptake of the multiple lesions in the chest. a 50-year - old male (patient 4) presented to the emergency department with shortness of breath and chest pain. his clinical history record showed an amputation of the right upper limb and ipsilateral scapula for soft tissue angiosarcoma of the right arm, two years earlier. ct scan of the chest showed bilateral hydropneumothorax with a large pleural effusion on the left side and smaller one on the right side. furthermore, several cystic lesions (measuring from 9 to 24 mm in size) and solid nodules (from 8 to 20 mm) were present in the lungs bilaterally, suspicious of metastatic lesions (fig. three weeks later, the patient had partial resolution of the hydropneumothorax and pneumomediastum, and some of the pulmonary pre - existent nodules had become cystic and the pre - existing cysts were bigger (fig. ct scan shows bilateral cysts, some of these with thick wall as in the anterior segment of left upper lobe. comparison between baseline ct scan and control 3 weeks later shows that the cystic lesions are bigger and more numerous than the prior, suggesting a rapid disease progression. moreover, nodules that were visible in the prior examination now have a cystic shape (blue circle). the key features of metastatic angiosarcoma in the lung can be summarized as high variability of radiologic manifestations dependent on the extent of growth in the vessel lumen, the extent of (typically perivascular) nodule production, and the presence and extent of associated (usually perinodular) alveolar hemorrhage. considering the findings described in the literature (table 3) and those that we have observed on ct scans in the 4 patients described, we can delineate a correlation with pathologic changes summarized in table 4 and as follows:1.ground glass opacity (ggo). the radiologic findings of a diffuse or patchy ground glass attenuation were histologically characterized by areas of extended or focal alveolar hemorrhage, as was present in patient 3, and, in the advanced phase of patient 2. this pattern may present clinically as a diffuse alveolar hemorrhage syndrome.2.nodules (often) with halo. metastatic pulmonary nodules with a halo sign corresponded to lesions with alveolar hemorrhage in the surrounding parenchyma. this finding also justified the presence of wide areas of ground glass attenuation surrounding also tiny (less than 6 mm) nodules. this appears to be the most common pattern in the literature ; some cases may mimic pulmonary emboli.3.consolidation. when solid metastatic lesions enlarge, like in patient 3, they can become consolidative usually without air bronchograms. furthermore, the focal dilatations of bronchi peripherally to the lesions could be related to a postobstructive bronchial dilatation. this finding is relatively uncommon, with a single report describing consolidation as a secondary lesion.4.cysts. in the relatively advanced lesions, this is particularly remarkable, if we consider patient 4, in which solid lesions evolved into cystic lesions in only 3 weeks. the immediate consequence of this evolution is represented by complications such as pneumothorax and pneumomediastinum, particularly when cysts are beneath the pleura. even though only few reports describe cysts as secondary lesions, they may explain pneumothorax as a manifestation of metastatic angiosarcoma.5.tree-in-bud. the diffuse tree - in - bud pattern was observed in our first case ; the airways were not involved as shown by transbronchial cryobiopsy. this is not surprising, if we consider that all the lesions were located inside the arteriolar lumen, suggesting a neoplastic trombotic microangiopathy. the anatomic localization of the primary tumor in the right atrium probably facilitated the endovascular spread of the neoplastic emboli. the literature includes only 1 report of metastatic angiosarcoma - induced thrombotic microangiopathy ; tree - in - bud change is not mentioned. it could be argued that this finding is just tumor emboli filling the arteries and not truly thrombotic in pathogenesis. the radiologic findings of a diffuse or patchy ground glass attenuation were histologically characterized by areas of extended or focal alveolar hemorrhage, as was present in patient 3, and, in the advanced phase of patient 2. metastatic pulmonary nodules with a halo sign corresponded to lesions with alveolar hemorrhage in the surrounding parenchyma. this finding also justified the presence of wide areas of ground glass attenuation surrounding also tiny (less than 6 mm) nodules. this appears to be the most common pattern in the literature ; some cases may mimic pulmonary emboli. consolidation. when solid metastatic lesions enlarge, like in patient 3, they can become consolidative usually without air bronchograms. furthermore, the focal dilatations of bronchi peripherally to the lesions could be related to a postobstructive bronchial dilatation. this finding is relatively uncommon, with a single report describing consolidation as a secondary lesion. cysts. in the relatively advanced lesions, this is particularly remarkable, if we consider patient 4, in which solid lesions evolved into cystic lesions in only 3 weeks. the immediate consequence of this evolution is represented by complications such as pneumothorax and pneumomediastinum, particularly when cysts are beneath the pleura. even though only few reports describe cysts as secondary lesions, they may explain pneumothorax as a manifestation of metastatic angiosarcoma. the diffuse tree - in - bud pattern was observed in our first case ; the airways were not involved as shown by transbronchial cryobiopsy. this is not surprising, if we consider that all the lesions were located inside the arteriolar lumen, suggesting a neoplastic trombotic microangiopathy. the anatomic localization of the primary tumor in the right atrium probably facilitated the endovascular spread of the neoplastic emboli. the literature includes only 1 report of metastatic angiosarcoma - induced thrombotic microangiopathy ; tree - in - bud change is not mentioned. it could be argued that this finding is just tumor emboli filling the arteries and not truly thrombotic in pathogenesis. angiosarcomas, although rare entities, represent the most common histologic subtypes of primary tumors in the heart, and tumors of the great vessels of the chest. because of the propensity of angiosarcoma to involve the right atrium, patients may present with right - sided heart failure and lung metastases. in addition, regardless of the primary site, the most frequent sites of metastatic spread from an angiosarcoma are in the chest. in this report, we have reviewed our experience of 4 cases of angiosarcoma in the chest, matching clinical, radiological, and pathological data and comparing them with the literature. from a review of literature (table 3) and of our group of patients, we have identified 4 main radiological patterns of lung metastasis : alveolar hemorrhage, nodules with halo sign, masses, and cysts. from these 4 findings, our observations show a concordance with larger series already published : significant representation of nodules with halo sign ; the halo sign representing surrounding alveolar hemorrhage from these highly vascular neoplasms involving pre - existing vessels in the lung. the halo sign is one of the most important findings for suspecting metastasis from angiosarcoma ; it may occur even around very small nodules as illustrated in patients 1, 2, and 3. moreover, bilateral patchy ground glass attenuation alone can be present, likely due to alveolar hemorrhage, as we observed in the advanced stage of the patient 2 or as focal lesion in patient 3. a relatively rare finding in metastatic angiosarcoma to the lung is represented by large consolidations ; in the literature, only 2 cases have been reported. in our case, we observed the coexistence of organizing pneumonia, as demonstrated in the first nondiagnostic biopsy in patient 3. case 3 showed an interesting ancillary finding : focal bronchial dilatation in the periphery of the lesions, likely related to postobstructive bronchiectasis. another uncommon finding in metastatic angiosarcomas to the chest is pleural involvement as was present in patient 2. only 1 case in literature reported pleural involvement. of anecdotal interest is the fact that pleural lesions showed a relative stability after radiotherapy and chemotherapy, for about 3 years, at which time the patient dramatically progressed in the bone and lung. with regard to the other radiologic findings, on the basis of patient 4 history, we may also infer the natural history of cystic metastases of angiosarcoma in the lung. secondary nodules with halo sign rapidly evolved to cysts, and cysts may rupture and lead to pneumothorax and pneumomediastiun, and if blood also leaks out, a hemorrhagic pleural effusion and hydropneumothorax may be encountered. pleural effusion without pneumothorax may also be seen as, in patient 2, and in the case described by chen. in the literature, 4 mechanisms have been proposed for the genesis of cystic metastases, including excavation of a solid nodules, infiltration of tumor of preexisting bullous lesions, distension through the ball - valve effect of the tumor, and tumor cell proliferation to form blood - filled cystic spaces. in our case 1, we identified small centrilobular nodules with tree - in bud pattern, which did not involve airways, and was consistent with of endovascular and perivascular spread / growth of small tumor emboli. with regard to metastatic angiosarcoma, this finding has been described in literature only by demirag who identified pulmonary thrombotic tumor microangiopathy (pttm) from metastatic epithelioid angiosarcoma solely as a histologic finding, in absence of tree - in - bud pattern on ct scan. the tumor thrombi within small arteries and arterioles and associated fibrocellular and fibromuscular intimal proliferation are histological features of pttm. their most common ct finding is represented by tree - in - bud pattern, as firstly described by franquet. the most common tumors associated with pttm are metastatic gastric adenocarcinomas and ovarian carcinoma. in summary, clinical and radiologic presentation of angiosarcoma in the chest can be pleomorphic and its rarity makes diagnosis challenging. bocklage enumerated several differential diagnoses in their series, including infection, metastatic tumor of unknown primary site, multiple pulmonary emboli, granulomatosis with angitis, goodpasture syndrome, and idiopathic pulmonary hemorrhage. in our series, differential diagnosis included several conditions. in the first case, the vascular tree - in - bud pattern was consistent with the hypothesis of neoplastic thrombotic microangiopaty ; gastrointestinal tract tumor, pancreatic cancer, and metastatic angiosarcoma were the most probable diagnosis. the second case had a radiologic presentation of localized pleural lesions. knowing the primary tumor of the foot, pleural findings were highly suggestive of metastatic lesions of the pleura. differential diagnosis included poliangiitis with granulomatosis, angioinvasive aspergillosis, adenocarcinoma, kaposi sarcoma, and angiosarcoma. absence of specific clinical settings except for hemoptysis should be helpful in the suspicion of angiosarcoma. the fourth case presented with bilateral hydropneumothorax associated with solid and cystic lesions in the lungs. these aspects are quite unusual and suggestive of a variety of neoplastic cystic lesions such as metastatic sarcomas, mesenchymal cystic hamartoma, and infections (cystic evolution of pneumocystis jirovecii pneumonia)., our series shows that metastatic angiosarcoma in the chest may present with a variety of ct scan features and that these aspects are the result of specific morphologic lesions.	abstractrationale : angiosarcomas are rare, malignant vascular tumors.patient concerns : they represents about 2% of all soft tissue sarcoma, which can often metastasize through the hematogenous route. the radiological features have been analyzed in 4 patients with metastatic angiosarcoma in the chest.diagnoses:the main radiologic findings included nodules, cysts, nodules with halo sign, and vascular tree - in - bud. morphologic features, as observed in the histologic specimen, have been correlated with radiologic appearance.lessons:metastatic angiosarcomas to the lung are characterized by a wide variety of radiologic appearances that can be very characteristic. computed tomographic findings observed include bilateral solid nodules, cystic, and bullous lesions sometimes associated with spontaneous hemopneumothoraces.
(1,2,3,4) they can present in the acute setting by causing complications such as cyst rupture or intestinal volvulus. (5,6) we are presenting a rare case of and infected dermoid cyst that presented with an acute abdomen. an eleven year old boy presented to the accident and emergency department complaining of pain in the lower abdomen. initially his signs and symptoms were felt to be consistent with a non - specific cause. he re - presented a week later with persistent localised pain in the right iliac fossa and was noted to have a tender palpable mass, suggestive of appendicular pathology. an ultrasound revealed a cystic lesion measuring 38 mm by 39 mm by 38 mm in the right iliac fossa containing complex debris. the wall of the cyst appeared to be multilayered, possibly consistent with a duplication cyst. the appendix was not visualised.(figure 1) ultrasound images showing a spherical cystic lesion with layering within diagnostic laparoscopy was performed expecting to find a duplication cyst. a right lower quadrant incision was used to mobilize the caecum and deliver the cyst out of the abdomen. a limited right hemicolectomy with ileocolic anastomosis was performed.(figure 2) intraoperative picture showing a spherical lesion at the ileocaecal junction histopathology of the specimen demonstrated an unilocular cyst lined by keratinising squamous epithelium with inflammatory changes. mesenteric cysts are a rare group of lesions that have been classified into six subtypes based on their histopathology.(1) these include (a) cysts of lymphatic origin (lymphangioma) ; (b) cysts of mesothelial origin (benign cystic mesothelioma, malignant cystic mesothelioma) ; (c) cysts of enteric origin (duplication cyst) ; (d) cysts of urogenital origin ; (e) mature cystic teratoma (dermoid cysts), and (f) pseudocysts (infectious and traumatic cysts). dermoid cysts are commonly described in the head, neck, gonads, mediastinum, retroperitoneal and sacrococcygeal regions. there are four case reports in english literature of dermoid cysts occurring in the mesentery of children. dermoid cysts are postulated to occur as a result of abnormal migration of primordial germ cells. (7) this is a complex and poorly understood process, reliant on a host of migratory and homing factors (8), making it difficult to explain why our patient s cyst occurred at the ileocaecal region. we can only postulate that these cells may have migrated from the dorsal mesogastrium, in the midline, and then traversed in the mesentery to the ileocaecal region. (3) they may cause vague gastrointestinal symptoms or present with acute pain due to cyst rupture or intestinal volvulus. (5,6) it has also been reported in a patient with autoimmune haemolytic anaemia. (4) ultrasound imaging identified a cystic lesion, but was not able to differentiate it from the more commonly presenting lymphangiomas and duplication cysts. this was in keeping with the more commonly occurring dermoid cyst in the head and neck regions e.g. external angular dermoid. our report is unique in that our patient presented in a way that mimicked appendicitis. to our knowledge	mesenteric dermoid cysts are a rare cause of abdominal pain. although dermoid cysts occur in many parts of the body there have been only a few case reports involving the bowel mesentery. we present a case of a symptomatic mesenteric dermoid cyst involving the ileocaecal junction in a child.
carbon nanotubes (cnts) consist of cylindrical graphene sheets with nanometer diameters and present many unique characteristics, such as large ratio of surface area to mass, high electrical conductivity, and remarkable mechanical strength. single - wall cnts (swcnts) are comprised of cylindrical graphite sheets of nanoscale diameter capped by hemispherical ends. multi - wall cnts (mwcnts) are comprised of several to tens of incommensurate concentric cylinders of these graphitic shells with a layer spacing of 0.30.4 nm. mwcnts tend to have diameters in the 2100 nm range and can be considered as a mesoscale graphite system. since their discovery in 1991, extensive applications have been found in the physical, chemical, and material science fields. the advantages of cnts, such as their high surface area, favorable electronic properties, and electrocatalytic effect, have recently attracted considerable attention for the construction of electrochemical biosensors. electrochemical biosensors, particularly enzyme electrodes, have benefited greatly from the ability of cnt - based transducers to promote the electron - transfer reactions of enzymatically generated species, such as hydrogen peroxide [3, 4 ] or nadh, and from the resistance to surface fouling of transducers. electrochemistry is a powerful tool for real - time detection compared to fluorescence and spectrophotometry, which involves expensive detection systems. a combination of enzymatic reactions with the electrochemical method of monitoring electroactive enzymatic products allowed the development of enzyme - based electrochemical biosensors for sensitive and rapid determination of important environmental pollutants. chlorphenvinphos is an organophosphate (op) compound used as an agricultural and household pesticide. the toxic action of chlorphenvinphos is based on its ability to irreversibly modify the catalytic serine residue in acetylcholinesterase (ache) and effectively prevent nerve transmission by blocking breakdown of the transmitter choline. for these reasons, the rapid determination and reliable quantification of trace levels of chlorphenvinphos are important for health and environmental reasons. biosensors based on the inhibition of ache have been widely used for the detection of op compounds. the methodology involves the measurement of the uninhibited activity of the enzyme, followed by an incubation period for the reaction between enzyme and the inhibitor, and the measurement of enzyme activity after the inhibition. recently, cnts have been used for the construction of biosensors based on the inhibition of ache activity for the determination of op compounds. such composite electrodes combine the ability of cnts to promote electron - transfer reactions with the attractive advantages of paste electrode materials. these materials allow easy immobilization, reproducible electrochemical behavior, and useful physical characteristics [911 ]. this study describes the preparation and application of acetylcholinesterase biosensors based on carbon nanotube paste (cnts - ache biosensor) in the amperometric detection of chlorphenvinphos. compared to other analytical techniques, such as gas and liquid chromatography, enzyme - based electrochemical biosensors represent good selectivity, sensitivity, rapid responses, and reduced sizes in the determination of pesticide. the multiwall carbon nanotubes (mwcnt, 2040 nm diameter, 515 m) came from schenzhen nanotech port co. ltd. acetylcholinesterase (ache) (0.3 u mg) came from bovine erythrocytes. acetylthiocoline iodide was purchased from aldrich, and a 1.2 10 m stock solution was prepared in phosphate buffer ph 7.4. chlorphenvinphos was purchased from aldrich, and a 1.0 10 m stock solution was prepared in methanol. the electrochemical measurements were performed using a model pgstat20 autolab (eco chemie, utrecht, netherlands) potentiostat / galvanostat coupled to a personal computer and controlled with gpes 5.8 software. the electrochemical impedance spectroscopy (eis) data were obtained using a pgstat30 autolab (eco chemie, utrecht, netherlands) potentiostat / galvanostat controlled with fra software. the electrochemical cell was assembled using a conventional three - electrode system : an ag / agcl in kcl (3 mol l) reference electrode, a pt counter electrode, and a cnts - ache biosensor working electrode (1.2 mm diameter). an nir cary model 5 g spectrophotometer was used for comparative method coupled to a personal computer and controlled with cary win uv software with a quartz cell (optical path of 1.00 cm). scanning electron microscopy was performed in an feg - vp zeiss supra 35 microscope, operated at 5 kv, at different magnitudes. in previous studies carried out by our group, the most successful carbon nanotube paste was found using 6/4 (w / w) cnts / nujol. therefore, this carbon nanotube paste composition was used in the present investigation for the construction of the biosensor using acetylcholinesterase enzyme. the carbon nanotube paste electrode modified with ache subsequently, 0.050 g of this mixture was modified by adding 6.75 mg of ache 250 un and mixing until a uniformly wetted paste was obtained. after this, the paste was packed into a glass tube (= 1.2 mm), and a copper wire was embedded in the paste for electrical connection. square wave voltammograms for 3.0 10 m acetylthiocoline iodide in phosphate buffer ph 7.4 and 0.14 u of acetylcholinesterase were obtained between 0 and 1.0 v at increments of 2 mv and a frequency 50 hz in order to evaluate the oxidation process of thiocholine, the product of the enzymatic reaction using carbon nanotube paste electrode 60% (w / w). amperometric analysis was performed using acetylthiocoline iodide 3.0 10 m in phosphate buffer ph 7.4 and cnts - ache biosensor. the potential applied was 0.3 v. for the determinations of chlorphenvinphos samples, experiments of standard addition were carried out using the amperometric method. insecticide samples were prepared by dissolving in methanol and diluting to volume with phosphate buffer ph 7.4. after this, an aliquot of this solution was transferred into the cell and amperometric measurements were recorded in triplicate. next, three successive additions of 100 l of a standard 2.5 10 m chlorphenvinphos solution in phosphate buffer ph 7.4 were performed. after each addition, amperometric measurements were recorded and the mean current was determined. a spectrophotometric method for the determinations of chlorphenvinphos the electrochemical impedance spectroscopy (eis) data were obtained for frequencies from 10,000 hz to 0.01 hz at an amplitude of 10 mv. the impedance spectra were obtained within the ac potential, in 5 mm potassium ferricyanide in 0.5 m kcl, in the format of nyquist plots. cnts have been known to promote electron transfer reactions due to their electronic structure, high electrical conductivity and redox active sites [12, 13 ]. the electrocatalytic action of cnts facilitates low - potential measurements of the product of enzymatic reaction. for this reason, the cnts - ache biosensor was prepared without introduction of redox mediators (rm), which are able to shuttle electrons between the active site of redox enzymes, and an electrode replacing the natural cosubstrate of the enzyme. the cnts - ache biosensor combines the ability of carbon nanotube paste to promote electron - transfer reactions with the attractive advantages of paste electrode materials. these materials allow easy enzyme immobilization, reproducible electrochemical behavior and useful physical characteristics. the use of cnts as a matrix for immobilization exhibits advantages for chemically modified electrodes, primarily in the diversity of preparation methods for sensors and biosensors. as cnt matrices are effective in the immobilization process as transducer material, they are used together in the composite production as carbon paste. the coupling of the biocatalytic material and the electrode surface can be promoted through the interaction between the functional groups of the materials and the enzyme through the terminal amino acids. for this reason, the cnts - ache biosensor was prepared without introduction of solid support for immobilization of the ache. micrographies of the carbon nanotube paste electrode (60% w / w) and cnts - ache biosensor surfaces after polishing with 600 grit sandpaper are presented in figure 1 for different magnitudes. the comparison of images shows a significant difference in the morphology of materials. in figure 1(d), it is possible to observe the presence of cnts and small lumps, due to the enzyme ache, which has a type of cauliflower formation. the eis experiments, from nyquist plots, allow the obtainment of charge transfer resistance values for the electrode process being studied. as such, the experiments were carried out in the following conditions : (a) carbon nanotube paste electrode (60% w / w), at 0.346 v (ac potential), (b) cnts - ache biosensor, at 0.192 v, all 5 mm fe(cn)6 in 0.5 m kcl solution. all responses (figure 2) presented typical semicircles at high frequencies and a straight line at low frequencies, corresponding to kinetic and diffusional processes, respectively. to fit the eis data, the corresponding spectra were modeled using randle 's equivalent circuits of mixed kinetic and diffusional control (insets in figure 2(a)), where r s is the electrolyte resistance, c the interface capacitance, and r ct the charge - transfer resistance (domain of the kinetic control), resulting from the diffusion of fe(cn)6 towards the electrode surface from the bulk of the electrolyte. as evidenced in the nyquist plots, the simulated symbols (cross) based on the model agree with the experimental results. the estimated parameters obtained by assuming randle 's model are listed in table 1. the value of r tc for the cnts - ache biosensor increased threefold compared to the carbon nanotube paste electrode for the reaction [fe(cn)6 ]. this is probably due to the presence of the ache enzyme at the electrode surface, as seen in micrographs, which is not conductive. in the current study, the voltammetric characteristics of acetylthiocholine iodide (substrate) and thiocholine (enzymatically - generated product) on the carbon nanotube paste electrode were investigated by square wave voltammetry in phosphate buffer. two oxidation processes (peaks i and ii) of thiocholine are observed at 0.045 and 0.250 v (versus ag / agcl) as observed by liu. for a glassy carbon electrode modified with carbon nanotube film. the anodic oxidation peak of acetylthiocholine iodide was observed at 0.620 v and its oxidation process begins at 0.500 v. as the potential of oxidation of thiocholine and the initial oxidation of acetylthiocholine iodide are close to one another, a study on the working potential was achieved using chronoamperometry. the optimum potential for biosensor operation and current - time responses were obtained in thiocholine on a carbon nanotube paste electrode. the potential range evaluated was from 0 to 350 mv and the chronoamperometric responses obtained are presented in figure 4. the maximum current responses were observed at 300 and 350 mv and the potential at 300 mv was selected for amperometric measurements of thiocholine with the cnts - ache biosensor. this potential was selected due to its greater substrate oxidation potential. in order to optimize the cnts - ache biosensor 's performance, the influence of substrate concentration on the biosensor response was studied, with the purpose of increasing the signal obtained for enzymatic reaction. thus, the effect of the substrate concentration on the cnts - ache biosensor 's response was investigated between 0.5 and 3.0 10 m acetylthiocholine iodide solution in phosphate buffer ph 7.4. the highest analytical signal was obtained at 3.0 10 m. following this, the incubation time was studied ranging from 2 to 20 min. incubation time is the time the biosensor remains immersed in the solution containing the pesticide and must be sufficiently extensive. however, the maximum value of the inhibition was not 100%, which can likely be attributed to the binding equilibrium between pesticide and binding sites in the enzyme. table 2 summarizes the range over which each variable was investigated and the optimum value found in the optimization of the proposed method. the repeatability of the cnts - ache biosensor was determined from five different measurements in the same solution containing 3.0 10 m acetylthiocholine iodide in phosphate buffer ph 7.4. the electrode surface was renewed after each determination resulting in a mean peak current of 1.199 0.013 10 a (n = 5). an acceptable reproducibility was obtained with a relative standard deviation of 10% for measurements carried out in 3.0 10 m of acetylthiocholine iodide in phosphate buffer ph 7.4. the stability and life span of the biosensor are very important parameters in analytical determinations. for this reason, these parameters were investigated for the proposed biosensor in consecutive measurements without surfacing for over 20 days. when the cnts - ache biosensor was stored at 4c with measurements taken every day for 20 days, no noticeable change was observed in the response obtained in 3.0 10 m of acetylthiocholine iodide in phosphate buffer ph 7.4. the electrochemical determination of chlorphenvinphos was performed through the inhibition of the reaction of ache with the substrate, acetylthiocholine, in order to allow the maximum inhibition to be achieved. the percentage of inhibition caused by chlorphenvinphos on the enzymatic activity of the biosensor was calculated using the following equation : (1)%i=i0i1i100, where i 0 and i 1 are the biosensor responses before and after the incubation procedure, respectively. under optimized conditions, a linear response between the percentages of inhibition as a function of chlorphenvinphos concentration was obtained in the range investigated : 4.90 104.76 10 m, with the limit of detection being 1.15 10 m. the curve was linear in the entire interval of chlorphenvinphos concentration, according to the following equation : (2)%i=21.81(%i)+4.91 [chlorphenvinphos ] (106 m),r=0.995 (n=6). the result is presented in figure 5, which shows the curve obtained with surface renewal between successive determinations. the favorable characteristics presented by the proposed biosensor allowed its application for the direct determination of chlorphenvinphos in real samples. consequently, the performance of the cnts - ache biosensor was tested by applying it for the determination of chlorphenvinphos in the insecticide sample using the standard addition method. the results obtained by the cnts - ache biosensor were compared with those obtained by the spectrophotometric method. the results are summarized in table 3. applying a paired t - test to the results obtained by this procedure and spectrophotometric method, it was found that all results are in agreement at a 95% confidence level and with a relative error lower than 3%. these results therefore suggest that the cnts - ache biosensor is suitable for the determination of chlorphenvinphos in an insecticide sample. in this paper, we have described the use of a cnts - ache biosensor for determination of chlorphenvinphos. it is therefore concluded that the cnts - ache biosensor presents easy preparation, fast response, sensitivity, durability, good repeatability and reproducibility. furthermore, this biosensor can be used in chronoamperometry for the determination of chlorphenvinphos in an insecticide sample, producing a relative error lower than 3%.	an amperometric biosensor for chlorphenvinphos (organophosphorus pesticide) based on carbon nanotube paste and acetylcholinesterase enzyme (cnts - ache biosensor) is described herein. this cnts - ache biosensor was characterized by scanning electron microscopy (sem) and electrochemical impedance spectroscopy (eis). the sem result shows the presence of cnts and small lumps, due to the enzyme ache, which has a type of cauliflower formation. from eis analysis is possible to observe increased r tc for cnts - ache biosensor when compared to the carbon nanotube paste electrode for the reaction [fe(cn)6]4/3. using a chronoamperometric procedure, a linear analytical curve was observed in the 4.90 1077.46 106 m range with limit of detection of 1.15 107 m. the determination of chlorphenvinphos in the insecticide sample proved to be in agreement with the standard spectrophotometric method, with a 95% confidence level and with a relative error lower than 3%. in this way, the cnts - ache biosensor presented easy preparation, fast response, sensitivity, durability, good repeatability, and reproducibility.
prior to discussing typical gaseous mediators, scfas are to be briefly considered. shortly after discovering scfas, they were often referred to as volatile fatty acids. the main reason for coining this term was their specific odor, not their volatility per se. scfas are synthesized by host cells and various microorganisms that predominantly inhabit the intestines (archaea, bacteroides, bifidobacterium, butyrivibrio, clostridium, collinsella, coprococcus, desulfovibrio, eubacterium, lactobacillus, prevotella, propionibacterium, roseburia, and others). representatives of these groups of microorganisms are equipped with a large armamentarium of glycoside hydrolases. this enables them to degrade complex polysaccharide and protein molecules of animal, plant, and microbial origins. the resulting smaller molecules are anaerobically metabolized to form terminal products, including scfas and a number of gases (h2, ch4, co2, h2s, and nh3) (29). scfas produce multifarious effects on physiological, metabolic, regulatory, and behavioral processes in the mammal host organism that take place both within the gi tract and beyond it. one of the causes of neurophysiological disorders is the disruption of scfas formation and metabolism, primarily because scfas are actively involved in providing the organism (including its nervous cells) with energy (4, 5, 29, 30). in addition, the activity of the sympathetic nervous system is subject to regulation by scfas (e.g. by propionate) via their interaction with g - protein - coupled receptors (gprs), such as the gpr41 and gpr43 receptors of the ganglia of the enteric nervous system. the activation of these receptors in sympathomimetic neurons involves signaling pathways, for example, the gplcmapk pathway that controls the organism 's energy budget and maintains metabolic homeostasis. changing the amount of scfas and ketone bodies formed from them in the liver, as well as their ratio, causes alterations in energy metabolism and the activity of the sympathetic nervous system (5, 12, 31). apart from providing the cells of the central nervous system with energy, scfas, such as propionate and butyrate, exert an influence on the intracellular potassium level, which implies the involvement of scfas in the operation of cell signaling systems. in particular, these scfas regulate the expression of the gene coding for tryptophan hydroxylase, the key enzyme of the serotonin biosynthesis pathway, and, therefore, produce an effect on brain neurochemistry (4). scfas decrease the activity of chromosome histone deacetylases (hdacs), thereby facilitating the access of repair enzymes to the dna. this promotes the improvement of the health state of patients with the excessive activity of these enzymes that is characteristic of parkinson 's disease, depression, and schizophrenia. inhibiting hdac activity ameliorates the state of patients suffering from malignant tumors of the nervous system. scfas as hdac inhibitors exert a beneficial influence on model animals with cerebral trauma, dementia, autoimmune encephalitis, and depression - like symptoms (4, 5, 12). in gi endocrine cells, scfas induce the synthesis of neuroactive compounds, including histamine, serotonin, 5-aminovaleric acid, -aminobutyric acid, -alanine, leptin, peptide yy (32, 33), glucagon - like peptide hormone-1 (glp-1) (29), catecholamines, and other hormones and neurotransmitters (34). for instance, introducing propionate into the ventricles of the brain causes autism - like behavioral changes in rats (34). children with autistic spectrum problems are characterized by elevated concentrations of scfas, especially of propionate, in the intestines. this is presumably due to the abnormally high activity of clostridia and the presence of an unusual microbial species, sutterella wadsworthensis, which seems to be typical of autism. the behavioral problems of autistic people may result from propionate 's capacity to modulate the expression of many autism - related genes, predominantly those associated with mitochondrial processes (35). studies with mice with autism - like disorders revealed that administering a probiotic based upon a special target - oriented bacteroides fragilis strain ameliorates the microbial ecology of the intestines, decreases their permeability, and mitigates anxiety - related stereotyped behavior (12). further research will enable us to develop novel microecological approaches for improving autism symptoms under clinical conditions (36). gases formed in the animal / human organism, including those produced via microbial fermentation in the gi tract, have received much attention from researchers and clinicians, starting in the 1970s. no, co, and h2s are among the most ancient gas molecules that can perform neuromediator functions. presumably, some other gases (h2, ch4, nh3, co2, and others) also exhibit neuromediator activities. both the host tissue - dependent and microbial synthesis of gases with proven neuromediator functions is carried out by specific enzymes. for example, the synthesis of no from arginine is catalyzed by no synthases (noss) while co synthesis by heme oxygenases (hos) causes heme degradation. h2s is predominantly synthesized from l - cysteine, and this reaction is catalyzed by at least three different enzymes (27, 37, 38). to reiterate, the composition and amount of gases synthesized in the human organism vary depending on the individual and gi area involved. gasotransmitters produce their effects on the cells that synthesize them, on adjacent cells, and on even remote tissues / organs. gaseous signal molecules do not bind to specific receptors on cell membranes and do not accumulate in synaptic vesicles ; upon their synthesis, they are usually released from the synthesizing cells (28, 39, 40). gases can sufficiently easily penetrate into the cells of the nervous, vascular, and immune systems, as well as into those of other systems. many host- or microbiota - produced gases are capable of post - translational modification of various proteins, which results in functional alterations. some of these alterations associated with oxidative stress, mitochondria imbalance, and other cell disorders cause damage to biological macromolecules and even cell death. nevertheless, there is much evidence that the interaction of some gases with some peptides does not result in cell death (41). all the aforementioned molecules are among the smallest bioactive molecules that perform universal functions related to the life - sustaining activities of both multicellular organisms and bacteria. the volume of intestinal gases that is produced per day varies between 400 and 1,200 ml. nitrogen, oxygen, hydrogen, methane, carbon dioxide, and h2s account for 2090%, 3.910%, 20.950%, 7.210%, 930%, and 0.00028% of the total volume, respectively. in addition, ammonia, carbon monoxide, nitrous oxide, acetaldehyde,1 and sulfur dioxide accumulate in the gi tract. these gaseous substances enter the gi tract with air and food ; in addition, they are formed by various eukaryotic and prokaryotic cells via enzymatic or non - enzymatic processes. most gas molecules are removed from the intestines ; they can also be absorbed and delivered to the bloodstream ; subsequently, they are released via the respiratory system. h2 and ch4 are only formed by microbial fermentation in the gi tract ; after entering the bloodstream, they reach the lungs and are exhaled. their quantity varies to a large extent, depending on a human individual 's diet. no is a small short - lived signal molecule that can modify the functions of diverse proteins both directly and indirectly, via post - translational modification caused by their binding to thiol groups and other amino acid sites (43). in the human organism, there are three isoforms of endogenic noss in the animal organism (three nos enzymes), which are nos1 (neuronal nos, nnos), nos2 (inducible nos, inos), and nos3 (endothelial nos, enos). inducible nos is activated under the influence of microbial metabolites and inflammatory cytokines released in response to infection and tissue damage (27, 28). these enzymes and some bacterial noss produce no from l - arginine in a process involving oxygen and nadh and resulting in l - citrulline formation (26, 27, 40, 45, 46). bacterial noss (bnoss) also catalyze no synthesis from arginine both in vitro and in vivo. they are present in various bacterial species (streptomycetes, bacilli, and so on) (45, 47), including human / animal pathogenic and symbiotic microorganisms that inhabit the intestines (44), the oral cavity (40, 48), and the vagina (49). the classical l - arginine - no pathway coexists with the alternative nitrate - nitrite - no pathway (46, 5053). the alternative pathway is characteristic of intestinal bacteria that obtain nitrate and nitrite from digested food (44). apart from synthesizing their own no, intestinal bacteria, including probiotic strains (such as lactobacteria, bifidobacteria, and escherichia coli nissle 1917), can stimulate no formation by host epithelial cells (40, 48, 5456). the probiotic strain - synthesized no is rapidly degraded by e. coli and staphylococcus aureus both in vitro and in the intestines of test animals (44, 57). the main targets of no and related compounds are proteins that contain iron (guanylate cyclase, nos enzymes, hemoglobin, and enzymes involved in the citric acid cycle and protein and dna synthesis) and sh groups. no also targets reactive oxygen species (ros) (28, 50, 58). in mammals, no is involved in regulating impulse transfer across synaptic clefts, regional blood flow, intestine peristalsis, and water and electrolyte transport. no influences the functioning of the immune and cardiovascular systems and regulates energy metabolism (40, 4446, 50, 52, 59, 60). the regulatory influence on the vital functions of the organism is exerted by pico- or nanomolar no concentrations. at these concentrations, no behaves as a neuromediator and mice with a defective nnos are characterized by elevated locomotive activity, virility that is retained for a long time, high fertility, and long - term depression (ltd). male mice lacking neuronal isoform (nos-1-/- or nnos-/-)-encoding genes are more aggressive than wild - type males (61). nnos - containing mice were more resistant to experimental stroke caused by ligaturing the middle cerebral artery. in an analogy to eukaryotes, no performs communicative and antioxidant functions in bacteria and is involved in biofilm formation / dispersal regulation and in expression of genes that are required for iron utilization (28, 45). no - dependent antibiotic resistance implicates chemical modification of antibiotics or attenuation of antibiotic - induced oxidative stress (28, 60) by stimulating bacterial catalase activity (45). bacteria can also inactivate no itself by converting it into nitrates (62) or, alternatively, by forming s - nitrosothiols (63). microbial no produces diverse effects on eukaryotic organisms. in the example of the worm caenorhabditis elegans, it was demonstrated that b. subtilis- and e. coli - synthesized no behaves as a transcription activator. it induces processes in the worm 's enterocytes that enhance its heat resistance and prolong the lifespan (64). no also affects the functions of ionotrophic glutamate receptors (iglurs) and acid - sensitive ion channels (asics) that are present in various areas of the central nervous system and in other mammalian tissues. no can modify iglurs and asics either directly, by s - nitrosylation of cysteine, or indirectly, via cgmp protein kinase g (pkg)-dependent phosphorylation (38). recently, the traditional opinion that nitrate and nitrite contained in food can cause stomach cancer has been called into question (65). moreover, the application of drugs or dietary strategies for modifying no metabolism in order to exploit the therapeutic potential of nitrates as no sources has been increasingly discussed in the literature (46, 66). it should be taken into account that, despite the predominantly regulatory role of nanomolar no concentrations, no applied at high (micro- to millimolar) concentrations generates toxic compounds, such as no2, n2o3, and especially onoo- (peroxynitrite) that impair thiol groups of organic molecules and react with protein tyrosyl groups and dna nitrogenous bases in various mammal cells and microbial symbionts (28, 50, 58, 67). carbon monoxide has long been considered as the most widespread air pollutant and a silent killer because of its high affinity for reduced iron in hemoglobin that transports oxygen to the tissues of the animal / human organism. various plants and animals, including humans, have been revealed to synthesize co as an intermediate product formed during heme degradation by heme oxygenases. currently, three kinds of heme oxygenases (ho-1 to 3) are known ; the inducible (ho-1) and the constitutive (ho-2) enzymes are of paramount importance in terms of endogenous co synthesis. ho-1 activity is induced by various stressors and widely spread in liver, kidney, and spleen cells as well as in aged erythrocytes. ho-2 is located in neurons, other brain cells, and the endothelial layer of blood vessels ; the enzyme is activated by ca - calmodulin, glucocorticoids, and opiates (37, 43). co is also formed by bacteria, including pathogens, plant and animal symbionts, and soil and marine species that contain heme oxygenases (28, 68). this enzyme is responsible for the anaerobic metabolism of co, which is the sole carbon source in a number of bacteria, for example, rhodospirillum rubrum (69). co is a sufficiently stable molecule that easily enters cells because it readily crosses the cell membrane ; inside a cell, it exerts its biological effects, including anti - apoptotic, anti - proliferative, anti - inflammatory, cytoprotective, and other activities, both at the co generation site and at a sufficiently large distance from it (43). co also regulates ion channels / transporters in various subtypes of epithelial cells (26, 27). recently, convincing evidence has been presented that co possesses all typical properties of a gasotransmitter / gasomodulator with a broad biological action spectrum, when applied at physiological concentrations (28, 70). the protective influence of co on the central nervous system was investigated in model systems. co inhalation (up to 250 ppm) protects test animals against i / r brain injury and ischemic stroke (71). the same co concentration was revealed to prevent neurological damage (neuronal apoptosis) in a pig model of deep hypothermic circulatory arrest. pretreatment of primary cultures of cerebral granular neurons with co (250 ppm) prevented apoptosis caused by oxidative stress or exogenous glutamate. the co - dependent protection implicates activation of guanylate cyclase and, subsequently, mitochondrial atp - sensitive k+ channels. this is accompanied by an increase in intracellular ros level and stimulation of no formation (72). in studies with hippocampal neurons, it was established that the key early stage of their apoptosis is activation of membrane k channels, which promotes the extrusion of potassium ions from the cytoplasm and initiates apoptosis. co can prevent apoptosis by directly inhibiting these channels (43, 72, 73). the neuroprotective effect of co in this system was attributed to its capacity to stimulate concomitant formation in neurons of two kinds of protective compounds, ros and no. nonetheless, studies with human neuroblastome cells (sh - sy5y) revealed that long - term treatment with a co donor (co - rm-2) induced symptoms of cell injury caused by lowering antioxidant activity or inhibiting no formation. the capacity of co at a concentration of 1,000 or 3,000 ppm (action time 40 min) to disrupt ca - dependent signaling pathways in sh - sy5 cells and in the homogenate of the total brain tissue of rats appears to be due to its modulating effect on the target protein, plasma lemma ca atpase (pmca) (74). intracellular h2o2 production in the brain tissue is increased at high co concentrations, which is accompanied by the enhanced production of hydroxyl radicals and a decrease in the ratio between the reductive and oxidative processes in mitochondria (72). hence, the action time and concentration of co at the target site determine whether its effect is beneficial or detrimental, in an analogy to the majority of drugs. currently, co is envisaged as a physiological signal molecule regulating the functions of membrane channel proteins and transporters (73). it has been established that the antimicrobial and anti - inflammatory effects of co and co - releasing molecules (co - rms) (e.g. metal carbonyl co - rm-3, ru(co)3cl, and glycinate) implicate the opening of k+/na+ channels in eukaryotic and bacterial cells, which decreases the proton motive force and disrupts ion transport for a short time. the mechanisms of protection of the nervous and cardiovascular systems in the presence of co - rms have not been completely elucidated yet. there is evidence that co - rm2 behaves as an inhibitor of voltage - activated potassium channels ; and mitochondria represent the main target of co. this does not rule out an additional effect of co - rms, the stimulation of ros production in mitochondria. it was established that the co released at low co - rm2 concentrations can produce a cardioprotective effect, due to its antioxidant properties. recently, increasing attention has been paid to the use of heme oxygenases, co inhalation, and co - rms for treating various infection and inflammation processes as well as cardiovascular and, potentially, neurophysiological problems (28, 70, 72, 73, 75, 76). hydrogen sulfide is a very water - soluble gas. at a concentration of 1 ppm, it can be recognized because of its rotten egg odor ; 4 ppm h2s causes a headache ; at still higher concentrations (500 ppm and above), h2s can produce a lethal effect (37, 39). the equilibrium ratio between its three forms (h2s, hs, and s) varies depending on medium ph. in the organism acute intoxication is due to h2s binding to the iron of cytochrome c oxidase, which inactivates the enzyme and abolishes oxidative phosphorylation in mitochondria (39). despite its toxic effect, h2s has recently been established to play a vital role in bacteria, plants, and invertebrate and vertebrate animals, including mammals. predominantly, the synthesis of this gas involves three enzymes : cystathionine--synthase (cbs), cystathionine--lyase (cse or cth), and 3-mercaptopyruvate sulfur transferase (3-mst) (43). h2s - synthesizing enzymes are expressed, to a different extent, in the cardiovascular, nervous, immune, urinary, respiratory, and gi systems (77). the microbiota of the large intestine, which includes more than 26 genera, is one of the key factors implicated in the metabolism of s - containing compounds and endogenous generation of h2s in the human organism. a red meat - enriched diet stimulates h2s synthesis by supplying the large intestine with a significant amount of sulfated proteins. the human large intestine contains considerable h2s amounts that predominantly result from h2s formation from inorganic (e.g. sulfates and sulfites) and organic (methionine, cysteine, taurine, sulfate - containing polysaccharides, and lipids) compounds (78, 79). sulfur - containing organic compounds, including those present in garlic, onion, and other food stuffs, supply the organism with its h2s pool (78). sources of microbial h2s include, for example, e. coli strains that possess two enzymes (l - cysteine transaminase and 3-mst), which catalyze its formation. the bacterial production of h2s can also involve cbs and cse or cth (80). some representatives of intestinal bacteria (prevotella, bacteroides, helicobacter, peptococcus, and akkermansia) produce glycosyl sulfatases or similar enzymes that promote production of sulfates from sulfomucins (78). sulfate - reducing bacteria compete with methanogenic microorganisms for h2 molecules both in vitro and in vivo. in the human large intestine, desulfovibrio vulgaris is predominantly responsible for h2s generation by reducing various sulfur - containing compounds, including sulfates and s - containing organic substances, e.g., cysteine. the bacteria of the large intestine ferment cysteine, yielding h2s, ammonia, and pyruvate. if the oxygen content is low (under microaerophilic conditions), h2s at millimolar concentrations can serve as an electron donor and an energy source. if food contains a limited amount of cysteine, endogenous and microbial cse activity and, therefore, h2s production are increased ; conversely, enriching food in cysteine or the chemical / genetical suppression of cse activity results in a decrease in h2s production (81). within the lower part of the mammal gi tract, h2s behaves either as a potential toxin or as a signal molecule, depending on its concentration (28, 82). at high (millimolar) concentrations, h2s is a highly toxic compound that causes a whole spectrum of pathological processes (78), including those brought about by inhibiting mitochondrial functions ; it also produces genotoxic effects by damaging the dna (78, 83). in contrast, when applied at low (micromolar) concentrations, h2s serves as an inorganic electron donor for mitochondria. h2s regulates a number of physiological processes, such as the inflammatory response, apoptosis, cell proliferation, neuronal impulse transfer, and smooth muscle tone (83). the effects of h2s applied at physiological concentration are mainly focused on the cardiovascular and the nervous systems (77). the varied regulatory effects of h2s are due to its capacity for modifying proteins via reducing disulfide (s = s) bonds or attaching a sulfur atom to a thiol group (sh). as a result these important post - translational processes change the conformation and functional activity of proteins responsible for transmembrane ion transport or enzymes involved in protein phosphorylation / dephosphorylation and synthesis of secondary metabolites and cofactors (39). intestinal h2s of microbial origin is predominantly degraded by intestinal epithelial enzymes (39). both free and sulfate - conjugated h2s is excreted from the organism, predominantly by the kidneys (38). the physiological effects of h2s are due to the influence of this molecule on various molecular targets in diverse tissues, including heme - containing proteins, ion channels, and signal proteins. in the presence of glutathione, cysteine, or dihydrolipoic acid, h2s is released from the lysate of cultured neurons and astrocytes at ph 8.08.4. when excited, neurons take up sodium ions and excrete potassium ions, which results in increasing the intracellular potassium concentration and depolarizing the membranes of adjacent astrocytes. the main h2s targets include atp - sensitive potassium channels as well as calcium and chloride channels. there is sufficient evidence that the (neuro)modulatory effect of h2s on cell functions and physiological processes is due to its interaction with several cell transporter systems. it was revealed that h2s enhances the activity of transporter systems by facilitating the release of antioxidants that are required for protecting the systems against exogenous toxic substance - caused damage. the h2s - transporter interactivity plays a major role in maintaining the redox potential of nervous cells. this is an additional mechanism of the neuroprotective and neuromodulatory activities of this gaseous substance. of special note is the impact of h2s on various types of k channels that are essential for the transfer of ions in epithelial cells. h2s is likely to indirectly affect na transfer by acting on the proteins of k+ channels and transporter molecules (26, 27). the influence of h2s, a biological signal molecule, on neuronal activity in the hippocampus, cerebellum, cortex, and brain stem has been researched during the course of more than 15 years. it was established that h2s is an active neuromodulator and neuroprotector in various brain cells (31, 37, 39, 84). at physiological concentrations, h2s functions as a synaptic activity modulator. cbs, which is present in the cells of various brain areas, is responsible for the generation of h2s. it activates transmembrane atp - associated channels (in neurons both inside and outside the brain) via modulating glutamate - dependent n - methyl - d - aspartate receptors. this gaseous modulator also regulates the activity of serotonergic neurons and induces the release of corticotrophin - releasing hormone (39, 84). two different forms of sulfur, acid - labile and bound sulfur, are stored in brain cells. acid - labile sulfur is incorporated in the iron - sulfur centers of mitochondrial enzymes involved in oxidative phosphorylation. significant amounts of bound sulfur are present in the cytoplasm of brain neurons and astrocytes. release of h2s from the lysate of cultures neurons and astrocytes proceeds at ph 8.08.4. the excitation of nervous cells results in sodium influx into and potassium efflux from the cells. a drastic increase in ambient potassium concentration results in the depolarization of the membranes of adjacent astrocytes. to abolish membrane depolarization, the proteins responsible for na+/hcotransfer are activated. taking up hco results in the alkalinization of the intracellular content of astrocytes, which causes h2s release from sulfur - binding compounds. unbound h2s (9.2 m) is retained for a longer time in the brain tissue than in liver and heart cells (84). in astrocytes, h2s also influences the intracellular level of calcium that plays a major role in intercellular communication. the intracellular calcium level rapidly increases upon the addition of h2s ; subsequently, it slowly decreases. these effects of h2s and various h2s donors were revealed in astrocyte cultures and in the glia of hippocampal sections (84). h2s was established to exert an influence on the operation of the peripheral nervous system, which involves modulating pain perception and the transfer of pain signals to the relevant brain areas (39). the neurotoxic effect of glutamate on brain tissue cultures is partly due to inhibiting the entry of cystine into the cells. h2s can mitigate the toxic effect by reversibly inhibiting cystine transfer by glutamate and, therefore, stimulating cystine influx into the cells (31, 33). there is a supplementary pathway of synthesizing h2s from d - cysteine, which involves 3-mst and d - amine oxidase (33). in contrast to the pathway of h2s synthesis from l - cysteine, the d - cysteine - dependent pathway predominantly functions in the cerebellum and the kidneys. studies with the primary cultures of cerebellar neurons revealed that the cerebellar tissue does not sustain hydrogen peroxide - induced oxidative stress if d - cysteine is available (33). the discovery of the d - cysteine - dependent pathway of synthesizing h2s provides foundations for a new therapeutic technique that is based on delivering this gas to target tissues (85). h2s and s - adenosyl - methionine impede the increase in the glucocorticoid concentration in blood plasma under stress. it was established that low h2s concentrations are capable of neutralizing reactive oxygen and nitrogen species (superoxide radical, hydrogen peroxide, peroxynitrite, hypochlorite, and so on) and of reversibly inhibiting the mitochondrial respiratory chain. it is the antioxidant effect of h2s that is responsible for its neuro- and cardioprotective activities (39). currently, it is widely accepted that h2s, like no and co, is an important neurotransmitter. the modulatory effect of h2s on cell functions and physiological processes is due to its interaction with several transporter systems. it has been established that h2s enhances their activity by releasing antioxidants that provide protection from exogenous toxic factors. the h2s - transporter systems interactivity plays a major role in maintaining the redox potential of nervous cells ; this is an additional mechanism of the neuroprotective and modulatory activity of this gaseous agent. an influence of h2s on human behavior was suggested for the first time in studies with human subjects with seizures, psychiatric disorders, or abnormal electroencephalograms ; most of the subjects lacked the enzymes (cbs) that are involved in h2s synthesis. subsequently, it was revealed that patients with down syndrome, in contrast, are characterized by abnormally high concentrations of these enzymes in the brain tissue (86). the h2s content in the brain tissue was decreased by over 50% in alzheimer patients, and this deficiency is apparently due to a drastic (70%) decrease in the concentration of s - adenosyl - methionine that activates cbs. prevented nervous cell damage and apoptosis in a model system in which this disease was caused by administering the toxin rotenone to test animals (39). there is evidence that h2s functions as a signal molecule in the visual system of mammals. h2s synthesis - catalyzing enzymes (cbs and cse) were detected in various kinds of eye cells, and h2s was found to regulate sympathetic and glutamatergic neurotransmission during the signal transduction processes in this system. further data on the regulatory influence of h2s on ion channels and transporters will contribute to our understanding of the role of h2s in relation to the risk of development of ocular neuropathies (87). even though the use of gaseous h2s for therapeutic purposes is hardly feasible, we can apply chemical compounds that release h2s in the human organism either rapidly (nahs) or slowly (gyy 4137). this gives grounds for the suggestion that h2s should be used for medical purposes (88). it is important that while the one - time use of nahs provided protection for neurons from oxidative stress, the repeated administration of this substance produced a toxic effect on these cells (84). nonetheless, h2s treatment is considered an efficient therapeutic technique in a number of diseases (e.g. lung cystic fibrosis and kidney problems in patients with hereditary hypertension) that are characterized by enhanced na+ influx into cells (81). in all likelihood, the employment of chemical donors or microbial producers of h2s (as an ion channel / transporter modulator) for medical purposes will hold much promise as a potential pharmacological approach to the treatment of a number of neurodegenerative diseases. ammonia is one of the end products of degradation of proteins, peptides, urea, and various amino acids. in the human organism, nh3 is predominantly formed by the intestinal microbiota and the cells of the gi tract, kidneys, the liver, and muscles. at least 410 g of nh3 are daily synthesized in the intestines of adult human individuals. among aerobes, gram - negative intestinal bacteria of the genera proteus, klebsiella, and pseudomonas as well as e. coli are the most active nh3 producers ; active nh3-producing anaerobes include the genera clostridia, ruminococcus, bacteroides, and some lacto- and bifidobacteria. peptidococci, ruminococci, coprococci, bifidobacteria, lactobacilli, clostridia, bacteroides, and some streptococci and enterococci exhibit significant urease activity. in healthy human individuals, up to 7 g of urea are degraded daily by microbial ureases (amounting to 50% of the total pool of this compound) (89, 90) and by those of fungi (candida albicans) (91). the involvement of microorganisms in ammonia metabolism is consistent with the fact that the intestines of germ - free animals contain 2030% less urea than their conventional counterparts. after administering carbon - labeled urea to these animals, intestinal urease - producing microorganisms form ammonia that is transferred via the portal vein to the liver, where it is reincorporated into urea. gi microorganisms also incorporate nh3 into amino acids are synthesized de novo using co2 or acetic, propionic, and other organic acids as carbon sources. unless utilized in biochemical processes in the large intestine, microbially produced nh3 rapidly passes through mucous membranes and spreads within the organism. for the most part, intestinal unbound nh3 reaches the liver via the portal vein ; in the liver, it is virtually completely converted into urea and glutamine via a series of reactions (the urea cycle). the unbound nh3 content in the blood of healthy adults is approximately 35 m (ca. unbound nh3 circulates in the organism ; it is excreted with urine and, to a lesser extent, with feces. ammonia - derived metabolites are also excreted with urine or, alternatively, used for synthesizing amino acids and other biological molecules. endogenous nh3 formed in the brain and in peripheral tissues is not transferred to the liver ; instead, it is transformed in these tissues into glutamine and alanine (89, 90). a genetic disruption of the biosynthesis of urea cycle enzymes, liver and kidney dysfunction, excessive nh3 formation in skeletal muscles caused by physical exertion or other kinds of stress, or an imbalance in the intestinal ecological system result in increasing nh3 content in the organism to a toxic level (hyperammonemia). for instance, liver cirrhosis is associated with the formation of a direct bypass between the portal vein system and the bloodstream ; this prevents the detoxification of harmful gi tract - produced compounds, including nh3, that reach the bloodstream. increased nh3 concentrations penetrate into the brain tissue, which is a major factor of pathogenesis of hepatic encephalopathy (he). it manifests itself in fatigue, ache, muscle weakness, loss of appetite, nausea, vomiting, diarrhea, pain in the back, sides, or the abdomen, and motor and cognitive disturbances. in the early 1970s, it was established that the genetic disruption of the urea cycle results in pathological changes in the brain of newborns. it was suggested that gaba is implicated in causing ammonia - induced toxicity in the nervous system. an increase in ammonia concentration in the brain tissue results in stimulating gaba - induced chloride channels in neurons and astrocytes. evidence was presented that liver dysfunction - induced hyperammonemia is accompanied by changes in cell energy metabolism and formation of excessive glutamine amounts in astrocytes. it is astrocytes that incorporate ammonia in glutamine molecules after it crosses the blood - brain barrier (bbb). hyperammonemia is also responsible for osmotic stress in the brain, which results in redistribution of cerebrospinal fluid and causes low - grade swelling in astrocytes and edema in the white matter as well as an increase in intracranial pressure (9295). ammonia also inhibits energy production in mitochondria, which is attributed to ammonia 's capacity to suppress ketoglutarate dehydrogenase activity and stimulate glycolysis (94). an increase in ammonia concentration in the arteries affects the expression of a number of genes that code for neuroglial proteins. these proteins regulate cell growth, mitochondrial functions, and transfer of neuroactive amino acids (95). when applied at supraphysiological concentrations, nh3 induces rapid release of glutamate from neurons. in humans, this results in the development of irritability, aggression, hyperexcitability, and even movement disorders. hyperammonemia also promotes removal of gaba from neurons, which frequently manifests itself in somnolence or lethargy ; these symptoms may ultimately cause the development of a comatose state (95, 96). apart from the direct neurotoxic effects, high nh3 concentrations increase the permeability of the bbb ; modulate the serotonergic and dopaminergic systems of the brain ; cause the accumulation of abnormal neurotransmitters, such as octopamine, in the brain ; and result in glucose intolerance and increased urinary output calcium and phosphate concentrations (12, 95). systemic inflammation that constantly accompanies acute and chronic liver dysfunction represents an important risk factor in terms of encephalopathic complications. hyperammonemia and the attendant neuroinflammatory response to liver cirrhosis cause microglia activation and monocyte recruitment ; the enhanced synthesis of proinflammatory cytokines (tnf, il-1, and il-6) ; the accumulation of ammonia, lactate, and manganese ; and an increased permeability of the bbb. encephalopathy may result from the synergistic effect of ammonia and cytokines (93, 95). astrocytes lose the capacity to adequately regulate their own volume, glutamine accumulation is increased, and a cascade of signaling processes is triggered. this results in enhanced ca accumulation and an increased formation of reactive oxygen and nitrogen species, which is due to the activation of nadph oxidase and nos (93). some patients suffering from hyperammonemia and the attendant gi dysfunction (constipation or diarrhea) exhibit psychotic symptoms and movement disorders that resemble those typical of genuine autism spectrum disorders (35, 91, 96). the important role of microbial nh3 with respect to the brain functions and life expectancy of he patients is consistent with the fact that the patients ' state is markedly improved after administering the antibiotic rifamycin, the prebiotic lactulose, and probiotics to them (95). the neuropsychic state, including cognitive capacities, is also improved by synbiotics, for example, by the bifidobacterium longum - fructo - oligosaccharide combination (12). neural inflammation, brain edema, and the resulting encephalopathy symptoms that are due to increased nh3 concentrations in the brain tissue can be mitigated by minocycline, an inhibitor of microglia activation, and n - acetylcysteine, as well as by mild hypothermia ; all these techniques produce neuro- and hepatoprotective effects (95). no is a small short - lived signal molecule that can modify the functions of diverse proteins both directly and indirectly, via post - translational modification caused by their binding to thiol groups and other amino acid sites (43). in the human organism, there are three isoforms of endogenic noss in the animal organism (three nos enzymes), which are nos1 (neuronal nos, nnos), nos2 (inducible nos, inos), and nos3 (endothelial nos, enos). inducible nos is activated under the influence of microbial metabolites and inflammatory cytokines released in response to infection and tissue damage (27, 28). these enzymes and some bacterial noss produce no from l - arginine in a process involving oxygen and nadh and resulting in l - citrulline formation (26, 27, 40, 45, 46). bacterial noss (bnoss) also catalyze no synthesis from arginine both in vitro and in vivo. they are present in various bacterial species (streptomycetes, bacilli, and so on) (45, 47), including human / animal pathogenic and symbiotic microorganisms that inhabit the intestines (44), the oral cavity (40, 48), and the vagina (49). the classical l - arginine - no pathway coexists with the alternative nitrate - nitrite - no pathway (46, 5053). the alternative pathway is characteristic of intestinal bacteria that obtain nitrate and nitrite from digested food (44). apart from synthesizing their own no, intestinal bacteria, including probiotic strains (such as lactobacteria, bifidobacteria, and escherichia coli nissle 1917), can stimulate no formation by host epithelial cells (40, 48, 5456). the probiotic strain - synthesized no is rapidly degraded by e. coli and staphylococcus aureus both in vitro and in the intestines of test animals (44, 57). the main targets of no and related compounds are proteins that contain iron (guanylate cyclase, nos enzymes, hemoglobin, and enzymes involved in the citric acid cycle and protein and dna synthesis) and sh groups. no also targets reactive oxygen species (ros) (28, 50, 58). in mammals, no is involved in regulating impulse transfer across synaptic clefts, regional blood flow, intestine peristalsis, and water and electrolyte transport. no influences the functioning of the immune and cardiovascular systems and regulates energy metabolism (40, 4446, 50, 52, 59, 60). the regulatory influence on the vital functions of the organism is exerted by pico- or nanomolar no concentrations. at these concentrations, no behaves as a neuromediator and mice with a defective nnos are characterized by elevated locomotive activity, virility that is retained for a long time, high fertility, and long - term depression (ltd). male mice lacking neuronal isoform (nos-1-/- or nnos-/-)-encoding genes are more aggressive than wild - type males (61). nnos - containing mice were more resistant to experimental stroke caused by ligaturing the middle cerebral artery. in an analogy to eukaryotes, no performs communicative and antioxidant functions in bacteria and is involved in biofilm formation / dispersal regulation and in expression of genes that are required for iron utilization (28, 45). no - dependent antibiotic resistance implicates chemical modification of antibiotics or attenuation of antibiotic - induced oxidative stress (28, 60) by stimulating bacterial catalase activity (45). bacteria can also inactivate no itself by converting it into nitrates (62) or, alternatively, by forming s - nitrosothiols (63). microbial no produces diverse effects on eukaryotic organisms. in the example of the worm caenorhabditis elegans, it was demonstrated that b. subtilis- and e. coli - synthesized no behaves as a transcription activator. it induces processes in the worm 's enterocytes that enhance its heat resistance and prolong the lifespan (64). no also affects the functions of ionotrophic glutamate receptors (iglurs) and acid - sensitive ion channels (asics) that are present in various areas of the central nervous system and in other mammalian tissues. no can modify iglurs and asics either directly, by s - nitrosylation of cysteine, or indirectly, via cgmp protein kinase g (pkg)-dependent phosphorylation (38). recently, the traditional opinion that nitrate and nitrite contained in food can cause stomach cancer has been called into question (65). moreover, the application of drugs or dietary strategies for modifying no metabolism in order to exploit the therapeutic potential of nitrates as no sources has been increasingly discussed in the literature (46, 66). it should be taken into account that, despite the predominantly regulatory role of nanomolar no concentrations, no applied at high (micro- to millimolar) concentrations generates toxic compounds, such as no2, n2o3, and especially onoo- (peroxynitrite) that impair thiol groups of organic molecules and react with protein tyrosyl groups and dna nitrogenous bases in various mammal cells and microbial symbionts (28, 50, 58, 67). carbon monoxide has long been considered as the most widespread air pollutant and a silent killer because of its high affinity for reduced iron in hemoglobin that transports oxygen to the tissues of the animal / human organism. various plants and animals, including humans, have been revealed to synthesize co as an intermediate product formed during heme degradation by heme oxygenases. currently, three kinds of heme oxygenases (ho-1 to 3) are known ; the inducible (ho-1) and the constitutive (ho-2) enzymes are of paramount importance in terms of endogenous co synthesis. ho-1 activity is induced by various stressors and widely spread in liver, kidney, and spleen cells as well as in aged erythrocytes. ho-2 is located in neurons, other brain cells, and the endothelial layer of blood vessels ; the enzyme is activated by ca - calmodulin, glucocorticoids, and opiates (37, 43). co is also formed by bacteria, including pathogens, plant and animal symbionts, and soil and marine species that contain heme oxygenases (28, 68). this enzyme is responsible for the anaerobic metabolism of co, which is the sole carbon source in a number of bacteria, for example, rhodospirillum rubrum (69). co is a sufficiently stable molecule that easily enters cells because it readily crosses the cell membrane ; inside a cell, it exerts its biological effects, including anti - apoptotic, anti - proliferative, anti - inflammatory, cytoprotective, and other activities, both at the co generation site and at a sufficiently large distance from it (43). co also regulates ion channels / transporters in various subtypes of epithelial cells (26, 27). recently, convincing evidence has been presented that co possesses all typical properties of a gasotransmitter / gasomodulator with a broad biological action spectrum, when applied at physiological concentrations (28, 70). the protective influence of co on the central nervous system was investigated in model systems. co inhalation (up to 250 ppm) protects test animals against i / r brain injury and ischemic stroke (71). the same co concentration was revealed to prevent neurological damage (neuronal apoptosis) in a pig model of deep hypothermic circulatory arrest. pretreatment of primary cultures of cerebral granular neurons with co (250 ppm) prevented apoptosis caused by oxidative stress or exogenous glutamate. the co - dependent protection implicates activation of guanylate cyclase and, subsequently, mitochondrial atp - sensitive k+ channels. this is accompanied by an increase in intracellular ros level and stimulation of no formation (72). in studies with hippocampal neurons, it was established that the key early stage of their apoptosis is activation of membrane k channels, which promotes the extrusion of potassium ions from the cytoplasm and initiates apoptosis. co can prevent apoptosis by directly inhibiting these channels (43, 72, 73). the neuroprotective effect of co in this system was attributed to its capacity to stimulate concomitant formation in neurons of two kinds of protective compounds, ros and no. nonetheless, studies with human neuroblastome cells (sh - sy5y) revealed that long - term treatment with a co donor (co - rm-2) induced symptoms of cell injury caused by lowering antioxidant activity or inhibiting no formation. the capacity of co at a concentration of 1,000 or 3,000 ppm (action time 40 min) to disrupt ca - dependent signaling pathways in sh - sy5 cells and in the homogenate of the total brain tissue of rats appears to be due to its modulating effect on the target protein, plasma lemma ca atpase (pmca) (74). intracellular h2o2 production in the brain tissue is increased at high co concentrations, which is accompanied by the enhanced production of hydroxyl radicals and a decrease in the ratio between the reductive and oxidative processes in mitochondria (72). hence, the action time and concentration of co at the target site determine whether its effect is beneficial or detrimental, in an analogy to the majority of drugs. currently, co is envisaged as a physiological signal molecule regulating the functions of membrane channel proteins and transporters (73). it has been established that the antimicrobial and anti - inflammatory effects of co and co - releasing molecules (co - rms) (e.g. metal carbonyl co - rm-3, ru(co)3cl, and glycinate) implicate the opening of k+/na+ channels in eukaryotic and bacterial cells, which decreases the proton motive force and disrupts ion transport for a short time. the mechanisms of protection of the nervous and cardiovascular systems in the presence of co - rms have not been completely elucidated yet. there is evidence that co - rm2 behaves as an inhibitor of voltage - activated potassium channels ; and mitochondria represent the main target of co. this does not rule out an additional effect of co - rms, the stimulation of ros production in mitochondria. it was established that the co released at low co - rm2 concentrations can produce a cardioprotective effect, due to its antioxidant properties. recently, increasing attention has been paid to the use of heme oxygenases, co inhalation, and co - rms for treating various infection and inflammation processes as well as cardiovascular and, potentially, neurophysiological problems (28, 70, 72, 73, 75, 76). hydrogen sulfide is a very water - soluble gas. at a concentration of 1 ppm, it can be recognized because of its rotten egg odor ; 4 ppm h2s causes a headache ; at still higher concentrations (500 ppm and above), h2s can produce a lethal effect (37, 39). the equilibrium ratio between its three forms (h2s, hs, and s) varies depending on medium ph. in the organism, this gas easily enters the cells via passive transfer across the membranes. acute intoxication is due to h2s binding to the iron of cytochrome c oxidase, which inactivates the enzyme and abolishes oxidative phosphorylation in mitochondria (39). despite its toxic effect, h2s has recently been established to play a vital role in bacteria, plants, and invertebrate and vertebrate animals, including mammals. predominantly, the synthesis of this gas involves three enzymes : cystathionine--synthase (cbs), cystathionine--lyase (cse or cth), and 3-mercaptopyruvate sulfur transferase (3-mst) (43). h2s - synthesizing enzymes are expressed, to a different extent, in the cardiovascular, nervous, immune, urinary, respiratory, and gi systems (77). the microbiota of the large intestine, which includes more than 26 genera, is one of the key factors implicated in the metabolism of s - containing compounds and endogenous generation of h2s in the human organism. a red meat - enriched diet stimulates h2s synthesis by supplying the large intestine with a significant amount of sulfated proteins. the human large intestine contains considerable h2s amounts that predominantly result from h2s formation from inorganic (e.g. sulfates and sulfites) and organic (methionine, cysteine, taurine, sulfate - containing polysaccharides, and lipids) compounds (78, 79). sulfur - containing organic compounds, including those present in garlic, onion, and other food stuffs, supply the organism with its h2s pool (78). sources of microbial h2s include, for example, e. coli strains that possess two enzymes (l - cysteine transaminase and 3-mst), which catalyze its formation. the bacterial production of h2s can also involve cbs and cse or cth (80). some representatives of intestinal bacteria (prevotella, bacteroides, helicobacter, peptococcus, and akkermansia) produce glycosyl sulfatases or similar enzymes that promote production of sulfates from sulfomucins (78). sulfate - reducing bacteria compete with methanogenic microorganisms for h2 molecules both in vitro and in vivo. in the human large intestine, desulfovibrio vulgaris is predominantly responsible for h2s generation by reducing various sulfur - containing compounds, including sulfates and s - containing organic substances, e.g., cysteine. the bacteria of the large intestine ferment cysteine, yielding h2s, ammonia, and pyruvate. if the oxygen content is low (under microaerophilic conditions), h2s at millimolar concentrations can serve as an electron donor and an energy source. if food contains a limited amount of cysteine, endogenous and microbial cse activity and, therefore, h2s production are increased ; conversely, enriching food in cysteine or the chemical / genetical suppression of cse activity results in a decrease in h2s production (81). within the lower part of the mammal gi tract, h2s behaves either as a potential toxin or as a signal molecule, depending on its concentration (28, 82). at high (millimolar) concentrations, h2s is a highly toxic compound that causes a whole spectrum of pathological processes (78), including those brought about by inhibiting mitochondrial functions ; it also produces genotoxic effects by damaging the dna (78, 83). in contrast, when applied at low (micromolar) concentrations, h2s serves as an inorganic electron donor for mitochondria. h2s regulates a number of physiological processes, such as the inflammatory response, apoptosis, cell proliferation, neuronal impulse transfer, and smooth muscle tone (83). the effects of h2s applied at physiological concentration are mainly focused on the cardiovascular and the nervous systems (77). the varied regulatory effects of h2s are due to its capacity for modifying proteins via reducing disulfide (s = s) bonds or attaching a sulfur atom to a thiol group (sh). as a result these important post - translational processes change the conformation and functional activity of proteins responsible for transmembrane ion transport or enzymes involved in protein phosphorylation / dephosphorylation and synthesis of secondary metabolites and cofactors (39). intestinal h2s of microbial origin is predominantly degraded by intestinal epithelial enzymes (39). both free and sulfate - conjugated h2s is excreted from the organism, predominantly by the kidneys (38). the physiological effects of h2s are due to the influence of this molecule on various molecular targets in diverse tissues, including heme - containing proteins, ion channels, and signal proteins. in the presence of glutathione, cysteine, or dihydrolipoic acid, h2s is released from the lysate of cultured neurons and astrocytes at ph 8.08.4. when excited, neurons take up sodium ions and excrete potassium ions, which results in increasing the intracellular potassium concentration and depolarizing the membranes of adjacent astrocytes. the main h2s targets include atp - sensitive potassium channels as well as calcium and chloride channels. there is sufficient evidence that the (neuro)modulatory effect of h2s on cell functions and physiological processes is due to its interaction with several cell transporter systems. it was revealed that h2s enhances the activity of transporter systems by facilitating the release of antioxidants that are required for protecting the systems against exogenous toxic substance - caused damage. the h2s - transporter interactivity plays a major role in maintaining the redox potential of nervous cells. this is an additional mechanism of the neuroprotective and neuromodulatory activities of this gaseous substance. of special note is the impact of h2s on various types of k channels that are essential for the transfer of ions in epithelial cells. h2s is likely to indirectly affect na transfer by acting on the proteins of k+ channels and transporter molecules (26, 27). the influence of h2s, a biological signal molecule, on neuronal activity in the hippocampus, cerebellum, cortex, and brain stem has been researched during the course of more than 15 years. it was established that h2s is an active neuromodulator and neuroprotector in various brain cells (31, 37, 39, 84). at physiological concentrations, h2s functions as a synaptic activity modulator. cbs, which is present in the cells of various brain areas, is responsible for the generation of h2s. it activates transmembrane atp - associated channels (in neurons both inside and outside the brain) via modulating glutamate - dependent n - methyl - d - aspartate receptors. this gaseous modulator also regulates the activity of serotonergic neurons and induces the release of corticotrophin - releasing hormone (39, 84). two different forms of sulfur, acid - labile and bound sulfur, are stored in brain cells. acid - labile sulfur is incorporated in the iron - sulfur centers of mitochondrial enzymes involved in oxidative phosphorylation. significant amounts of bound sulfur are present in the cytoplasm of brain neurons and astrocytes. release of h2s from the lysate of cultures neurons and astrocytes proceeds at ph 8.08.4. the excitation of nervous cells results in sodium influx into and potassium efflux from the cells. a drastic increase in ambient potassium concentration results in the depolarization of the membranes of adjacent astrocytes. to abolish membrane depolarization, the proteins responsible for na+/hcotransfer are activated. taking up hco results in the alkalinization of the intracellular content of astrocytes, which causes h2s release from sulfur - binding compounds. unbound h2s (9.2 m) is retained for a longer time in the brain tissue than in liver and heart cells (84). in astrocytes, h2s also influences the intracellular level of calcium that plays a major role in intercellular communication. the intracellular calcium level rapidly increases upon the addition of h2s ; subsequently, it slowly decreases. these effects of h2s and various h2s donors were revealed in astrocyte cultures and in the glia of hippocampal sections (84). h2s was established to exert an influence on the operation of the peripheral nervous system, which involves modulating pain perception and the transfer of pain signals to the relevant brain areas (39). the neurotoxic effect of glutamate on brain tissue cultures is partly due to inhibiting the entry of cystine into the cells. h2s can mitigate the toxic effect by reversibly inhibiting cystine transfer by glutamate and, therefore, stimulating cystine influx into the cells (31, 33). there is a supplementary pathway of synthesizing h2s from d - cysteine, which involves 3-mst and d - amine oxidase (33). in contrast to the pathway of h2s synthesis from l - cysteine, the d - cysteine - dependent pathway predominantly functions in the cerebellum and the kidneys. studies with the primary cultures of cerebellar neurons revealed that the cerebellar tissue does not sustain hydrogen peroxide - induced oxidative stress if d - cysteine is available (33). the discovery of the d - cysteine - dependent pathway of synthesizing h2s provides foundations for a new therapeutic technique that is based on delivering this gas to target tissues (85). h2s and s - adenosyl - methionine impede the increase in the glucocorticoid concentration in blood plasma under stress. it was established that low h2s concentrations are capable of neutralizing reactive oxygen and nitrogen species (superoxide radical, hydrogen peroxide, peroxynitrite, hypochlorite, and so on) and of reversibly inhibiting the mitochondrial respiratory chain. it is the antioxidant effect of h2s that is responsible for its neuro- and cardioprotective activities (39). currently, it is widely accepted that h2s, like no and co, is an important neurotransmitter. the modulatory effect of h2s on cell functions and physiological processes is due to its interaction with several transporter systems. it has been established that h2s enhances their activity by releasing antioxidants that provide protection from exogenous toxic factors. the h2s - transporter systems interactivity plays a major role in maintaining the redox potential of nervous cells ; this is an additional mechanism of the neuroprotective and modulatory activity of this gaseous agent. an influence of h2s on human behavior was suggested for the first time in studies with human subjects with seizures, psychiatric disorders, or abnormal electroencephalograms ; most of the subjects lacked the enzymes (cbs) that are involved in h2s synthesis. subsequently, it was revealed that patients with down syndrome, in contrast, are characterized by abnormally high concentrations of these enzymes in the brain tissue (86). the h2s content in the brain tissue was decreased by over 50% in alzheimer patients, and this deficiency is apparently due to a drastic (70%) decrease in the concentration of s - adenosyl - methionine that activates cbs. prevented nervous cell damage and apoptosis in a model system in which this disease was caused by administering the toxin rotenone to test animals (39). there is evidence that h2s functions as a signal molecule in the visual system of mammals. h2s synthesis - catalyzing enzymes (cbs and cse) were detected in various kinds of eye cells, and h2s was found to regulate sympathetic and glutamatergic neurotransmission during the signal transduction processes in this system. further data on the regulatory influence of h2s on ion channels and transporters will contribute to our understanding of the role of h2s in relation to the risk of development of ocular neuropathies (87). even though the use of gaseous h2s for therapeutic purposes is hardly feasible, we can apply chemical compounds that release h2s in the human organism either rapidly (nahs) or slowly (gyy 4137). this gives grounds for the suggestion that h2s should be used for medical purposes (88). it is important that while the one - time use of nahs provided protection for neurons from oxidative stress, the repeated administration of this substance produced a toxic effect on these cells (84). nonetheless, h2s treatment is considered an efficient therapeutic technique in a number of diseases (e.g. lung cystic fibrosis and kidney problems in patients with hereditary hypertension) that are characterized by enhanced na+ influx into cells (81). in all likelihood, the employment of chemical donors or microbial producers of h2s (as an ion channel / transporter modulator) for medical purposes will hold much promise as a potential pharmacological approach to the treatment of a number of neurodegenerative diseases. ammonia is one of the end products of degradation of proteins, peptides, urea, and various amino acids. in the human organism, nh3 is predominantly formed by the intestinal microbiota and the cells of the gi tract, kidneys, the liver, and muscles. at least 410 g of nh3 are daily synthesized in the intestines of adult human individuals. among aerobes, gram - negative intestinal bacteria of the genera proteus, klebsiella, and pseudomonas as well as e. coli are the most active nh3 producers ; active nh3-producing anaerobes include the genera clostridia, ruminococcus, bacteroides, and some lacto- and bifidobacteria. peptidococci, ruminococci, coprococci, bifidobacteria, lactobacilli, clostridia, bacteroides, and some streptococci and enterococci exhibit significant urease activity. in healthy human individuals, up to 7 g of urea are degraded daily by microbial ureases (amounting to 50% of the total pool of this compound) (89, 90) and by those of fungi (candida albicans) (91). the involvement of microorganisms in ammonia metabolism is consistent with the fact that the intestines of germ - free animals contain 2030% less urea than their conventional counterparts. after administering carbon - labeled urea to these animals, intestinal urease - producing microorganisms form ammonia that is transferred via the portal vein to the liver, where it is reincorporated into urea. gi microorganisms also incorporate nh3 into amino acids are synthesized de novo using co2 or acetic, propionic, and other organic acids as carbon sources. unless utilized in biochemical processes in the large intestine, microbially produced nh3 rapidly passes through mucous membranes and spreads within the organism. for the most part, intestinal unbound nh3 reaches the liver via the portal vein ; in the liver, it is virtually completely converted into urea and glutamine via a series of reactions (the urea cycle). the unbound nh3 content in the blood of healthy adults is approximately 35 m (ca. unbound nh3 circulates in the organism ; it is excreted with urine and, to a lesser extent, with feces. ammonia - derived metabolites are also excreted with urine or, alternatively, used for synthesizing amino acids and other biological molecules. endogenous nh3 formed in the brain and in peripheral tissues is not transferred to the liver ; instead, it is transformed in these tissues into glutamine and alanine (89, 90). a genetic disruption of the biosynthesis of urea cycle enzymes, liver and kidney dysfunction, excessive nh3 formation in skeletal muscles caused by physical exertion or other kinds of stress, or an imbalance in the intestinal ecological system result in increasing nh3 content in the organism to a toxic level (hyperammonemia). for instance, liver cirrhosis is associated with the formation of a direct bypass between the portal vein system and the bloodstream ; this prevents the detoxification of harmful gi tract - produced compounds, including nh3, that reach the bloodstream. increased nh3 concentrations penetrate into the brain tissue, which is a major factor of pathogenesis of hepatic encephalopathy (he). it manifests itself in fatigue, ache, muscle weakness, loss of appetite, nausea, vomiting, diarrhea, pain in the back, sides, or the abdomen, and motor and cognitive disturbances. in the early 1970s, it was established that the genetic disruption of the urea cycle results in pathological changes in the brain of newborns. it was suggested that gaba is implicated in causing ammonia - induced toxicity in the nervous system. an increase in ammonia concentration in the brain tissue results in stimulating gaba - induced chloride channels in neurons and astrocytes. evidence was presented that liver dysfunction - induced hyperammonemia is accompanied by changes in cell energy metabolism and formation of excessive glutamine amounts in astrocytes. it is astrocytes that incorporate ammonia in glutamine molecules after it crosses the blood - brain barrier (bbb). hyperammonemia is also responsible for osmotic stress in the brain, which results in redistribution of cerebrospinal fluid and causes low - grade swelling in astrocytes and edema in the white matter as well as an increase in intracranial pressure (9295). ammonia also inhibits energy production in mitochondria, which is attributed to ammonia 's capacity to suppress ketoglutarate dehydrogenase activity and stimulate glycolysis (94). an increase in ammonia concentration in the arteries affects the expression of a number of genes that code for neuroglial proteins. these proteins regulate cell growth, mitochondrial functions, and transfer of neuroactive amino acids (95). when applied at supraphysiological concentrations, nh3 induces rapid release of glutamate from neurons. in humans, this results in the development of irritability, aggression, hyperexcitability, and even movement disorders. hyperammonemia also promotes removal of gaba from neurons, which frequently manifests itself in somnolence or lethargy ; these symptoms may ultimately cause the development of a comatose state (95, 96). apart from the direct neurotoxic effects, high nh3 concentrations increase the permeability of the bbb ; modulate the serotonergic and dopaminergic systems of the brain ; cause the accumulation of abnormal neurotransmitters, such as octopamine, in the brain ; and result in glucose intolerance and increased urinary output calcium and phosphate concentrations (12, 95). systemic inflammation that constantly accompanies acute and chronic liver dysfunction represents an important risk factor in terms of encephalopathic complications. hyperammonemia and the attendant neuroinflammatory response to liver cirrhosis cause microglia activation and monocyte recruitment ; the enhanced synthesis of proinflammatory cytokines (tnf, il-1, and il-6) ; the accumulation of ammonia, lactate, and manganese ; and an increased permeability of the bbb. encephalopathy may result from the synergistic effect of ammonia and cytokines (93, 95). astrocytes lose the capacity to adequately regulate their own volume, glutamine accumulation is increased, and a cascade of signaling processes is triggered. this results in enhanced ca accumulation and an increased formation of reactive oxygen and nitrogen species, which is due to the activation of nadph oxidase and nos (93). some patients suffering from hyperammonemia and the attendant gi dysfunction (constipation or diarrhea) exhibit psychotic symptoms and movement disorders that resemble those typical of genuine autism spectrum disorders (35, 91, 96). the important role of microbial nh3 with respect to the brain functions and life expectancy of he patients is consistent with the fact that the patients ' state is markedly improved after administering the antibiotic rifamycin, the prebiotic lactulose, and probiotics to them (95). the neuropsychic state, including cognitive capacities, is also improved by synbiotics, for example, by the bifidobacterium longum - fructo - oligosaccharide combination (12). neural inflammation, brain edema, and the resulting encephalopathy symptoms that are due to increased nh3 concentrations in the brain tissue can be mitigated by minocycline, an inhibitor of microglia activation, and n - acetylcysteine, as well as by mild hypothermia ; all these techniques produce neuro- and hepatoprotective effects (95). the data presented in this review provide compelling evidence that a whole gamut of gaseous endogenous and microbial products perform important functions in neurophysiological, biochemical, microbiological, and medical terms. the (patho)physiological effects produced by most of these gases in the organs and tissues of the organism have been studied in detail. unfortunately, determining the concentrations of these gases in target cells presents serious difficulties that are due to the limitations of currently available measurement techniques and the gases ' high reactivity and short lifetime. most endogenous and microbially produced scfas and gases readily pass through the mucosa layer and cell membranes and exert toxic effects on mammal cells if applied at high concentrations. in contrast, many gases behave as broad action spectrum regulators within the physiological concentration range. in particular, they influence the functions of the nervous system by serving as nutrients, metabolites, or regulators involved in the operation of various kinds of nervous cells. this is the reason for classifying them as gasotransmitters and/or gasomodulators. in microorganisms, the aforementioned gaseous products function as intermediates in denitrification (the role of no) and oxygen - free sulfate respiration (the function of h2s) or as non - conventional carbon and energy sources (the role of co).. the gases can promote their survival and metabolic activity under various conditions, and specifically in the presence of antibiotics and in the host microbiota system. formation of gasotransmitters / gasomodulators by intestinal bacteria and host cells provides an illustrative example of (patho)physiological effects of both the microbiota and the diet (which influences gas production by the host and the microbiota). the synthesis and biological activities of each of the aforementioned gases are, to an extent, influenced by other gases. therefore, most gas molecules are to be regarded as cooperatively functioning low molecular weight agents that control a specific function or a whole complex of functions (25, 28, 43, 95, 97). gases differ in stability and, therefore, can produce their effect either directly on the site at which they are formed (h2s) or on remote tissues / organs (e.g. co and nh3) (43). no molecules can diffuse and influence metabolic processes within a distance of several cell diameters (50). owing to their high reactivity, gases do not accumulate locally ; instead, they rapidly reach their target cells where they interact with intracellular enzymes, transporters, and ion channel proteins (26, 27, 40). a large family of genes coding for receptors in cation channels (trp channels) has been revealed. they function as polymodal detectors (sensors) of a wide spectrum of extra- and intracellular signals. six similar protein families were detected ; they form trp channels (trpc, trpv, trpm, trpa, trpp, and trpml) (98). a large number of gas molecules, for example, o2, no, co, h2s, and ch4, can penetrate cell membranes, which is a prerequisite for performing the aforementioned auto-, para-, or endocrine functions. the gases ' capacity to influence cell - environment interaction, electrolyte homeostasis, and cell - cell electrochemical communication via changes in redox signaling, gap junction channels, and ion channel / transporter regulation enables them to regulate a plethora of physiological processes in cells and tissues (26, 27, 99). many gases can form covalent bonds with prosthetic metal complexes in receptor proteins or non - covalent bonds with protein regulatory subunits. they occupy the space within and around a protein, which prevents other gases from accessing its functionally active sites (98). ion channels, for example, trpv1 and trpa1, can behave as sensory protein molecules that transduce gas - conveyed signals into electric signals in neurons, including those of nervus vagus. the transmission of these signals allows the nervous system to initiate processes that enable, for example, pain perception. at this point, it should be noted that behavioral analysis, monitoring neurotransmission in various pathways, exploring synaptic plasticity in models such as long - term potentiation (ltp) and ltd, are the bread and butter of present - day neuroscience. studies with animals revealed that some gasotransmitters or their precursors (at appropriate concentrations) are therapeutically efficient and safe, and it seems feasible to use them for treating a number of pathological processes in the human organism. in fact, some of the aforementioned gases have already been employed for therapeutic purposes (25, 28, 43, 56, 83). we suggest using the recently coined term psychobiotics (16) to denote probiotic bacterial strains that are employed as biologically active additives or functional nutrients for optimizing the gasotransmitter pools of the human organism and for beneficially influencing brain processes and behaviors that are subject to regulation by scfas, nitric oxide, carbon monoxide, h2s, and ammonia.further comparative studies using germ - free and conventional animal models as well as tissue cultures (100) will enable us to elucidate in detail the molecular mechanisms and cell targets of the recently discovered world of gasotransmitters, regardless of whether they represent host - produced, microbial, or diet - derived substances. the authors have declared that they have no interests that might be perceived as posing a conflict or bias.	the symbiotic gut microbiota plays an important role in the development and homeostasis of the host organism. its physiological, biochemical, behavioral, and communicative effects are mediated by multiple low molecular weight compounds. recent data on small molecules produced by gut microbiota in mammalian organisms demonstrate the paramount importance of these biologically active molecules in terms of biology and medicine. many of these molecules are pleiotropic mediators exerting effects on various tissues and organs. this review is focused on the functional roles of gaseous molecules that perform neuromediator and/or endocrine functions. the molecular mechanisms that underlie the effects of microbial fermentation - derived gaseous metabolites are not well understood. it is possible that these metabolites produce their effects via immunological, biochemical, and neuroendocrine mechanisms that involve endogenous and microbial modulators and transmitters ; of considerable importance are also changes in epigenetic transcriptional factors, protein post - translational modification, lipid and mitochondrial metabolism, redox signaling, and ion channel / gap junction / transporter regulation. recent findings have revealed that interactivity among such modulators / transmitters is a prerequisite for the ongoing dialog between microbial cells and host cells, including neurons. using simple reliable methods for the detection and measurement of short - chain fatty acids (scfas) and small gaseous molecules in eukaryotic tissues and prokaryotic cells, selective inhibitors of enzymes that participate in their synthesis, as well as safe chemical and microbial donors of pleiotropic mediators and modulators of host intestinal microbial ecology, should enable us to apply these chemicals as novel therapeutics and medical research tools.
acute gastrointestinal (gi) bleeding is a common emergency condition and an important cause of mortality. furthermore, the gi bleeding source is not found in approximately 3 - 5% of patients by esophagogastroduodenoscopy (egd) and colonoscopy. in these cases, most lesions responsible are found in the small bowel. however, gastrointestinal bleeding originating in the small bowel is often difficult to diagnose and treat. hemangiomas originating from the small bowelare uncommon benign tumors, and may cause massive or occult gi bleeding. although manytools can be used to diagnose tumors in the small intestine, hemangiomas with a small intestine origin are difficult to differentiate from other more common entities. recently, single incisional laparoscopic surgery has been utilized to treat various benign conditions in the abdomen. here, we report a case of jejunal polypoid hemangioma, causing recurrent gi bleeding and subsequent life - threatening anemia, which was treated using a single incisional laparoscopic approach. an 81-year - old female patient complaining of intermitt ent melena for 3 months, and of nausea and dizziness, which were aggravated just days before presentation, was found to have severe anemia. at presentation on emergency room, her initial hemoglobin and hematocrit levels were 4.7 g / dl and 15.8%, respectively. however, because her vital signs and performance status were stable, we went ahead the diagnostic evaluations with blood transfusions instead of the emergency operation. she had a medication history of hypertension and diabetes mellitus, but had not taken aspirin. colonoscopic findings were unremarkable except for a 0.3 cm polyp in sigmoid colon, which also had no evidence of bleeding. an emergency abdominal ct scan was performed, and demonstrated a highly enhancing polypoid tumor in the distal ileum (fig. 1). diagnostic considerations included ; adenocarcinoma or lymphoma, a polyp, a carcinoid tumor, or a vascular lesion. however, a gi contrast study failed to demonstrate any bleeding focus or mass lesion. surgery was performed using a single incisional laparoscopic approach with a surgical glove and a wound protector. the patient was placed in the supine position under general anesthesia with an endotracheal tube, and a 2-cm vertical transumbilical incision was made (fig. 2) and the abdominal cavity accessed. a small allexis wound retractor (applied medical, rancho santa margarita, ca, usa) was placed, and a size 6 surgical glove was installed over the external ring. the thumb and middle finger of the glove were partially cut and 10-mm trocar for a videoscope and 5 mm trocar for a working device was placed and tied. when the intra - abdominal cavity was explored, an intussusceptum was found at the distal jejunal level (fig. the small bowel, including the intussusceptum, was taken out of the abdominal cavity, and segmental resection of the small bowel and end - to - end anastomosis by hand sewing were performed extracorporeally. the anastomosed small bowel was then placed in the abdominal cavity, and the wound was closed after saline irrigation. the resected mass was soft, pale brown, and 2 cm in size (fig. 4), and was histologically confirmed to be a jejunal polypoid hemangioma (fig. 5). the patient was discharged on postoperative day 7 without any complication. hemangiomas of the gi tract are uncommon, and account for only 0.05% of all intestinal neoplasms and 7 to 10% of all benign tumors of the small bowel. ninety percent of hemangiomas are clinically evident, and present with symptoms, such as, acute or chronic gi hemorrhage, anemia, or obstruction, and rarely with platelet sequestration. other potentially serious complications of hemangiomas of the gi tract, such as, intussusception, small bowel obstruction, perforation, malabsorption, and bleeding from other sites of involvement, may also occur. bleeding is one of the symptoms associated with a small bowel neoplasm, and is usually occult and requires an extensive gi evaluation before a diagnosis is obtained. however, the diagnosis and localization of small bowel tumors remains a clinical challenge, because of the inaccessibility of this region to conventional diagnostic modalities. ct is frequently used as a front line tool for the evaluation of abdominal symptoms, especially in critically ill patients. ct scans show transluminal thickening of the wall of involved bowel loops with non - homogenous and persistent lesion contrast enhancement. double contrast studies demonstrate a nodular defect, which may change in configuration after compression or distension, which suggests a soft, possibly vascular tumor. the detection of this pathologic finding by double contrast study depends on the size of lesion and on the presence of active intestinal peristalsis. livengood and associates described the feasibility of the angiographic localization of hemangioma of the small bowel. they performed angiography with methylene blue, which allowed lesions to be identified from an extraluminal vantage point. this method avoids the guesswork involved in transillumination and palpation for tumor localization during laparoscopy. in our case, we performed egd, colonoscopy, an abdominal ct scan, and a double contrast study to indentify the bleeding focus, and with the exception of abdominal ct, these modalities did not indenty the problematic lesion. laparoscopic small bowel resection is an established technique and is performed by exteriorizing the diseased bowel segment and using traditional resection and anastomotic techniques. recently, multiple attempts have been made to reduce parietal trauma and visible scar formation even after laparoscopic surgery, and patient satisfaction has become a rapidly evolving issue, particularly in terms of single incisional laparoscopic surgery. this issue reflects the importance of cosmesis and body image trauma associated with surgical procedures, and many surgeons have devised " scarless " surgical procedures using standard laparoscopic instruments. in the described case, bleeding that originates from the small bowel presents challenges in terms of diagnosis, localization, and treatment. in addition to its superior cosmetic results, a single incisional laparoscopic approach causes less morbidity by minimizing skin incisions. however, some bleeding lesions in the small bowel may be manifestations of a malignant process, and thus, it is essential that the surgeon has multiport laparoscopic skills, because these are vital for safe and effective single incisional laparoscopic surgery.	bleeding lesions in the small bowel are a much more significant challenge in terms of detection and treatment than those of the stomach or the large bowel, and require extensive gastrointestinal evaluation before a diagnosis can be made. the authors report the case of an 81-year - old female patient who underwent small bowel segmental resection by single incisional laparoscopic approach for distal jejunalhemangioma, which caused severe anemia. an abdominal computed tomography scan demonstrated a highly enhancing polypoid tumor in the distal ileum. during the single incisional laparoscopic exploration using a 2 cm sized skin incision, jejuno - jejunal intussusceptions and a jejunal tumor were noted. single incisional laparoscopy was performed to assist the jejunal segmental resection. pathologic reports confirmed the lesion to be a jejunalhemangioma. the authors report an unusual case of jejunalhemangioma caused by intussusception and gastrointestinal hemorrhage, which was treated by single incisional laparoscopic surgery.
chronic urticaria not responding to high - dose antihistamines is a therapeutic challenge, and in such cases other systemic treatment options should be considered. the literature is scarce in defining effective immunosuppressive drugs that may be used for long - term treatment. systemic corticosteroids are usually effective but are not feasible as maintenance therapy, and other immunosuppressive drugs such as azathioprine, methotrexate, oral tacrolimus, and mycophenolate mofetil have only been used in case reports or small patient series. in two randomised, double - blind, and placebo - controlled trials cyclosporine a was found to be effective in controlling recalcitrant chronic urticaria [4, 5 ]. finally, recent reports also point to omalizumab, a recombinant monoclonal antibody that inhibits the high - affinity fc receptor of ige, as an effective agent in patients with refractory chronic urticaria [68 ]. tumour necrosis factor alpha (tnf - alpha) inhibitors have so far only been used to treat a total of eight patients with chronic urticaria according to available publications [911 ]. here we present our experience in 20 adult patients with severe refractory chronic urticaria who were received with either adalimumab or etanercept and thereby significantly expand our knowledge of the use of tnf - alpha inhibitors for this indication. the patients described herein were a retrospective sample of patients with chronic urticaria (duration of urticaria ranged from seven months to 46 years with a mean of 13 months) seen in the outpatient clinic of a tertiary dermatological referral centre. twenty adult patients with severe chronic urticaria with or without angioedema that was refractory to high - dose antihistamines and at least one immunosuppressive agent were offered off - label monotherapy with either adalimumab 40 mg twice monthly or etanercept 50 mg once weekly. for the main part of the patients, adalimumab was chosen over etanercept as first choice therapy, but this choice was not based on a predefined belief of superiority of this drug over the other. previous therapy with high dose antihistamines up to four times daily of cetirizine 10 mg, loratadine 10 mg, desloratadine 5 mg, or fexofenadine 180 mg, prednisolone up to 25 mg once daily, azathioprine up to 100 mg daily, cyclosporine a up to 3 mg / kg daily, mycophenolate mofetil up to 500 mg twice daily, dapsone up to 50 mg twice daily, colchicine up to 0.5 mg twice daily, or omalizumab 300 mg once every four weeks was either ineffective or associated with unacceptable side effects, and therefore alternative therapy was considered appropriate. urticaria patients were screened for signs of systemic disease or chronic infection with a clinical interview, and urine analysis and culture, throat swab for streptococci, and an ice cube test for cold - induced urticaria were performed. further evaluations were performed as appropriate including urea breath test for the diagnosis of helicobacter pylori, stool culture, chest and sinus x - rays, and skin prick tests for common aero- or food - allergens. blood samples were taken including complete blood count, electrolytes, thyroid stimulating hormone, antinuclear antibodies, c - reactive protein, hepatitis b and hepatitis c screening, immunoglobulins a, e, g, and m, and kidney and liver function. furthermore, a serum - induced basophil histamine release test, hr - urticaria test, was performed (reflab, copenhagen, denmark). if the hr - urticaria test was found positive (> 16.5% of total histamine content), patients were categorised as having chronic autoimmune urticaria (cau). in total, only two patients had cau. if the hr - urticaria test was found negative (50% reduction in symptoms and signs), and no / limited response. furthermore, the duration of therapy and any signs of adverse effects of adalimumab and etanercept were recorded. a total of 12 patients (60%) obtained resolution of urticaria and/or angioedema after initiation of therapy with either adalimumab or etanercept (table 1). none of the patients worsened during treatment, but five patients had no benefit from the treatment. these patients were not offered treatment with another tnf - alpha inhibitor. among responders, response to treatment was observed within the first month after initiating therapy with either adalimumab or etanercept. however, to achieve long - term relief of urticaria symptoms, continuous treatment with tnf - alpha inhibitors was needed, as intermission led to return of symptoms within a few weeks. therefore, therapy with tnf - alpha inhibitors was not regarded as curative. the duration of treatment among responders ranged between three and 30 months with a mean of 11 months. patients were allowed to continue antihistamines during treatment with tnf - alpha inhibitors. however, the patients who had benefit from treatment were able to reduce or discontinue antihistamines. six patients (30%) developed adverse reactions during treatment with adalimumab or etanercept. in five patients, these were restricted to increased frequency of mild upper respiratory infection not requiring antibiotics or hospitalization, whereas one patient, a 31-year - old female, was hospitalized due to severe headache, dizziness, vertigo, and decreased muscle strength of the upper extremities three months after initiating treatment with adalimumab. in this patient a lumbar puncture showed cerebrospinal lymphocytosis and increased protein concentration indicating a cns toxic reaction. however, an mr scan of the cerebrum did not reveal any abnormalities and her symptoms gradually disappeared within six months following discontinuation of adalimumab. among 20 patients with chronic urticaria who were unresponsive to treatment with high - dose antihistamines and one or more standard immunosuppressive therapies, we observed complete or almost complete resolution of symptoms in 60% and partial resolution in a further 15% of the patients with the tnf - alpha inhibitors adalimumab or etanercept. responders had csu or cau with and without angioedema, whereas patients with dpu or nu did not respond to treatment with tnf - alpha inhibitors. it is of interest that five of the 12 patients who had significant improvement with tnf - alpha inhibitors previously had failed treatment with omalizumab due to lack of efficacy or an anaphylactoid reaction. we and others have recently shown that omalizumab is effective in a significant proportion of patients with severe chronic urticaria [68 ], but this study clearly indicates that a subgroup of patients who can not be sufficiently treated with omalizumab may obtain resolution of symptoms with a tnf - alpha inhibitor. however, it is our present position that the preferred therapy for chronic recalcitrant urticaria not responding to high - dose antihistamines and/or standard immunosuppressive drugs should be omalizumab, and that tnf - alpha inhibitors should be recommended as a second - line therapeutic option. this is based primarily on the observation that omalizumab, in comparison with tnf - alpha inhibitors for this indication, has significantly fewer adverse effects during prolonged treatment, and with a more sustained and reliable efficacy. a literature search identified three publications including a total of eight patients with chronic urticaria who have received monotherapy with tnf - alpha inhibitors [911 ]. treatment of one patient with cold urticaria and one patient with dpu was successful. in one small series of six patients with refractory csu and chronic urticarial vasculitis, therapy with etanercept, adalimumab, or infliximab resulted in marked rapid improvement in all patients. the pathogenesis of chronic urticaria involves mediator release, including tnf - alpha, which exists preformed in the mast cells and which is known to be newly synthesized upon mast cell activation. the theoretical basis for the use of tnf - alpha targeting therapy is supported by a study that has shown that tnf - alpha is upregulated in patients with chronic urticaria compared with healthy controls. in addition it has been shown that tnf - alpha is expressed throughout the epidermis in both lesional and nonlesional skin of patients with chronic urticaria but not in healthy controls. interestingly the cytokine profile in the skin of patients with chronic urticaria mimics that found in patients with psoriasis, psoriatic arthritis, and rheumatoid arthritis with increased expression of tnf - alpha and il-10 and decreased expression of il-2 and interferon gamma. in conclusion this study suggests that tnf - alpha inhibitors could be considered in patients with severe chronic urticaria where other treatment options are contraindicated have been unsuccessful or have been associated with unacceptable adverse reactions. as this was only a small explorative cohort with no prespecified primary outcome, validated scoring system of response to treatment, or treatment algorithm for adalimumab and etanercept, it was not possible to draw any firm conclusions about superiority of one drug over the other, selective response in any urticaria subtype or with certain clinical, or paraclinical characteristics. this is a limitation of the study. larger controlled trials with longer followup are needed to confirm the efficacy and safety of tnf - alpha inhibitors in the management of patients with severe chronic urticaria.	patients with severe chronic urticaria may not respond to antihistamines, and other systemic treatment options may either be ineffective or associated with unacceptable side effects. we present data on efficacy and safety of adalimumab and etanercept in 20 adult patients with chronic urticaria. twelve (60%) patients obtained complete or almost complete resolution of urticaria after onset of therapy with either adalimumab or etanercept. further three patients (15%) experienced partial response. duration of treatment ranged between 2 and 39 months. those responding completely or almost completely had a durable response with a mean of 11 months. six patients (30%) experienced side effects and five patients had mild recurrent upper respiratory infections, whereas one patient experienced severe cns toxicity that could be related to treatment with tnf - alpha inhibitor. adalimumab and etanercept may be effective and relatively safe treatment options in a significant proportion of patients with chronic urticaria who do not respond sufficiently to high - dose antihistamines or in whom standard immunosuppressive drugs are ineffective or associated with unacceptable side effects.
congenital intrascleral cysts are unusual clinical conditions with uncertain etiology. the lesions are usually located at the limbus with corneal and scleral components. in this report, we present a case with progressively enlarging congenital intrascleral cyst without corneal involvement in a 30-month - old boy, and describe its clinical and histopathologic features and surgical management. a 30-month - old boy presented to the clinic with a bulbar subconjunctival cystic mass of the right eye (figure 1a). a thin - walled, multilocular scleral cyst, measuring 5 mm 8 mm in its largest dimensions, was identified at the inferior nasal quadrant, extending from the medial canthus to the limbus of the right eye (figure 1a, b, and c). the rest of the ocular examination in the right eye and the left eye was normal. during surgery, saline hydrodissection was carried out with a 30-gauge needle to separate the underlying cyst wall from conjunctiva, confirming that the lesion was intrascleral. a conjunctival incision was made through the upper margin of the mass from the 3 oclock position to the inner canthus to expose the anterior portion of the lesion by blunt dissection without perforating the cyst. the posterior aspect of the cyst was then gently cleaned with a weck - cell sponge to be sure that no residual epithelium was left behind. after the total removal of the cyst, the sclera at the posterior aspect was noted to be very thin and the underlying choroid was clearly visible. this thin area was patched with preserved fascia lata using 8.0 vicryl sutures to prevent staphyloma formation (figure 1b). conjunctiva above the patch was also closed with 8.0 vicryl sutures. at the end of the surgery, histopathologic examination revealed that the cyst wall was lined with multiple layers of nonkeratinized epithelial cells. no other complications or recurrence took place during the 6 years postoperatively (figure 1d). congenital intrascleral cysts are seen less frequently than their acquired counterparts, which typically occur after ocular surgery or trauma.1 these congenital lesions are considered to be developmental defects that usually enlarge at the first year of life, as was the case in our patient. generally, they are located at the limbus at birth as a nidus, but expansion of the cyst into the cornea may develop later in childhood.2,3 vision is often affected due to the distortion and scarring caused by the lesion on and around the limbus. in our case, the vision was normal because of the absence of the corneal component. in one reported case, mahmood and awad3 stated that corneal involvement may not occur in patients up to 3 years of age, but the cyst may enlarge in later years to disturb visual acuity. considering this possibility and due to the large size of the cyst in our patient, we operated early. histopathologic examination revealed that the cyst wall was lined with nonkeratinized epithelial cells without goblet cells. the presence of goblet cells is said to favor congenital origin.3 rao reported the presence of glandular structures simulating lacrimal gland tissue and stated that this indicates the developmental nature of the lesion. careful removal of the cyst wall, denaturation of remnants with saline and distilled water, and chemical coterization with trichloroacatic acid and tetracyclin injection are advised to prevent recurrence.4 in limbal cysts, peripheral lamellar keratoplasty is known to be effective to prevent the development of staphyloma and recurrence.5 in order to prevent staphyloma formation in our case, we utilized banked fascia lata, which worked well without complications. based on our experience in this case, we would suggest that banked fascia lata may be considered as an alternative grafting material in defect restoration when banked human sclera is not readily available. in conclusion, we report our experience with a congenital intrascleral cyst that was surgically repaired with the use of banked fascia lata and had an uncomplicated postoperative course of 6 years.	congenital intrascleral cysts are rare. they are mostly located at the limbus with corneal involvement. we report a case of a 30-month - old boy with a bulber conjunctival cyst noticed at birth. the lesion enlarged over the following months but did not involve the cornea. during surgery the cyst proved to be intrascleral and a complete excision was carried out. the remaining defect was repaired with banked fascia lata. the histopathology revealed a scleral cyst wall lined by nonkeratinizing squamous epithelium with no goblet cells. we conclude that congenital intrascleral epithelial cysts are rare but should be considered in differential diagnosis of external eye cystic lesions. in our case, early excision and repair with fascia lata led to an uncomplicated postoperative course of 6 years.
a 26-year - old male presented with painless decrease of vision in the right eye following trauma with an iron particle 4 days prior to presentation. on examination, the visual acuity in the right eye was 20/60, intraocular pressure (iop) was 12mmhg and there was localized conjunctival congestion superonasally [fig anterior segment was unremarkable, while on fundus evaluation there was clear media with normal disc, few internal limiting membrane striae in the macular area, areas of retinal vasculitis associated with isolated white - centered retinal hemorrhages, and perivascular white - colored retinal exudates localized inferiorly [fig. 1 ]. the protruding vitreous knuckle present beneath the insertion of the medial rectus muscle at its superior margin was cut with vitrector and the 2-mm - long scleral tear was sutured with 7 - 0-vicryl. after taking an undiluted vitreous biopsy with a 23-gauge vitrectomy cutter, intravitreal antibiotics, based on the microbiological culture, biochemical tests and mini api i d 32 staph strip, s. epidermidis was isolated. antibiotic susceptibility testing of the isolate was done by the kirby - bauer disk diffusion method as per clinical and laboratory standards institute guidelines (clsi, 2009). the organism was found to be resistant to ciprofloxacin, ofloxacin, cefuroxime, oxacillin and cefoxitin (methicillin), and sensitive to amikacin, cefazoline, gentamicin, vancomicin, gatifloxacin, moxifloxacin and chloramphenicol. postoperative treatment with tablet gatifloxacin (400 mg stat followed by 200 mg twice daily for 7 days), eye drop gatifloxacin 0.3% (12 times per day) and eye drop prednisolone acetate 1% (2 hourly initially, followed by tapering doses) resolved the infection. final uncorrected visual acuity was 20/20 at 6 weeks postoperatively with resolution of all retinal signs [fig. 2 ]. (a) area of occult scleral injury marked by localized congestion and chemosis ; (b) fundus photograph showing macular internal limiting membrane striae ; (c) white - centered retinal hemorrhages ; (d) midperipheral and peripheral retinal vasculitis postoperative fundus photograph showing resolution of retinal signs. various authors[357 ] have documented the appearance of hemorrhages and vasculitis as an early feature of bacterial endophthalmitis. a case of fungal endophthalmitis with vasculitis experimental inoculation of bacteria into the vitreous cavity revealed retinal periphlebitis to be the first clinical feature observed. the pathogenesis of hemorrhages and periphlebitis is attributed to the breakdown of blood retinal barrier, and thus perivascular exudation of plasma proteins and white blood cells. this could be because of the lack of awareness, rarity of this sign, more fulminant cases or delayed diagnosis or delayed presentation leading to a poor view of fundus. a medline search revealed only eight similar cases reported so far [table 1 ]. these findings may mimic the picture of retinal vein occlusion, eales disease, behcet 's, sarcoidosis, pars planitis, infectious retinochoroiditis and non - specific uveitis, but a history of recent open globe trauma or surgery or systemic infection should indicate the possibility of an infectious etiology. in post - surgical eyes with increased anterior chamber reaction, dilated inferior retinal examination should be done to look for such white - centered retinal hemorrhages and vasculitis to rule out early endophthalmitis. a delay in the diagnosis and treatment of endophthalmitis awareness about retinal vasculitis being an early presenting feature could lead to early diagnosis of endophthalmitis and good visual outcome.	the article reports a case and review of the literature of endophthalmitis presenting as isolated retinal vasculitis. a 26-year - old male was observed to have white - centered retinal hemorrhages and retinal vasculitis following an occult scleral perforation. at presentation, the visual acuity was 20/60. with clinical suspicion of early endophthalmitis, he underwent wound exploration, scleral tear repair, vitreous biopsy and administration of intravitreal antibiotics. microbiology evaluation revealed significant presence of methicillin - resistant coagulase - negative staphylococcus epidermidis. final visual acuity improved to 20/20 at 6 weeks postoperatively. literature search revealed eight similar cases, all of them due to staphylococcus species. retinal vasculitis and white - centered retinal hemorrhages can be a presenting sign of early endophthalmitis, especially with non - fulminant pathogens like s. epidermidis.
it is well documented that as the glomerular filtration rate declines, patient mortality increases. studies have consistently shown that patients with end - stage kidney disease (eskd) have a shortened life expectancy. although some patients have a gradual steady decline, punctuated by periods of acute illness, many exhibit a gradual deterioration in their functional status before a more abrupt decline in the final weeks of life. demonstrated that the functional status of the eskd patient managed conservatively is maintained until the last 12 months of life, when an abrupt decline is observed. patients with eskd appear to have a shorter duration of terminal illness than that described for many other chronic conditions. the major causes of death amongst dialysis patients are cardiovascular disease, infection and withdrawal from dialysis. cardiovascular death includes sudden cardiac death and cardiac arrest, and is estimated to account for between 22 and 27% of deaths in dialysis patients [6, 7 ]. despite the shortened life expectancy amongst dialysis patients, advance directives are underutilized by this population and many dialysis patients feel they are inadequately educated about their options in terms of withdrawal from dialysis and end - of - life (eol) care. dialysis patients have been found to suffer a high burden of symptoms and pain, comparable with cancer sufferers [10, 11 ]. specialist palliative care (spc) services can have an integral role in guiding nephrologists in the management of these symptoms as well as in eol care. there are very few studies looking at the utilization of palliative care services and the implementation of eol care by nephrologists. the purpose of this study was to retrospectively investigate the eol care of dialysis patients in a dublin teaching hospital. our aim was to review our utilization of spc services and assess the degree to which the patient was involved in the decision - making process, particularly looking at the use of advance care planning (acp). a retrospective chart review of patients undergoing renal replacement therapy (rrt) (haemodialysis and peritoneal dialysis) at a large university teaching hospital was performed. the study location is a tertiary referral centre in dublin, with a catchment population of 600 000. it is and one of four in - centre haemodialysis units in the region. using the institution 's renal database, patients who underwent dialysis and died between 1 january 2005 and 30 december 2009 were identified. in order to exclude patients with acute kidney injury, who have a higher mortality rate, only patients who had received either form of rrt for a minimum of 90 days were included. data extraction focused on patient characteristics including age at commencement of dialysis, age at death, cause of eskd, previous modalities of rrt and the charlson comorbidity index (cci). the cci is a method of predicting mortality in various disease subgroups, based on the weighting of 19 specified comorbid conditions [12, 13 ]. the cci used in this study included a point for every decade over 40 years. to focus on the role of the spc service, referral patterns were recorded including date of referral and the interval between referral and death. patient resuscitation status was also reviewed, including the presence of an advance care plan (acp). we considered an acp to be a documented discussion about future care between the patient and their care provider. it should include discussions regarding the individual 's wishes and their personal goals for care. this definition is based on the nhs national end - of - life care programme guidelines. to review the eol care decision process, we investigated if such decisions were made by the medical team alone or if the patient, family member / next of kin (nok) or both were involved. we recorded if a decision had been made to withdraw dialysis and whether a documented discussion took place with the patient or a family member / nok. in cases of withdrawal from dialysis the place of death was categorized as hospital, inpatient hospice, home or elsewhere. continuous variables are reporting as mean / median with measures of central tendency and categorical variables are reported as proportions. comparisons were performed using the, independent samples t - test and one - way analysis of variance, where relevant and a two - sided p - value of < 0.05 was considered statistically significant. the modality of rrt prior to death was haemodialysis for 102 (77.9%) patients and peritoneal dialysis for 29 (21.9%). eighty - eight patients were male (67.2%) and 43 were female (32.8%). the mean age at death was 63.2 15.1 years after a median of 27 months of dialysis (range 3318). cci and age were significantly correlated (r = 0.564, p < 0.001). no differences between those referred to spc and those not referred to spc were observed (table 1). table 1.baseline demographicspredictor variableall (n = 31)palliative care involvement (n = 48)no palliative care involvement (n = 83)p - valueage at death, years (mean sd)63.2 15.164.0 13.962.7 15.70.625male gender88 (67.2%)32 (66.7%)56 (67.5%)0.925female gender43 (32.8%)16 (33.3%)27 (32.5%)haemodialysis102 (77.9%)38 (79.2%)64 (77.1%)0.785peritoneal dialysis29 (22.1%)10 (20.8%)19 (22.9%)cci (mean sd)7.9 2.58.4 2.77.6 2.40.081age at dialysis start, years (mean sd)59.9 15.660.8 14.059.3 16.60.506dialysis vintage, months (median, range)27 (3318)21 (3170)34.5 (3318)0.237 baseline demographics a referral to spc services was placed in 48 (36.7%) cases, with a median duration of involvement of 12 days before death (range 0907). two patients were referred to spc services over 1 year before their death, the first, referred 907 days prior to death, was for control of symptoms associated with chronic liver disease and chronic kidney disease and not in anticipation of an imminent decline. the second, referred 612 days prior to death, was for the management of a complex pain syndrome. the majority, 104 patients (78.5%), died in an acute hospital, 19 (14.5%) at home and 3 (2.3%) in an inpatient hospice (table 2). mean cci was 7.9 2.5 for those who died in an acute hospital, 7.6 2.9 in those who died at home and 11.0 3.5 in those who died in an inpatient hospice (p = 0.185). the mean age of those who died in an acute hospital was 63.4 + 15.5 years, 60.6 15.4 years in those who died at home and 68.0 4.6 years in those who died in an inpatient hospice (p = 0.788). table 2.eol care issuesall (n = 131)palliative care involvement (n = 48)no palliative care involvement (n = 83)p - valuedialysis withdrawn50 (38.2%)3515<0.001duration of palliative involvement in days (median, range)12 (1907)12 (1907)dnr documented68 (51.9%)38 (79.2%)30 (36.1%)0.005place of death home19 (14.5%)3 (6.2%)16 (19.3%)0.041 hospital104 (79.5%)42 (87.5%)62 (74.7%) hospice3 (2.3%)3 (6.2%)0n / a elsewhere5 (3.8%)05 (6.0%)n / a dialysis was withdrawn in 50 cases, the majority of whom died in an acute hospital setting (79.5%). the median time from the withdrawal of dialysis to death was 6 days (0105 days). spc services were involved in the eol care of 35 of these cases (70%). dialysis was more likely to be withdrawn in cases where spc was involved (p < 0.001) (table 3). table 3.withdrawal of dialysispalliative care involved (n = 48)palliative care not involved (n = 83)p - valuedialysis withdrawn35 (72.9%)15 (18.1%)<0.001documentation of discussion prior to withdrawal of dialysis30 (85.7%)13 (86.7%)ns withdrawal of dialysis we collected further data on the decision - making process (as documented in the medical records) for the 50 cases of dialysis withdrawal. a discussion with either the patient or a family member / nok regarding the withdrawal of dialysis was documented in 43 (86.0%) of these cases. according to the medical records, this discussion was held with both the patient and the family in 9 (18.0%) cases. there was a documented discussion with the patient 's family but not with the patient in 32 (64.0%) cases. there was a documented discussion regarding the withdrawal of dialysis with the patient but not their family in two (4.0%) cases. dialysis was withdrawn with no documented evidence of a discussion having taken place with the patient themselves in 39 cases (78.0%). there was documentation of underlying cognitive impairment in the case of nine (23.7%) of these patients. we could find no documented discussion regarding the decision to withdraw dialysis for seven (14.0%) patients. we could not determine whether a discussion was held but not documented, or whether this represented a best interest decision. a do not resuscitate (dnr) order was documented in the chart of 68 patients (51.9%). this was more likely to be documented in the case of those referred to spc services (36.1 versus 79.2%, p = 0.005). a referral to spc services was placed in 48 (36.7%) cases, with a median duration of involvement of 12 days before death (range 0907). two patients were referred to spc services over 1 year before their death, the first, referred 907 days prior to death, was for control of symptoms associated with chronic liver disease and chronic kidney disease and not in anticipation of an imminent decline. the second, referred 612 days prior to death, was for the management of a complex pain syndrome. the majority, 104 patients (78.5%), died in an acute hospital, 19 (14.5%) at home and 3 (2.3%) in an inpatient hospice (table 2). mean cci was 7.9 2.5 for those who died in an acute hospital, 7.6 2.9 in those who died at home and 11.0 3.5 in those who died in an inpatient hospice (p = 0.185). the mean age of those who died in an acute hospital was 63.4 + 15.5 years, 60.6 15.4 years in those who died at home and 68.0 4.6 years in those who died in an inpatient hospice (p = 0.788). table 2.eol care issuesall (n = 131)palliative care involvement (n = 48)no palliative care involvement (n = 83)p - valuedialysis withdrawn50 (38.2%)3515<0.001duration of palliative involvement in days (median, range)12 (1907)12 (1907)dnr documented68 (51.9%)38 (79.2%)30 (36.1%)0.005place of death home19 (14.5%)3 (6.2%)16 (19.3%)0.041 hospital104 (79.5%)42 (87.5%)62 (74.7%) hospice3 (2.3%)3 (6.2%)0n / a elsewhere5 (3.8%)05 (6.0%)n / a dialysis was withdrawn in 50 cases, the majority of whom died in an acute hospital setting (79.5%). the median time from the withdrawal of dialysis to death was 6 days (0105 days). spc services were involved in the eol care of 35 of these cases (70%). dialysis was more likely to be withdrawn in cases where spc was involved (p < 0.001) (table 3). table 3.withdrawal of dialysispalliative care involved (n = 48)palliative care not involved (n = 83)p - valuedialysis withdrawn35 (72.9%)15 (18.1%)<0.001documentation of discussion prior to withdrawal of dialysis30 (85.7%)13 (86.7%)ns withdrawal of dialysis we collected further data on the decision - making process (as documented in the medical records) for the 50 cases of dialysis withdrawal. a discussion with either the patient or a family member / nok regarding the withdrawal of dialysis was documented in 43 (86.0%) of these cases. according to the medical records, this discussion was held with both the patient and the family in 9 (18.0%) cases. there was a documented discussion with the patient 's family but not with the patient in 32 (64.0%) cases. there was a documented discussion regarding the withdrawal of dialysis with the patient but not their family in two (4.0%) cases. dialysis was withdrawn with no documented evidence of a discussion having taken place with the patient themselves in 39 cases (78.0%). there was documentation of underlying cognitive impairment in the case of nine (23.7%) of these patients. we could find no documented discussion regarding the decision to withdraw dialysis for seven (14.0%) patients. we could not determine whether a discussion was held but not documented, or whether this represented a best interest decision. a do not resuscitate (dnr) order was documented in the chart of 68 patients (51.9%). this was more likely to be documented in the case of those referred to spc services (36.1 versus 79.2%, p = 0.005). the medical community is becoming increasingly aware of the important role that spc services play in the management of patients with life - limiting, non - malignant disease, such as eskd. eskd and dialysis place a significant burden of physical and psychological symptoms on a patient. worldwide, there are escalating numbers of elderly and multimorbid patients initiating dialysis [16, 17 ]. this 5-year study is the first to analyse the use of spc services and eol care issues amongst dialysis patients in ireland. there are data to suggest that up to 50% of dialysis patients experience chronic pain. fatigue, sleep disturbance, pruritus, poor appetite and nausea are also frequently reported [9, 19 ]. despite the high symptom burden of eskd, referral rates dialysis patients have complex and unique palliative care needs and the need for spc involvement may arise years before death. we have identified an underutilization of spc services as well as a general trend towards late referral of dialysis patients. almost 80% of our study population died in an acute hospital, regardless of the final modality of rrt. a small minority of our study population died in an inpatient hospice and only 14.5% died at home. by comparison, a study of lung cancer patients in ireland found that 50% of deaths occurred in an acute hospital, 29% occurred at home and 12% in inpatient hospices. our population of dialysis patients was more likely to die in hospital and less likely to die at home than patients with a solid organ malignancy. this difference may reflect the fact that dialysis patients have unique medical needs and often require ongoing nephrology care, even at the eol. fears and reservations about caring for an ailing dialysis - dependent patient at home can influence the decision to transfer them out of an acute hospital. patients often opt to remain in the hospital where their renal care has been conducted for comfort reasons as well as for ease of access to ongoing therapies. we were surprised to find that only three patients in our study population died in an inpatient hospice. the duration of time from the withdrawal of dialysis to death is usually a number of days rather than weeks and unfortunately, the waiting period for an inpatient hospice bed often exceeds this timeframe. it is advisable to determine the patient 's preferred place of death prior to the withdrawal of dialysis. if their preference is for a hospice death, this should be organized in advance of dialysis withdrawal. results from a uk study suggest that 50% of patients with advanced chronic kidney disease have a preference to die in hospital, but a study by davison found that this was the preference of approximately a quarter of patients. owing to the retrospective nature of our study however, it is more likely that this is a reflection of the high level of comorbid illness of the dialysis population coupled with suboptimal acp. further investigation is required to determine why the majority of eskd patients die in acute hospitals rather than at home. withdrawal of dialysis was found to be an important cause of death in our study population. there was a documented decision to withdraw dialysis in over one - third of patients. the proportion of deaths occurring in the setting of the withdrawal of dialysis is higher in our study than reported in other countries [2328 ]. we have not differentiated between the withdrawal of dialysis as a cause of death and withdrawal of dialysis before death. neither have we differentiated between cases in whom dialysis was withdrawn despite it being technically feasible and those in whom it was withdrawn due to factors which limited the ability to perform dialysis (e.g. hypotension and access difficulties). the median time to death after the withdrawal of dialysis was 6 days, with no statistically significant difference between haemodialysis and peritoneal dialysis patients. once dialysis is discontinued, the time available to ensure optimal eol care is short, highlighting the importance of acp and ideally, referral to spc services prior to making the decision to withdraw dialysis. in concordance with previous irish data, only a minority of the study population had an advance care plan in place [29, 30 ]. irish people are not familiar with certain terminology used in relation to eol care, with one survey reporting that 71% of respondents had never heard the term evidently, efforts to improve public awareness are required but it is also imperative that physicians are mindful of the fact that patients and their families may not understand the terminology being used during acp or eol discussions. in the majority of cases, patients did not appear to be involved in discussions regarding eol care. in contrast to this was the finding that family members / nok were frequently involved in the decision - making process. for example, there was a documented discussion with the family / nok regarding the decision to withdraw dialysis in over 80% of cases. by comparison, documentation of the same discussion taking place with the patient themself undoubtedly, family members play an important role in decision - making at the eol ; however, the patient is the major stakeholder in their own eol decisions and their views must be respected. we can not explain why eol discussions were preferentially held with family members, rather than the patient. one possible explanation is that physicians have concerns regarding the ability of a patient to understand or accept the decision being discussed with them. irish people (particularly older people) place a great amount of trust in doctors when decisions regarding their medical care are required and this finding may represent a feature of irish healthcare. timely acp provides the patient with a forum to clearly document their wishes well in advance of a decline in their health. the main limitations are its retrospective design and reliance on documentation in the medical record. these effects were minimized by adoption of a systematic approach to patient identification and data extraction. there is likely to be regional variation in the availability of spc and our results may not be truly representative of the overall irish practice patterns. as we were unable to determine patient preference regarding the place of death, we can not determine if our results mirror patients ' wishes. a certain proportion of the home deaths may have been sudden in nature and therefore not reflective of a patient who is in a phase of anticipated decline in health. we encountered difficulties accessing all data in some cases, particularly those patients who died outside of the acute hospital setting. despite its limitations, this study is the largest of its kind to analyse eol care issues and the use of spc services in an irish eskd population and provides an important insight into current eol care practices. there are data to suggest that up to 50% of dialysis patients experience chronic pain. fatigue, sleep disturbance, pruritus, poor appetite and nausea are also frequently reported [9, 19 ]. despite the high symptom burden of eskd, referral rates dialysis patients have complex and unique palliative care needs and the need for spc involvement may arise years before death. we have identified an underutilization of spc services as well as a general trend towards late referral of dialysis patients. almost 80% of our study population died in an acute hospital, regardless of the final modality of rrt. a small minority of our study population died in an inpatient hospice and only 14.5% died at home. by comparison, a study of lung cancer patients in ireland found that 50% of deaths occurred in an acute hospital, 29% occurred at home and 12% in inpatient hospices. our population of dialysis patients was more likely to die in hospital and less likely to die at home than patients with a solid organ malignancy. this difference may reflect the fact that dialysis patients have unique medical needs and often require ongoing nephrology care, even at the eol. fears and reservations about caring for an ailing dialysis - dependent patient at home can influence the decision to transfer them out of an acute hospital. patients often opt to remain in the hospital where their renal care has been conducted for comfort reasons as well as for ease of access to ongoing therapies. we were surprised to find that only three patients in our study population died in an inpatient hospice. the duration of time from the withdrawal of dialysis to death is usually a number of days rather than weeks and unfortunately, the waiting period for an inpatient hospice bed often exceeds this timeframe. it is advisable to determine the patient 's preferred place of death prior to the withdrawal of dialysis. if their preference is for a hospice death, this should be organized in advance of dialysis withdrawal. results from a uk study suggest that 50% of patients with advanced chronic kidney disease have a preference to die in hospital, but a study by davison found that this was the preference of approximately a quarter of patients. owing to the retrospective nature of our study, however, it is more likely that this is a reflection of the high level of comorbid illness of the dialysis population coupled with suboptimal acp. further investigation is required to determine why the majority of eskd patients die in acute hospitals rather than at home. withdrawal of dialysis was found to be an important cause of death in our study population. there was a documented decision to withdraw dialysis in over one - third of patients. the proportion of deaths occurring in the setting of the withdrawal of dialysis is higher in our study than reported in other countries [2328 ]. we have not differentiated between the withdrawal of dialysis as a cause of death and withdrawal of dialysis before death. neither have we differentiated between cases in whom dialysis was withdrawn despite it being technically feasible and those in whom it was withdrawn due to factors which limited the ability to perform dialysis (e.g. hypotension and access difficulties). the median time to death after the withdrawal of dialysis was 6 days, with no statistically significant difference between haemodialysis and peritoneal dialysis patients. once dialysis is discontinued, the time available to ensure optimal eol care is short, highlighting the importance of acp and ideally, referral to spc services prior to making the decision to withdraw dialysis. in concordance with previous irish data, only a minority of the study population had an advance care plan in place [29, 30 ]. there is currently no legislation in ireland to recognize or enforce advance care directives. irish people are not familiar with certain terminology used in relation to eol care, with one survey reporting that 71% of respondents had never heard the term evidently, efforts to improve public awareness are required but it is also imperative that physicians are mindful of the fact that patients and their families may not understand the terminology being used during acp or eol discussions. in the majority of cases, patients did not appear to be involved in discussions regarding eol care. in contrast to this was the finding that family members / nok were frequently involved in the decision - making process. for example, there was a documented discussion with the family / nok regarding the decision to withdraw dialysis in over 80% of cases. by comparison, documentation of the same discussion taking place with the patient themself undoubtedly, family members play an important role in decision - making at the eol ; however, the patient is the major stakeholder in their own eol decisions and their views must be respected. we can not explain why eol discussions were preferentially held with family members, rather than the patient. one possible explanation is that physicians have concerns regarding the ability of a patient to understand or accept the decision being discussed with them. irish people (particularly older people) place a great amount of trust in doctors when decisions regarding their medical care are required and this finding may represent a feature of irish healthcare. timely acp provides the patient with a forum to clearly document their wishes well in advance of a decline in their health. the main limitations are its retrospective design and reliance on documentation in the medical record. these effects were minimized by adoption of a systematic approach to patient identification and data extraction. there is likely to be regional variation in the availability of spc and our results may not be truly representative of the overall irish practice patterns. as we were unable to determine patient preference regarding the place of death, we can not determine if our results mirror patients ' wishes. a certain proportion of the home deaths may have been sudden in nature and therefore not reflective of a patient who is in a phase of anticipated decline in health. we encountered difficulties accessing all data in some cases, particularly those patients who died outside of the acute hospital setting. despite its limitations, this study is the largest of its kind to analyse eol care issues and the use of spc services in an irish eskd population and provides an important insight into current eol care practices. this study examines a population of established eskd (predominantly haemodialysis) patients with a broad spectrum of baseline renal pathologies. the majority of patients died in an acute hospital without involvement from spc services and dialysis was withdrawn in over one - third of patients. nok or family members played an important role in the decision - making process in relation to eol care, highlighting an important cultural trend in ireland. it appears that the patient was less likely to be involved in decision - making, however, this may be attributable to poor documentation of discussions held by physicians. this study highlights the need to structure eol care planning on an individual patient basis. the cornerstone of such a strategy is the recognition of those patients who are at a high risk of death within the next 612 months and the identification of patients with a high burden of symptoms. tools are available for both prognostication and symptom assessment in dialysis patients [3134 ]. universal screening of dialysis patients to determine their symptom burden and palliative care needs would be a helpful strategy to guide both nephrology and palliative care teams. risk patients require early referral to spc services, who can provide valuable guidance in the management of many of the problematic symptoms associated with eskd, even before the patient reached a terminal phase. we suggest that nephrologists hold a discussion with each patient regarding prognosis, goals of care and acp. important issues to address include patient preferences regarding future hospitalizations (including the specific circumstances under which a patient would wish to be hospitalized), place of death, the withdrawal of dialysis and resuscitation. coordinated input from both nephrology and spc teams will ultimately result in improved eol care for eskd patients.	backgroundalthough patients with end - stage kidney disease (eskd) have a shortened life expectancy, their end - of - life (eol) care is suboptimal. the aim of this study was to review the utilization of specialist palliative care (spc) in patients with eskd in dublin, ireland.methodswe conducted a retrospective chart review of prevalent patients with eskd who died between january 2005 and december 2009 at a tertiary referral centre. we recorded spc referrals, modality of renal replacement therapy, age and place of death.resultsof 131 included patients, 88 (67.2%) were male, mean age at death was 63.2 15.1 years and 102 (77.9%) were treated with haemodialysis. forty - eight patients (36.7%) were referred to spc, who were involved in the patients ' management for a median of 12 days (range 0907) before death. a total 104 patients (79.4%) died in an acute hospital, 19 (14.5%) died at home, 3 (2.3%) died in an inpatient hospice and the place of death was unknown for 5 patients (3.8%). dialysis was withdrawn prior to death in 50 patients (38.1%), with a median time to death after withdrawal of dialysis of 6 days (0105 days). a discussion regarding the withdrawal of dialysis was more frequently held with family member(s) rather than the patient.conclusionsspc was involved in the antemortem care of 1/3 of the patients with the majority of referrals placed at a late stage. given the short timeframe until death once dialysis is withdrawn, it is imperative that appropriate eol care is instituted. this study identifies an underutilization of spc and improved integration of palliative care and nephrology services may optimize eol care for patients with eskd.
the loss of teeth from the posterior maxilla will certainly result in the loss of bone depth of the residual ridge ; the depth decrease becoming more profound with time. additionally, with time, the pneumatization of the sinus is to be expected. a profound reduction in bone volume will certainly complicate the rehabilitation of the posterior edentulous maxilla with implant - supported prostheses. however, where the residual bone height is > 5 mm and primary stability of an introduced dental implant has been achieved, it has been shown that the simple elevation of the sinus lining can be sufficient to regenerate new bone : the periosteum itself having sufficient osteo - regenerative capacity to repair, regenerate and remodel the sinus floor. where the residual bone depth is < 5 mm, various sinus grafting procedures have been used to increase the bone depth and volume by raising the anatomical floor of the sinus cavity at the site of a prospective dental implant osteotomy by using autogenous bone and/or a biomaterial. essentially, there are three methods of raising the sinus floor, either to simply raise the sinus lining or to additionally introduce bone regenerative material under the lining : there is, (1) the lateral window approach (lwa). (2) the bone - added osteotome sinus floor elevation (baosfe). in addition, the sinus floor can simply be penetrated, thus, allowing full usage of all the available bone at the osteotomy site with no direct augmentation ; this is the simple perforation. in the last 30 years many articles have been published describing the successful use of different augmentation materials that can be placed under the sinus lining : aghaloo and moy, in a systematic review, where 5128 implants were placed with a follow - up ranging from 12 to 102 months, implant survival was 92% for implants placed into autogenous bone and autogenous / composite grafts, 93.3% for implants placed in allogenic / nonautogenous composite grafts, 81% for implants placed in alloplast and alloplast / xenograft materials, and 95.6% for implants placed into xenograft material alone. other studies have also compared the delayed placement of dental implants into the sinus graft against immediate placement. in a systematic review of implant survival rates in the grafted sinus that took into account the influence of implant surface, graft material and implant placement timing, it was found that simultaneous and delayed procedures had similar outcomes. thus, with the success of the various surgical techniques, the sinus grafting materials and the simultaneous placement of the implants, hatano. went on to assess the long term changes in sinus - graft height after maxillary sinus floor augmentation (lwa) and simultaneous placement of implants : a total of 191 patients undergoing sinus floor augmentation with 2:1 mixture of autogenous bone / xenograft (bio - oss) were radiographically followed - up for up to 10 years. changes in sinus - graft height were calculated with respect to implant length and original sinus floor depth. after 2 - 3 years, the grafted sinus floor was level with or slightly below the implant apex. the results showed that the sinus - graft height decreased significantly and approached the original sinus height. the number of patients having the sinus floor below the implant apex reached a maximum after 3 years. the clinical survival rate was 94%, with implant losses occurring within 3 years of augmentation. they concluded that progressive sinus pneumatisation occurs after augmentation with 2:1 autogenous bone / xenograft mixture and the long term stability of the sinus - graft height influences implant success. more recently, trombelli. in a randomized study of 30 sites in 30 patients compared the use of deproteinized bovine bone mineral, deproteinized bovine bone material (dbbm) (n = 15) and synthetic hydroxyapatite (s - ha) in a collagen matrix, s - ha (n = 15) as grafting materials when using the transcrestal sinus floor elevation. the extent of the sinus lift and the height of the graft above the implant apex were assessed on periapical radiographs taken immediately after surgery and at 6 months following surgery. the results showed that both materials resulted in a substantial increase in sinus floor depth and produced a substantial height of graft maintained graft material above the implant apex at 6 months postsurgery. however, the measurements obtained with the s - ha were superior to those with dbbm. when multiple implants are to be placed, and a pneumatised sinus exists, the published reports suggest that an lwa is favoured for sinus floor augmentation. at the same time, if a transcrestal sinus floor augmentation has been carried out (the baosfe), the reports are restricted to single implant placement at any site. the aim of this preliminary study was to evaluate the clinical and radiographic outcome of adjacent transcrestal sinus augmentation grafts using dbbm, dbbm1, with the immediate placement of submerged adjacent implants,2 and thus determine the fate of the graft material. a male caucasion, 62 years of age presenting at a private practice devoted to implant and periodontal therapy, and advanced restorative treatment. teeth 16 and 15 (federation dentaire internationale - notation) were missing, having been extracted 8 months previously [figure 1 ]. a periapical radiograph [figure 2 ], with ball - bearing in place (5 mm in diameter) showed that there was < 5 mm of residual bone depth available for implant placement. preextraction : 1 h prior to surgery the patient received systemic coverage 2 g. amoxicillin, 400 mg ibuprofen, 2 mg dexamethasone. in addition, mouthrinse of chlorhexidine gluconate 2%, 20 min prior to surgery. under intravenous sedation and local anaesthesia, at the chosen implant sites, a crestal incision was made over the proposed transalveolar osteotomy sites, that is, at the inferior border of the maxillary sinus. the two osteotomies were prepared with palatal displacement of their axis and taken to the accessed depth approximately 1 - 2 mm coronal from the sinus floor. an accompanying trans - socket sinus penetration / lift facilitated with osteotomes3 was then carried out at each separate site. following this minimal penetration / fracture, the sinus lining was checked circumferentially for integrity with a specified socket probe.4 the procedure elevated the sinus (schneiderian) membrane, creating a classical tent. in this way, space was provided for bone graft placement or blood clot formation. particulate xenograft, 0.25 - 1 mm,5 mixed with sterile water was introduced incrementally into each osteotomy. the condensation pressure from the osteotomes, graft material and the trapped fluids exerted hydraulic pressure on the sinus membrane causing it to elevate. engelke and deckwer have shown in an endoscopic study that the membrane can be elevated by 5 mm without perforation. the volume and hydrostatic pressure from each osteotomy site caused the eventual coalescence of the two osteotomies. dental implants6 were immediately placed into the osteotomies [figure 3 ] : both implants were sandblasted and acid - etched -surface titanium, self - tapping screws. both implants were inserted mechanically with an initial seating torque = 50 ncm, determined by precalibration of the implant drive unit;7 final seating was with a hand wrench in excess of 50 ncm. cover screws were fitted and the wound site closed with 4.0 vicryl resorbable sutures.8 postoperative medication was 2 mg dexamethasone once daily for 3 days and 400 mg ibuprofen 4 times daily for 3 days. periapical radiograph taken immediately following the adjacent transcrestal (osteotomies) sinus grafts (using deproteinised bovine bone material) and the immediate placement of the implants at 6 months postsurgery, the implants were uncovered via a simple crestal incision displaced to the palatal aspect and the buccal mucoperiosteal tissue displaced to the buccal aspect, the abrams roll. 4 mm high gingival formers (healing caps) were fitted to each implant with a seating torque of 20 ncm. at 3 weeks postuncovering, impressions were taken of the implants. using a standardized rinn film - holder9 in the long - cone parallel technique, periapical radiographs were taken at crown fit, and at 6 and 12 months postcrown fit [figures 4 - 6 and 9 ]. periapical radiograph taken at same appointment as the fit of the cement - retained porcelain - bonded crowns. 7.5 months postsurgery periapical radiograph taken at 13.5 months postsurgery periapical radiograph taken at 19.5 months postsurgery all radiographs were photographed and digitized., san jose, ca, usa) and methodology already reported, specific bone - implant - contact sites and graft region above the original sinus floor - were investigated for their pixel density ; that recorded pixel - density inferring the relative bone - density values at those sites [figure 7 ]. additionally, the mid - points of the graft opposing the original sinus floor and the new highest point of the augmented sinus floor. the average of five recordings of the bone density at each site under scrutiny was given as a percentage of its control. radiograph displaying the actual areas at which the pixel - density was measured on each periapical radiograph. the numbers correlate to the rows on the tables using the known actual length of the inserted implants, the following measurements were determined on each individual implant [figure 8 ] : schematic of the measurements taken from each radiograph : graft height above implant a, distance ab, graft height above implant b, distance ef, graft height at lowest point between implants a and b (position d), at a tangent to implant a (position c) clinical situation 12 months postloading graft height above implant agraft height above implant bgraft height at lowest point between implants a and b, at a tangent to implant a. graft height above implant a graft height above implant b graft height at lowest point between implants a and b, at a tangent to implant a. to evaluate the changes in graft height, these measurements were repeated on each follow - up radiograph [figure 8 ]. all radiographs were photographed and digitized. using adobe photoshop elements nine (adobe systems incorp., san jose, ca, usa) and methodology already reported, specific bone - implant - contact sites and graft region above the original sinus floor - were investigated for their pixel density ; that recorded pixel - density inferring the relative bone - density values at those sites [figure 7 ]. additionally, the mid - points of the graft opposing the original sinus floor and the new highest point of the augmented sinus floor. the average of five recordings of the bone density at each site under scrutiny was given as a percentage of its control. radiograph displaying the actual areas at which the pixel - density was measured on each periapical radiograph. the numbers correlate to the rows on the tables using the known actual length of the inserted implants, the following measurements were determined on each individual implant [figure 8 ] : schematic of the measurements taken from each radiograph : graft height above implant a, distance ab, graft height above implant b, distance ef, graft height at lowest point between implants a and b (position d), at a tangent to implant a (position c) clinical situation 12 months postloading graft height above implant agraft height above implant bgraft height at lowest point between implants a and b, at a tangent to implant a. graft height above implant a graft height above implant b graft height at lowest point between implants a and b, at a tangent to implant a. to evaluate the changes in graft height, these measurements were repeated on each follow - up radiograph [figure 8 ]. over a period of 19.5 months from base - line (day of surgery) there was progressive loss of sinus floor height over the apex of each implant, whilst the sinus floor height between the implants showed a gain over that same period. the overall pixel density of the sinus graft increased from baseline to 6 months, however, with 6 months of loading that pixel density decreased progressively to 19.5 months [tables 1 - 5 ]. evaluation of radiograph, figure 3, taken at surgery end, base - line evaluation of radiograph, figure 4 taken at crown fit, 7.5 months after surgery (base - line) evaluation of radiograph, figure 5 taken 6 months postloading, 13.5 months from base - line evaluation of radiograph, figure 6 taken 12 months postloading, 19.5 months from base - line pixel density value change with time, months where implants are planned for placement into pneumatised sinuses, inadequate depths of implant beds are often augmented. where multiple implants are planned, lateral open - window (lw) surgical sites facilitate the placing of a large volume of sinus graft material, dependant on the achievement of high primary stability ; implants will be placed immediately or at a later date. if a single implant is to be placed into a deficient bone depth site, the transcrestal sinus graft technique is often employed (the baosfe), the implant being placed immediately or at a later date. the novel method investigated here was the use of the baosfe technique at adjacent osteotomies, thus allowing the introduction of a large volume of graft material similar to the lateral window technique but in a less invasive manner, and place multiple implants immediately. the progressive loss of graft material from around the apex of the implants is the expectation when either the lateral - window technique or the baosfe are used. the preliminary report of this coalescence method produced the same result as the lw and baosfe methods the explanation of this phenomena is already given : during this early period graft material can be resorbed by basic multicellular units and becomes surrounded by de novo bone which is then remodelled by secondary bone formation as a result of loading. changes in graft height over a period of 19.5 months, mm the progressive loss of the inter - implant sinus bone graft is also reported for both the lw and baosfe techniques. however, this novel coalescence method has shown a progressive increase in the inter - implant sinus graft region, thus, inferring a positive bony regeneration and remodelling at the region - table 6. the maintenance of bone height in the inter - implant region of a sinus graft will be conducive to the long term stability of the implants adjacent to the region. the results of this preliminary investigation indicate that the carrying out of a large - scale study is warranted to confirm the efficacy of this novel technique.	when multiple implants are to be placed, and a pneumatized sinus exists, the published reports suggest that the lateral window approach (lwa) is favored for sinus floor augmentation. simultaneously, if a transcrestal sinus floor augmentation has been carried out (bone - added osteotome sinus floor elevation), the reports are restricted to single implant placement at any site. the aim of this study was to evaluate the clinical and radiographic outcomes at adjacent transcrestal sinus augmentation grafts using deproteinized bovine bone material, with the immediate placement of submerged adjacent implants, and so determining the fate of the graft material. the progressive loss of the inter - implant graft is reported for the lwa technique. however, this novel coalescence method has shown a progressive increase in the inter - implant graft region, thus inferring a positive bony regeneration and remodelling at the region. these results indicate that the carrying out of a large scale study is warranted to confirm the efficacy of this technique.
granulomatosis with polyangiitis (wegener 's) (gpa) is an immune - mediated systemic necrotizing vasculitis often affecting the upper respiratory tract, lung, and kidney. involvement of gpa is limited to the upper respiratory tract and/or the lung in some cases although virtually anybody site can be involved in gpa such as the eye, skin, joints, heart, and the central nervous system. necrotizing vasculitis and irregular basophilic parenchymal necrosis with associated palisading granuloma comprise the main histologic characteristics of gpa. also, neutrophilic microabscesses and fibrosis are commonly found in a background mixed inflammatory infiltrate composed of neutrophils, lymphocytes, plasma cells, multinucleated giant cells, and macrophages [1, 2 ]. on histologic examination, gpa can mimic igg4-related disease (igg4-rd) since the inflammatory background in gpa may be rich in plasma cells and accompanied by fibrosis and/or obliterated blood vessels as in igg4-rd [1, 3, 4 ]. some biopsies of gpa cases (especially from the upper respiratory tract and orbit) may lack classic morphologic features such as necrotizing vasculitis, parenchymal necrosis, and palisading granuloma [5, 6 ]. igg4 immunostain is now often performed in this context for evaluating the possibility of igg4-rd. however, the prevalence of igg4-positive (igg4 +) cells in gpa has not been widely reported in the literature. therefore, we sought to assess the prevalence of igg4 + cells in gpa cases that have been confirmed by a thorough clinical and pathologic assessment. cases with the diagnosis of gpa were identified via electronic search from our surgical pathology file. we initially retrieved 36 biopsies from various body sites obtained during a period of 19992011. the sites of biopsies included sinus / nasopharynx (n = 16), oral cavity (n = 1), orbital region (n = 11), lung / pleural (n = 6), kidney (n = 1), and dura (n = 1). glass slides from each biopsy were reviewed and reassessed for the following histologic features : geographic necrosis, necrotizing granulomas, vasculitis, multinucleated giant cells, microabscesses, and fibrosis. based on these histologic parameters, each case was scored as to the confidence of histopathologic diagnosis of gpa as follows : 0 = nondiagnostic biopsy or not gpa with some findings against the diagnosis of gpa such as infection, 1 = nonspecific (one feature present) ; 2 = suggestive of gpa (two features present), or 3 = consistent with gpa (three or more features present). a definitive clinicopathologic diagnosis of gpa was made if a case had met the modified clinical criteria of the american college of rheumatology (acr) for gpa [7, 8 ] (modified to include assessment of anca status) and a pathology diagnostic score of 1, 2, or 3. twenty - six of the initial 36 cases were confirmed as the diagnosis of gpa and retained for this study. immunohistochemical staining for igg4 and igg was performed on all 26 cases using the dako dual multimer system or dako advance 2 stops multimer system (dako, carpinteria, ca, usa). monoclonal igg4 antibody (clone hp6025, dilution 1 : 100 ; zymed, san francisco, ca, usa) and polyclonal rabbit antihuman igg antibody (dilution 1 : 15,000, dako, carpinteria, ca, usa) were used for the study. hot - spot were identified at low power magnification. using 40x objective lens (olympus bx50 microscope, 40x objective, 10x eye piece, olympus dp70 camera, on - screen capture field diameter of 0.34 mm and field area of 0.0884 mm), three high - power fields (hpfs) of igg4 + hot spots, and their corresponding areas of igg immunostain were photographed. all photographs were printed for a manual counting of the absolute number of igg4 + and igg+ plasma cells in each hpf. averages of igg4 + and igg+ cells in three hpfs were used to determine the igg4 + cell count and igg4+/igg+ ratio for each case. increase in igg4 + cells was defined as the average igg4 + cells greater than 30 per hpf with the igg4+/igg+ cell ratio greater than 40%. patients ' medical records from our institution were reviewed for their serum levels of c - reactive protein (crp) and immunoglobulin subclasses. eight of 26 biopsies (31%) showed increased igg4 + cells with an average igg4 + cell count and an igg4+/igg+ cell ratio ranging from 37 to 137/hpf and from 44 to 83%, respectively (figure 1). these 8 biopsies were from sinonasal (n = 4) and orbital / periorbital (n = 4). the igg4 + cell count ranged from 0 to 28/hpf in the remaining 18 biopsies that were from sinonasal / oral cavity / nasopharynx (n = 10), orbit / periorbital (n = 3), lung / pleura (n = 3), iliac fossa / kidney (n = 1), and dura (n = 1). the igg4 + cell counts and igg4/igg ratios in the cases without increase in igg4 + cells ranged from 0 to 28 per hpf and from 0 to 39%, respectively (table 1). there was no significant difference in the distribution of age at diagnosis or gender between the cases with and without increased igg4 + cells. elevated titers of antineutrophil cytoplasmic autoantibodies (anca) were present in 25 of 26 cases in this study. all 8 cases with increased igg4 + cells were positive for anca ; seven cases were c - anca positive, 6 of which were also positive for proteinase 3 (pr-3) anca by elisa. one case was positive for p - anca confirmed as myeloperoxidase (mpo) anca by elisa. on histopathologic evaluation, most cases showed characteristic histologic findings of gpa (figure 2) ; 2 of 8 cases were graded as pathologic score 3, 2 as score 2, and the remaining 4 as score 1. the status of serum crp level was assessed at the time of the biopsy in 18 patients, and it was elevated in 15 of them (83%) (table 1). immunoglobulin levels were evaluated in only three patients (case 7, 9, and 17) and did not reveal polyclonal hypergammaglobulinemia. the eight biopsies with increased igg4 + cells were taken from seven patients, five of whom had elevated crp levels (table 1). the two patients with normal crp levels demonstrated classic histopathologic features including geographic necrosis, palisading granulomas, vasculitis, or microabscesses, which supported the clinicopathologic diagnosis of gpa despite the normal crp level. in this study, we sought to determine the prevalence of increased igg4 + plasma cells in gpa in order to address the role of this finding in the differential diagnosis with igg4-rd. we believe that this is the largest series of gpa cases to examine igg4 + cells with application of current criteria and method for evaluation of increased igg4 + cells in the setting of igg4-rd. an increase in igg4 + cells in gpa has been suggested in a recent study by vaglio. in which they reviewed tissue igg4/igg ratio in 10 gpa cases along with 9 cases with the churg - strauss syndrome (css) and 22 cases with chronic sinusitis. however, they did not provide the details on the counting method or results of igg4 + cells. in our study, we applied the current criteria and methods in counting igg4 + cells, which should provide useful and important information in routine diagnostic surgical pathology practice. also, we made an extra effort to ensure the diagnosis of gpa by a thorough clinicopathologic evaluation and only included the cases with irrefutable diagnosis of gpa. all but one case showed positivity for c- or p - anca confirmed with elisa for pr3 or mpo ; the single anca negative case demonstrated definite histopathologic findings (pathologic diagnosis score 3) as well as clinical features for gpa, which supported our diagnosis of gpa. moreover, all 8 cases with increased igg4 + cells in this study were positive for anca, which reiterated our point. the cutoff number of increased igg4 + plasma cells has not been well established, and many studies have suggested different cutoff points. zhang. have used a cutoff point of > 30 per hpf [10, 11 ] (40x objective lens) while deshpande. and dhall. used > 50 hpf (20x or 40x objectives, resp.), and they have reached high sensitivity and specificity in diagnosing autoimmune pancreatitis [12, 13 ]. in addition to this variable thresholds, the method for counting igg4 + cells has not been well standardized. we used the same method as in our previous study by enumerating cells on printed images and using averages of three hot spots in order to ensure accuracy and reproducibility. we also applied a higher threshold for increased igg4 + cells by using both the igg4 + cell count per hpf at > 30 and the ratio of igg4+/igg+ cells at > 40%. have proposed that both absolute number of igg4 + cell higher than 50 per hpf and igg4+/igg+ ratio greater than 40% should be present in order to make the diagnosis of igg4-rd involving lymph nodes and other extranodal sites. although our cutoffs for igg4 + status may be slightly lower than the histologic criteria proposed by cheuk. (which also used averages of three different hpfs), the latter used a wider field area than the one in our study (0.196 mm versus 0.088 mm, resp.). therefore, the threshold used in this study would be comparable to the one of the most stringent ones in the literature. the most recent comprehensive diagnostic criteria for igg4-rd published by umehara. included two major components : (1) serum igg4 concentration of > 135 mg / dl and (2) > 10 igg4 + plasma cells / hpf with a ratio of igg4+/igg+ cells > 40%. igg4 serum level was not increased in the 3 cases tested in our study. instead, most of our gpa cases showed elevated serum crp which is unusual for igg4-rd. based on their criterion for igg4 + cell count as > 10, however, potentially additional five cases in our study would have been considered to have sufficiently increased igg4 + cells, which will make 50% of our gpa cases with increased igg4 + cells. positive anca has been reported in patients with grave 's disease receiving antithyroidal medication such as propylthiouracil (ptu) [16, 17 ] and may even mimic gpa clinically. however, none of our patients had a history of either graves 's disease or treatment with ptu. the possibility of positive anca in patients with igg4-rd has not been addressed in previous studies, and whether this can occur has yet to be determined. a further study with testing for anca in igg4-rd previous studies have reported the presence of inflammatory infiltrates rich in igg4 + plasma cells in clinical settings other than igg4-rd [1921 ], usually as an isolated finding. in kidneys, several studies have highlighted a frequent association of igg4 + plasma cell - rich infiltrates with the glomerular lesions that are typically seen in anca - associated angiitis, namely, pauci - immune necrotizing and crescentic glomerulonephritis [19, 20 ]. in midst of all these findings, questions regarding the nature of relationship between igg4 + plasma cells and interestingly, there have been some data suggesting that anca of igg4 subclass possibly plays a role in the pathogenesis of anca - related small vessel vasculitis [2224 ]. the predominance of igg4 as well as igg1 subclasses of anca was first reported in patients with gpa and other clinically related disorders by brouwer. in 1991. holland. later suggested a possible pathogenic role for the igg4 subclass in gpa. in vitro, anca activated neutrophils by colligating pr3 and fcriia / iiib receptors. anca are predominantly of the igg isotype, and igg1, igg3, and igg4 subclasses are particularly represented. igg4 subclass isolated from anca - positive sera demonstrated varying abilities to stimulate release of superoxide, which was unrelated to pr3-anca titer, neutrophil donor used in their in vitro test, or neutrophil fcri expression. this study suggested that igg4 was capable of activating neutrophils via constitutively expressed fcriia / iiib or colligation of other unidentified cell surface molecules. liu. have reported that mpo - anca igg4 subclass might play a role in the development of gpa. they reported that the titers of anti - mpo igg4 subclass in patients with gpa was significantly higher than those with microscopic polyangiitis (mpa). the mpo - anca in gpa and mpa might recognize overlapping but different epitopes on native mpo molecule. the difference in immunological characteristics of mpo - anca might have contributed to different disease entities such as gpa and mpa. another recent study reported that serum igg4 levels were markedly elevated in active css and also correlated with the disease activity and the number of involved organs. given these serological and immunological findings, one can postulate a possible role of tissue infiltrating igg4 + cells in the pathogenesis of gpa. however, the underlying cause or precise mechanism for increased igg4 + cells in the tissue as seen in some of our gpa cases has not been completely elucidated and further study would be needed in the future. also, whether there is any pathogenetic relationship between gpa and igg4-rd is not entirely clear. although there have been studies reporting elevated igg4 in the setting of anca - associated systemic diseases, no study demonstrating elevated anca in igg4-rd exists in the english literature to our knowledge. our anecdotal experiences also indicate that the anca is generally not elevated in igg4-rd. in conclusion, one should be aware of the fact that igg4 + cells can be remarkably increased in biopsies of gpa of the sinonasal and orbital / periorbital regions. since the morphologic and clinical manifestations of gpa and igg4-rd may overlap, it could be a significant diagnostic pitfall in the differential diagnosis of these two entities. further study is needed to confirm our observation in a larger number of cases, and a further exploration of potential pathogenetic relationship between gpa and igg4-rd might be of interest as well.	granulomatosis with polyangiitis (wegener 's) (gpa) may mimic igg4-related disease (igg4-rd) on histologic examination of some biopsies, especially those from head and neck sites. igg4 immunostain is often performed in this context for differential diagnosis with igg4-rd. however, the prevalence of igg4 + cells in gpa has not been explored. we examined the igg4 + cells in 26 cases confirmed as gpa by a thorough clinical and pathologic assessment. twenty - six biopsies consisted of 14 sinonasal / oral cavity / nasopharynx, 7 orbit / periorbital, 3 lung / pleura, 1 iliac fossa / kidney, and 1 dura specimens. eight of 26 (31%) biopsies revealed increased igg4 + cells (> 30/hpf and > 40% in igg4+/igg+ ratio). the igg4 + cells and igg4+/igg+ ratio ranged 37137/hpf and 4483%, respectively. eight biopsies with increased igg4 + cells were from sinonasal (n = 4) or orbital / periorbital (n = 4) sites. in conclusion, increased igg4 + cells are not uncommonly seen in sinonasal or orbital / periorbital biopsies of gpa, which could pose as a diagnostic pitfall.
in 2013, as part of a study of fmdv transmission between wildlife, especially african buffalo (syncerus caffer), and domestic animals, 20 long - horned ankole cattle (6 months of age) were introduced as sentinel animals into nyakatonzi (kasese district), in close proximity to the qenp. at the time they were transported, these animals, originating from another area where fmd outbreaks had not been reported for 10 years, had no circulating antibodies against fmdv nonstructural proteins (nsps) (measured by using the priocheck fmdv ns elisa kit [prionics, schlieren - zurich, switzerland ]). blood and probang samples (comprising oropharyngeal scrapings and fluid) were obtained from individual animals at 2-week intervals after their entry to the farm from which they moved regularly into the qenp for pasture and water. these sentinel animals freely mixed and grazed with buffalo (observed within a few meters of each other) and other local cattle. more than 6,000 buffalo no clinical signs of fmd were observed in the sentinel cattle, but serum samples were assayed for antibodies against fmdv nsps (serotype independent) ; rna was extracted from the probang samples and analyzed for fmdv genomes by using pan - serotypic real - time reverse transcription pcr (rt - qpcr) (7). fmdv rna was clearly detected (cycle threshold 21) in the probang sample from animal no. antibodies against fmdv nsps had developed in this animal (table 1), and we detected high - titer antibodies against both fmdv sat 1 and sat 3 antigens using solid - phase blocking elisas (spbes) (table 1) ; hence this calf simultaneously had fmdv rna in the oropharynx and antibodies against fmdv in serum. fmdv is maintained in the oropharynx of cattle for 10 days after infection (8) and continues after viremia has resolved coincident with the production of antibodies against fmdv. infectious fmdv (albeit at low levels) can be maintained within the oropharynx after infection for up to 3 and 5 years in carrier cattle and buffalo, respectively (9). 33) by 30 days after introduction to the qenp (table 1). in the spbes, however, during the early stage of seroconversion, cross - reactivity occurs between the serotypes in these assays (k. tjrnehj, unpub. data). ct, cycle threshold ; elisa, enzyme - linked immunosorbant assay ; fmdv, foot - and - mouth disease virus ; pi, percentage inhibition values in the nsp elisa (values > 50% are considered positive and are indicated in boldface) ; nsp, nonstructural protein ; rt - qpcr, real - time reverse transcription pcr ; sat, southern african territories ; spbe, solid - phase blocking elisa ;, negative sample. day 0 was defined as the day the sentinel animals arrived at the farm in nyakatonzi (kasese district). an in - house serotype - specific spbe was used ; samples positive at 1:10 dilution were titrated ; values are reciprocals of highest positive dilution. fmdv rna in probang samples was assayed by rt - qpcr, and virus isolation was attempted from probang samples with detectable rna and some other selected samples. the isolation of sat 3 fmdv from the rt - qpcr positive probang sample from calf no. we observed cytopathic effects within 48 hours and assayed the virus harvest using serotype - specific antigen elisas. we observed a strong signal (optical density 1.385), indicating sat 3 fmdv with no significant signal for other serotypes (data not shown). the presence of fmdv rna in the cell harvest was demonstrated by using rt - qpcr (cycle threshold 11), indicating isolation and growth of fmdv ; the isolate was named sat 3 uga/1/13. to characterize this strain, we amplified the rna region encoding viral protein (vp) 1 by rt - pcr ; the amplicon (821 bp) was sequenced with a bigdye terminator v. 3.1 cycle sequencing kit and an abi prism 3730dna analyzer (applied biosystems, foster city, ca, usa). using blast (http://blast.st-va.ncbi.nlm.nih.gov/blast.cgi) in mega5 (10), we found the sequence to be most closely related (81% identity) to the ugandan sat 3 buffalo isolate (uga/2/97) (5) (figure 1, panel a). the range of divergence was 19%36% in this region of the genome (published vp1 coding sequences are mainly incomplete [5,1113 ] ; thus we analyzed only 390 nt from the 648 nt encoding vp1). the uga/1/13 strain is most closely related to earlier uganda sat 3 viruses but is within a different lineage and is more divergent from sat 3 viruses from southern africa. the uga/27/70 and uga/2/97 viruses were assigned to topotypes v and vi, respectively (5) ; because the uga/1/13 is 20% different from these strains, it could be designated as a new topotype. however, it seems better to classify these uganda sat 3 strains within a single topotype (v). the genome sequence (8,268 nt) of this sat 3 virus was determined (genbank accession no. only 3 other full - genome sequences for sat 3 fmdvs have been published (13) ; these are from southern african isolates obtained 50 years ago. these sequences are 80% identical to the uga/1/13 isolate ; this degree of diversity is not unexpected considering the geographic and temporal separation between them. relationships between the sat 3 viruses were examined for different regions of the genome (table 2). phylogenetic relationships between known sat 3 p1 coding sequences were determined (figure 1, panel b). alignment of the predicted amino acid sequences for vp1 (figure 2) indicates high levels of variation within important antigenic regions of the virus (especially the g neighbor - joining trees for other regions of the genome showed very similar relationships (data not shown), indicating that these viruses are monophyletic. neighbor - joining trees showing the relationships between a) the partial vp1 coding sequences (390 nt) and b) the complete p1 capsid protein coding sequence (2223 nt) from the sat 3 fmdv uga/1/13 isolate (marked with) and other sat 3 fmdvs within the indicated topotypes defined previously (5 ; http://www.wrlfmd.org/fmdv_seqs/fmdv-sat3_seq.aspx). sequences, other than for uga/1/13, were obtained from genbank and have been published previously (5,1113). bec, bechuanaland (former name for botswana) ; fmdv, foot - and - mouth disease virus ; knp, kruger national park (in south africa) ; p1, precursor for the 4 capsid proteins ; sat, southern african territories ; sar, republic of south africa ; uga, uganda ; vp, viral protein ; zam, zambia ; zim, zimbabwe ; i v, topotypes. bec, bechuanaland (former name for botswana) ; fmdv, foot - and - mouth disease virus ; l, leader protein coding sequence ; sat, southern african territories ; uga, uganda ; utr, untranslated region. the region from the poly(c) tract to the first initiation codon was used for the comparison. excluding the poly(a) tail. predicted vp1 aa sequences of the 8 sat 3 fmdvs used in the phylogenetic comparison in figure 1, panel b. clear similarities between the uga/02/97 and uga/1/13 viruses are apparent. bec, bechuanaland (former name for botswana) ; fmdv, foot - and - mouth disease virus ; knp, kruger national park (in south africa) ; sar, republic of south africa ; sat, southern african territories ; uga, uganda ; vp, viral protein ; zam, zambia ; zim, zimbabwe. approximately 16 years after the most recent isolation of a sat 3 fmdv from buffalo in uganda, a new isolate (uga/1/13) was obtained from an apparently healthy long - horned ankole calf that was newly introduced into the qenp. to our knowledge, this is the first isolation of sat 3 fmdv from cattle in east africa. the vp1 coding sequence was 20% different from the most closely related virus strains within uganda and up to 36% divergent from sat 3 viruses from southern africa. the ugandan sat 3 viruses should be classified within a single topotype (v), but this requires modification of the topotype definition used previously (5). studies are needed to determine the consequences of infection of intensively farmed cattle by this virus.	after a 16-year interval, foot - and - mouth disease virus serotype sat 3 was isolated in 2013 from an apparently healthy long - horned ankole calf that grazed close to buffalo in uganda. the emergent virus strain is 20% different in nucleotide sequence (encoding vp1 [viral protein 1 ]) from its closest relatives isolated previously from buffalo in uganda.
supplies used for the sterile surgical procedure for each rat include 1 drape to be layed out as a sterile surface for the placement of instruments, one drape with a pre - cut hole about 0.5 in x 1.5 in in the center to be placed over the rat, and gauze. the pre - cut hole on the drape will align with the incision made on the rat. instruments needed for the surgery include 1 standard pair of scissors to be used to cut suture material, two forceps and a fine needle holder for handling and gripping of the suture, and a scalpel with a blade. we have listed the common tools used by our laboratory in the table of specific surgical materials and tools. place a 100 ml 0.9% sterile saline bag under a heating pad to ensure that the saline that comes in contact with the mesentery tissue is pre - warmed to approximately 37 c. as an alternative to saline, you can use ringer 's solution or another physiological buffer. lay out sterilized surgical instruments and supplies on a sterile drape for easy access during the exteriorization procedure. also, you will need sterile cotton tip applicators and the appropriate 3 types of sutures (4 - 0, 5 - 0, and 7 - 0). makes sure packages are pre - opened so that the materials can be accessed with sterile handling. finally, disinfect the pre - modified plastic stage by immersing it in 100% ethanol for at least 5 minutes. the stage is a 100 mm petri dish with an elliptical hole cut in the center (figure 1). a dremel tool can be used to initially make and, if necessary, widen the hole. after the hole is made, the edges of the cut plastic are made smooth using sand paper of different grains. the stage is then washed and, finally, either inert modeling clay (purchased from a local crafts store) or silicon glue is added to the hole 's edge in order to provide a raised, smooth surface. prior to contact with the tissue, the stage will need to be adequately rinsed with sterile saline. for this model of angiogenesis, we typically use adult male wistar rats (350 25 g). other rat strains and ages can be used. in our laboratory, rats are anesthetized via intramuscular injection with ketamine (80 mg / kg b.w.), xylazine (8 mg / kg b.w.), and atropine (0.08 mg / kg b.w.). after approximately 5 mins, position the anesthetized rat on its back on a heating pad to maintain body temperature. aseptic techniques are used during the surgical procedure. wear sterile gloves and do not allow instruments and supplies to contact non - sterile surfaces other than the rat tissues. clean abdominal area with alternating wipes using the sterile gauze soaked in 70% isopropyl and iodine. using the scalpel blade, make a small (approximately 0.75 inch) longitudinal incision along the skin and then the linea alba approximately 1 inch below the sternum. place the pre - cut drape over the abdominal section so that the opening aligns with the incision. this usually will result in exposure of the small intestine enough for it to be easily identified. using the cotton tip applicators, gently pull out a section of the small intestine and locate the ileum. as the mesentery is pulled out, it should be gently layed out in the plastic stage (figure 2). a mesenteric window is defined as the thin translucent membrane in between the artery / vein pairs feeding the small intestine (figure 2). leave the mesentery section exteriorized for 20 minutes. during the exteriorization period, use a sterile syringe (5 ml) to intermittently replenish the saline in the petri dish to ensure that the tissue remains immersed and does not dry out. during the 20-minute exposure time, mark the 2 centrally located mesenteric windows with sterile 7 - 0 suture. wipe the sutured area with alternating wipes using the sterile gauze soaked in 70% isopropoyl and iodine. the purpose of lubricating the eye is to prevent desiccation of the cornea during surgical procedure and recovery. the application of eye lubricant should be applied prior to the start of surgical intervention. if necessary, re - apply eye lubricant prior to returning the rat to its cage for recovery. on the day of interest post stimulation, rats are anesthetized via intramuscular injection with ketamine (80 mg / kg b.w.), xylazine (8 mg / kg b.w.), and atropine (0.8 mg / kg b.w.) and then euthanized via intracardiac injection of beuthanasia. beuthanasia is a pentobarbital sodium / phenytoin solution that can be used for rapid and painless euthanasia. per rat re - open the abdominal cavity and gently remove the small intestine and locate the two - marked windows. cut out each of the exteriorized mesenteric windows using forceps and micro - scissors. including a border of fat per window is advantageous for later tissue spreading onto microscope slides. immediately when cutting, try to avoid cutting through artery / vein pairs within the fat border of the windows to minimize potential blood filling of the windows. also, minimize cutting through the bowel that results in intestinal content contacting the windows. allow the tissues to partially dry and using a scalpel blade remove the excess fat., we commonly fix the slides in methanol (-20 c) for 30 min. given our interest in identifying the roles of vascular pericytes during angiogenesis, our laboratory commonly labels for endothelial and perivascular cells. useful labels to identify endothelial cells along the hierarchy of the microvascular networks are an anti - pecam antibody or bsi - lectin. perivascular cell markers used in this tissue include ng2, desmin, sm- actin, pdfgr, and class iii -tubulin. below, in section 3.3, we have listed protocols for colorimetric and fluorescent pecam immunolabeling protocols. prior to the initial primary antibody incubation, also, tissues are initially outlined with a wax pen to prevent uncontrolled flow of antibody solutions away from the tissue. endothelial cell antibody labeling procedures : pecam colorimetric lableing primary antibody incubation : drip approximately 100 - 200 ml primary antibody solution (1:200 mouse monoclonal biotinylated cd31 antibody diluted in antibody buffer (0.1% saponin in pbs + 2% bsa)) on top to each tissue ; make sure the whole tissue is covered with antibody solution. incubation for 1 hour at room temperature.wash tissues with pbs + 0.1% saponin for 30 minutes.secondary antibody incubation : drip secondary antibody solution (vectastain elite abc solution from vector laboratories ; a streptavidin peroxidase secondary antibody solution) on top of tissues. incubate at room temperature for 1 hour.wash tissues with pbs + 0.1% saponin for 30 minutes.incubate tissues with vector nova red for 15 minutes. then rise tissues with water.mount slides : dip slides in 95% ethanol 10 times. immerse slides in another 100% ethanol solution for 2 mins. then immerse slides in consecutive 100% xylene solutions for 2 minutes each. allow to dry and cover tissues with a thin layer of vectamount and a coverslip. pecam fluorescent labeling primary antibody incubation : drip approximately 100 - 200 ml primary antibody solution (1:200 mouse monoclonal biotinylated cd31 antibody diluted in antibody buffer (0.1% saponin in pbs + 2% bsa)) on top to each tissue ; make sure the whole tissue is covered with antibody solution. incubation for 1 hour at room temperature.wash tissues with pbs + 0.1% saponin for 30 minutes.secondary antibody incubation : drip secondary antibody solution ((1:100 streptavidin - cy3 diluted in antibody buffer (0.1% saponin in pbs + 2% bsa)) on top of tissues. incubate at room temperature for 1 hour.wash tissues with pbs + 0.1% saponin for 30 minutes.mount slides : drip 50:50 pbs with glycerol on top of tissues. then, use nail polish to seal the gap between cover slip and slide. primary antibody incubation : drip approximately 100 - 200 ml primary antibody solution (1:200 mouse monoclonal biotinylated cd31 antibody diluted in antibody buffer (0.1% saponin in pbs + 2% bsa)) on top to each tissue ; make sure the whole tissue is covered with antibody solution. secondary antibody incubation : drip secondary antibody solution (vectastain elite abc solution from vector laboratories ; a streptavidin peroxidase secondary antibody solution) on top of tissues. allow to dry and cover tissues with a thin layer of vectamount and a coverslip. primary antibody incubation : drip approximately 100 - 200 ml primary antibody solution (1:200 mouse monoclonal biotinylated cd31 antibody diluted in antibody buffer (0.1% saponin in pbs + 2% bsa)) on top to each tissue ; make sure the whole tissue is covered with antibody solution. secondary antibody incubation : drip secondary antibody solution ((1:100 streptavidin - cy3 diluted in antibody buffer (0.1% saponin in pbs + 2% bsa)) on top of tissues. representative images of rat mesentery tissues immunohistochemically labeled for pecam are displayed in figure 3. pecam labeling identifies all vessel types along the hierarchy of remodeling microvascular networks and can be used to quantify angiogenic metrics at specific time points post stimulation. feeding arterioles typically exhibit smaller diameters and elongated endothelial cell morphology compared to paired venules (figure 4). capillaries and capillary sprouts can be identified based on their vessel diameter and relative position within a network. typical characteristics of remodeling networks include increased capillary sprouting, vessel density, vascularized area and venular tortuosity. quantification of various angiogenic metrics identifies the time course of network growth (figure 5). capillary sprouting from pre - existing vessels, peaks between day 3 and day 5 and returns to the unstimulated level by day 10. this transient increase in sprouting is followed by increases in vascular density and vascularized area. as evidence for the remodeling of larger vessels in this model, the number of arteriole and venule segments also increases over the time course. in our laboratory, this model has been used to identify cellular phenotypic changes at specific time point during this remodeling process. for example, class iii -tubulin identifies pericytes along angiogenic vessels (figure 6). in unstimulated tissues, in contrast, during the peak of capillary sprouting, class iii -tubulin is expressed by perivascular cells. this type of result highlights the use of this simple and robust angiogenic model to identify novel cell types involved in the angiogenic process. modifications include an elliptical hole cut in the center and the subsequent addition of modeling clay or silicon glue to the hole 's edge for the creation of a raised, smooth surface. mesenteric windows are defined as the thin translucent membranes between the artery / vein pairs feeding the small intestine. during the exteriorization duration, the mesenteric region is laid out and immersed in saline inside a modified petri dish. inert yellow modeling clay provides a smooth surface for the mesentery to be pulled through the pre - cut hole. representative images of mesenteric microvascular networks from unstimulated tissues and tissues at 3 and 10 days post exteriorization of the mesentery. pecam labeling identified the hierarchy of microvascular networks including, arterioles (a), venules (v) and capillaries (c). post stimulation, microvascular networks displayed an increase in capillary sprouting (arrow heads) and vessel density. representative images of arteriole / venule pairs within adult rat mesenteric microvascular networks. in both images, arterioles (a) can be differentiated from venules (v) based on a smaller relative diameter and elongated endothelial cell morphology. representative quantification of angiogenic metrics over the time course of microvascular growth post mesentery exteriorization. statistical comparisons were made using a one - way anova followed by dunn 's test. representative fluorescent images of mesenteric microvascular networks from unstimulated tissues and tissues at 3 and 10 days post exteriorization of the mesentery. immunofluorescent pecam labeling (red) identifies endothelial cells, and class iii -tubulin labeling (green) identifies nerves (arrow heads) and perivascular cells (arrows). perivascular cells transiently upregulate class iii -tubulin during capillary sprouting. in unstimulated microvascular networks, 3 days post stimulation, class iii -tubulin positively labels perivascular cells along microvessels. by day 10 images supporting the feasibility of tracking pre - labeled locally applied cells during microvascular network growth stimulated by the mesentery exteriorization. cells were superfused over mesenteric windows during the 20-minute exteriorization period. 1 day post - surgery, dii labeled cells (red) were observed in the dame focal plane with pecam positive microvessels (green). a, b) examples of dii labeled bone marrow cells exhibiting rounded and elongated morphologies. in some cases (arrows) cells were elongated along microvessels. c) example of a cluster of dii labeled mesenchymal stem cells near the tip of a capillary sprout (arrow). scale bars are 50 m (a), and 20 m (b, c). supplies used for the sterile surgical procedure for each rat include 1 drape to be layed out as a sterile surface for the placement of instruments, one drape with a pre - cut hole about 0.5 in x 1.5 in in the center to be placed over the rat, and gauze. the pre - cut hole on the drape will align with the incision made on the rat. instruments needed for the surgery include 1 standard pair of scissors to be used to cut suture material, two forceps and a fine needle holder for handling and gripping of the suture, and a scalpel with a blade. we have listed the common tools used by our laboratory in the table of specific surgical materials and tools. place a 100 ml 0.9% sterile saline bag under a heating pad to ensure that the saline that comes in contact with the mesentery tissue is pre - warmed to approximately 37 c. as an alternative to saline, you can use ringer 's solution or another physiological buffer. lay out sterilized surgical instruments and supplies on a sterile drape for easy access during the exteriorization procedure. also, you will need sterile cotton tip applicators and the appropriate 3 types of sutures (4 - 0, 5 - 0, and 7 - 0). makes sure packages are pre - opened so that the materials can be accessed with sterile handling. finally, disinfect the pre - modified plastic stage by immersing it in 100% ethanol for at least 5 minutes. the stage is a 100 mm petri dish with an elliptical hole cut in the center (figure 1). a dremel tool can be used to initially make and, if necessary, widen the hole. after the hole is made, the edges of the cut plastic are made smooth using sand paper of different grains. the stage is then washed and, finally, either inert modeling clay (purchased from a local crafts store) or silicon glue is added to the hole 's edge in order to provide a raised, smooth surface. prior to contact with the tissue, the stage will need to be adequately rinsed with sterile saline. for this model of angiogenesis, we typically use adult male wistar rats (350 25 g). other rat strains and ages can be used. in our laboratory, rats are anesthetized via intramuscular injection with ketamine (80 mg / kg b.w.), xylazine (8 mg / kg b.w.), and atropine (0.08 mg / kg b.w.). after approximately 5 mins, position the anesthetized rat on its back on a heating pad to maintain body temperature. aseptic techniques are used during the surgical procedure. wear sterile gloves and do not allow instruments and supplies to contact non - sterile surfaces other than the rat tissues. clean abdominal area with alternating wipes using the sterile gauze soaked in 70% isopropyl and iodine. using the scalpel blade, make a small (approximately 0.75 inch) longitudinal incision along the skin and then the linea alba approximately 1 inch below the sternum. place the pre - cut drape over the abdominal section so that the opening aligns with the incision. gently apply pressure around the incision. this usually will result in exposure of the small intestine enough for it to be easily identified. using the cotton tip applicators, gently pull out a section of the small intestine and locate the ileum. as the mesentery is pulled out, it should be gently layed out in the plastic stage (figure 2). a mesenteric window is defined as the thin translucent membrane in between the artery / vein pairs feeding the small intestine (figure 2). use a sterile syringe (5 ml) to intermittently replenish the saline in the petri dish to ensure that the tissue remains immersed and does not dry out. during the 20-minute exposure time, mark the 2 centrally located mesenteric windows with sterile 7 - 0 suture. wipe the sutured area with alternating wipes using the sterile gauze soaked in 70% isopropoyl and iodine. the purpose of lubricating the eye is to prevent desiccation of the cornea during surgical procedure and recovery. the application of eye lubricant should be applied prior to the start of surgical intervention. if necessary, re - apply eye lubricant prior to returning the rat to its cage for recovery. on the day of interest post stimulation, anesthetize and euthanize the rat. in our laboratory, rats are anesthetized via intramuscular injection with ketamine (80 mg / kg b.w.), xylazine (8 mg / kg b.w.), and atropine (0.8 mg / kg b.w.) and then euthanized via intracardiac injection of beuthanasia. beuthanasia is a pentobarbital sodium / phenytoin solution that can be used for rapid and painless euthanasia. per rat re - open the abdominal cavity and gently remove the small intestine and locate the two - marked windows. cut out each of the exteriorized mesenteric windows using forceps and micro - scissors. including a border of fat per window is advantageous for later tissue spreading onto microscope slides. immediately when cutting, try to avoid cutting through artery / vein pairs within the fat border of the windows to minimize potential blood filling of the windows. also, minimize cutting through the bowel that results in intestinal content contacting the windows. allow the tissues to partially dry and using a scalpel blade remove the excess fat., we commonly fix the slides in methanol (-20 c) for 30 min. given our interest in identifying the roles of vascular pericytes during angiogenesis, our laboratory commonly labels for endothelial and perivascular cells. useful labels to identify endothelial cells along the hierarchy of the microvascular networks are an anti - pecam antibody or bsi - lectin. perivascular cell markers used in this tissue include ng2, desmin, sm- actin, pdfgr, and class iii -tubulin. below, in section 3.3, we have listed protocols for colorimetric and fluorescent pecam immunolabeling protocols. prior to the initial primary antibody incubation, also, tissues are initially outlined with a wax pen to prevent uncontrolled flow of antibody solutions away from the tissue. endothelial cell antibody labeling procedures : pecam colorimetric lableing primary antibody incubation : drip approximately 100 - 200 ml primary antibody solution (1:200 mouse monoclonal biotinylated cd31 antibody diluted in antibody buffer (0.1% saponin in pbs + 2% bsa)) on top to each tissue ; make sure the whole tissue is covered with antibody solution. incubation for 1 hour at room temperature.wash tissues with pbs + 0.1% saponin for 30 minutes.secondary antibody incubation : drip secondary antibody solution (vectastain elite abc solution from vector laboratories ; a streptavidin peroxidase secondary antibody solution) on top of tissues. incubate at room temperature for 1 hour.wash tissues with pbs + 0.1% saponin for 30 minutes.incubate tissues with vector nova red for 15 minutes. then rise tissues with water.mount slides : dip slides in 95% ethanol 10 times. immerse slides in another 100% ethanol solution for 2 mins. then immerse slides in consecutive 100% xylene solutions for 2 minutes each. allow to dry and cover tissues with a thin layer of vectamount and a coverslip. pecam fluorescent labeling primary antibody incubation : drip approximately 100 - 200 ml primary antibody solution (1:200 mouse monoclonal biotinylated cd31 antibody diluted in antibody buffer (0.1% saponin in pbs + 2% bsa)) on top to each tissue ; make sure the whole tissue is covered with antibody solution. incubation for 1 hour at room temperature.wash tissues with pbs + 0.1% saponin for 30 minutes.secondary antibody incubation : drip secondary antibody solution ((1:100 streptavidin - cy3 diluted in antibody buffer (0.1% saponin in pbs + 2% bsa)) on top of tissues. incubate at room temperature for 1 hour.wash tissues with pbs + 0.1% saponin for 30 minutes.mount slides : drip 50:50 pbs with glycerol on top of tissues. then, use nail polish to seal the gap between cover slip and slide. primary antibody incubation : drip approximately 100 - 200 ml primary antibody solution (1:200 mouse monoclonal biotinylated cd31 antibody diluted in antibody buffer (0.1% saponin in pbs + 2% bsa)) on top to each tissue ; make sure the whole tissue is covered with antibody solution. secondary antibody incubation : drip secondary antibody solution (vectastain elite abc solution from vector laboratories ; a streptavidin peroxidase secondary antibody solution) on top of tissues. incubate at room temperature for 1 hour. wash tissues with pbs + 0.1% saponin for 30 minutes. incubate tissues with vector nova red for 15 minutes. then rise tissues with water. immerse slides in another 100% ethanol solution for 2 mins. then immerse slides in consecutive 100% xylene solutions for 2 minutes each. allow to dry and cover tissues with a thin layer of vectamount and a coverslip. primary antibody incubation : drip approximately 100 - 200 ml primary antibody solution (1:200 mouse monoclonal biotinylated cd31 antibody diluted in antibody buffer (0.1% saponin in pbs + 2% bsa)) on top to each tissue ; make sure the whole tissue is covered with antibody solution. secondary antibody incubation : drip secondary antibody solution ((1:100 streptavidin - cy3 diluted in antibody buffer (0.1% saponin in pbs + 2% bsa)) on top of tissues. representative images of rat mesentery tissues immunohistochemically labeled for pecam are displayed in figure 3. pecam labeling identifies all vessel types along the hierarchy of remodeling microvascular networks and can be used to quantify angiogenic metrics at specific time points post stimulation. feeding arterioles typically exhibit smaller diameters and elongated endothelial cell morphology compared to paired venules (figure 4). capillaries and capillary sprouts can be identified based on their vessel diameter and relative position within a network. typical characteristics of remodeling networks include increased capillary sprouting, vessel density, vascularized area and venular tortuosity. quantification of various angiogenic metrics identifies the time course of network growth (figure 5). capillary sprouting from pre - existing vessels, peaks between day 3 and day 5 and returns to the unstimulated level by day 10. this transient increase in sprouting is followed by increases in vascular density and vascularized area. as evidence for the remodeling of larger vessels in this model, the number of arteriole and venule segments also increases over the time course. in our laboratory, this model has been used to identify cellular phenotypic changes at specific time point during this remodeling process. for example, class iii -tubulin identifies pericytes along angiogenic vessels (figure 6). in unstimulated tissues, in contrast, during the peak of capillary sprouting, class iii -tubulin is expressed by perivascular cells. this type of result highlights the use of this simple and robust angiogenic model to identify novel cell types involved in the angiogenic process. modifications include an elliptical hole cut in the center and the subsequent addition of modeling clay or silicon glue to the hole 's edge for the creation of a raised, smooth surface. mesenteric windows are defined as the thin translucent membranes between the artery / vein pairs feeding the small intestine. during the exteriorization duration, the mesenteric region is laid out and immersed in saline inside a modified petri dish. inert yellow modeling clay provides a smooth surface for the mesentery to be pulled through the pre - cut hole. representative images of mesenteric microvascular networks from unstimulated tissues and tissues at 3 and 10 days post exteriorization of the mesentery. pecam labeling identified the hierarchy of microvascular networks including, arterioles (a), venules (v) and capillaries (c). post stimulation, microvascular networks displayed an increase in capillary sprouting (arrow heads) and vessel density. representative images of arteriole / venule pairs within adult rat mesenteric microvascular networks. in both images, arterioles (a) can be differentiated from venules (v) based on a smaller relative diameter and elongated endothelial cell morphology. representative quantification of angiogenic metrics over the time course of microvascular growth post mesentery exteriorization. statistical comparisons were made using a one - way anova followed by dunn 's test. representative fluorescent images of mesenteric microvascular networks from unstimulated tissues and tissues at 3 and 10 days post exteriorization of the mesentery. immunofluorescent pecam labeling (red) identifies endothelial cells, and class iii -tubulin labeling (green) identifies nerves (arrow heads) and perivascular cells (arrows). in unstimulated microvascular networks, class iii -tubulin is nerve specific and does not identify perivascular cells. 3 days post stimulation, class iii -tubulin positively labels perivascular cells along microvessels. by day 10 images supporting the feasibility of tracking pre - labeled locally applied cells during microvascular network growth stimulated by the mesentery exteriorization. cells were superfused over mesenteric windows during the 20-minute exteriorization period. 1 day post - surgery, dii labeled cells (red) were observed in the dame focal plane with pecam positive microvessels (green). a, b) examples of dii labeled bone marrow cells exhibiting rounded and elongated morphologies. in some cases (arrows) cells were elongated along microvessels. c) example of a cluster of dii labeled mesenchymal stem cells near the tip of a capillary sprout (arrow). scale bars are 50 m (a), and 20 m (b, c). the exteriorization model was reported in 2006 and is adapted from previous mechanical injury rat mesentery models of angiogenesis and produces similar results to well established i.p. the 20 minute exteriorization time was experimentally determined to produce a robust angiogenic response. while this time period could be varied, it does allow for local application of angiogenic inhibitors for mechanistic studies and direct application of exogenous cells for cell lineage studies. feasibility of cell incorporation into remodeling mesenteric tissue is supported by preliminary studies in our laboratory using pre - labeled bone marrow cells and mesenchymal stem cells (figure 7), and by the success of investigating the fate of human adipose - derived stromal cells injection i.p.. in our laboratory, we have used this model to identify pericyte phenotypic changes over the time course of the angiogenic response and to assess the angiogenic potential during pathological conditions, such as hypertension. the angiogenic response and cell phenotype changes associated with this model can also be observed in other rat mesenteric angiogenic models including chronic hypoxia exposure. a limitation of the exteriorization model is that the exact triggering mechanisms of angiogenesis are unknown. exteriorization of the mesentery has been linked to mast cell degranulation and increased histamine levels, yet further investigation is required to gain more insight. the angiogenic stimulus is undoubtedly multi - factorial, producing a robust remodeling response across the hierarchy of a microvascular network. while the unknown mechanisms remain a major critique of this model, its reproducibility and simplicity make it attractive for identifying cellular dynamics involved in the inherently complex capillary sprouting process. the reproducibility of the model is supported by comparable angiogenic metrics over the time course of microvascular network growth across multiple rat strains (male wistar and female sprague - dawley) in previously published studies from our laboratory. since, the majority of adult rat mesenteric tissues are vascularized, the model also allows for multiple tissues to be examined per animal. unfortunately, this model is not obviously applicable to genetic mouse models as mouse mesenteric windows have less native vascularization and, in our experience, commonly lack observable branching networks. future applications include the investigation of vessel functionality during angiogenesis using intra - vital microscopy at specific time points and the investigation of related cellular dynamics involved in lymphangiogenesis and neurogenesis. though the extent of native vascularization per mesenteric window seems to be roughly proportional with age, we have observed branching microvascular networks in male wistar rats as young as 4 - 5 weeks old. these observations suggest that the exteriorization model could also be applied to compare the angiogenic differences across ages.	microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre - existing vessels. the angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. this information is critical for the development of therapies aimed at manipulating vessel growth.the exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. it was adapted from wound - healing models in the rat mesentery4 - 7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro - angiogenic agents8, 9. the exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two - dimensional visualization of entire microvascular networks down to single cell level. the stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.
long glucocorticoid - induced leucine zipper mouse double minute 2 homolog p53 upregulated modulator of apoptosis the transcription factor tumor protein p53 (tp53, best known as p53) is the main tumor suppressor regulating the expression of genes crucial for maintaining cellular homeostasis and ensuring genomic stability. upon activation by diverse oncogenic stress stimuli, p53 triggers cellular responses through regulation of its transcriptional targets to help repair or eliminate cells with damaged dna, i.e., potential cancers. indeed, most cancer cells carry mutation(s) of tp53, thereby growing and metastasizing by escaping p53 regulation, whereas normal cells maintain low levels of p53. therefore, it is not surprising that p53 expression is tightly regulated through feedback mechanisms involving partner molecules that continuously monitor p53 expression / degradation to allow the appropriate biological responses. the most prominent negative regulator of p53 is mouse double minute 2 homolog (mdm2), which binds p53 and interferes with p53-mediated transcription, promotes p53 nuclear export, and induces ubiquitin - mediated proteasomal degradation. p53 was discovered based on its antitumorigenic properties, although today it is known to exhibit a much broader spectrum of cellular functions. since its discovery, enormous resources have been invested in basic research on the physiological and pharmacological regulation of p53, which has resulted in significant advances in cancer research (for a review see refs). our recent paper in cell death and differentiation reveals new details of the regulation of p53 activation. long glucocorticoid (gc)-induced leucine zipper (l - gilz), a newly identified isoform of the better - characterized gilz, forms dimers with mdm2 and p53 (although it has greater affinity for the former) and inhibits the p53/mdm2 interaction. as a consequence of these events p53 the ubiquitination / proteasome machinery appears to be involved in l - gilz induced activation of p53, as l - gilz decreases ubiquitination of p53 and increases that of mdm2 (fig. 1). as expected, l - gilz activation of p53 triggers the antiproliferative and apoptotic pathways of p53, which involve increased expression of p21 and p53 upregulated modulator of apoptosis (puma) with consequent inhibition of cell proliferation in vitro and tumor growth in vivo. accordingly, silencing of l - gilz reverses the antiproliferative activity of dexamethasone (dex). hence, this new player, l - gilz, fits into the intricate network of signals activating p53 and pathways activated by p53 to further control cell fate. the mechanism of l - gilz action has not yet been fully elucidated, but it resembles that of other p53 and/or mdm2 molecular partners that regulate p53 activation by dissociating mdm2/p53 complexes and/or by acting on the ubiquitination machinery. however, unlike other binding partners, l - gilz is upregulated by dex in breast cancer cells, and this offers one more intriguing avenue to explore. we will now review what we know as the basis for what we still need to discover. l - gilz / p53 binding requires nuclear export of p53 and occurs in the cytoplasm, where l - gilz / mdm2 binding and l - gilz - induced dissociation of the p53/mdm2 complex also occur (fig., we do not yet know why l - gilz binds preferentially to mdm2, but also forms cytoplasmic dimers with p53. we also do not know whether the binding of l - gilz to p53 and/or mdm2 is required for altered ubiquitination of p53 and mdm2. we are aware that there could be mechanisms other than ubiquitination underlying l - gilz induced p53 activation. other unexplored avenues are possible differences in binding of l - gilz to p53 and/or mdm2 under basal conditions and in the presence of stress stimuli that normally activate p53. if such differences exist, the diverse interactions of l - gilz with p53/mdm2 may assume functional significance, for example to maintain low expression levels or activate p53, depending on l - gilz, p53, and mdm2 expression levels. understanding the molecular mechanism underlying p53 activation by l - gilz is essential for planning pharmacological manipulation of l - gilz when p53 activation is required. long glucocorticoid - induced leucine zipper (l - gilz) interacts directly with p53 and mouse double minute 2 homolog (mdm2) in the cytosol, dissociating the p53/mdm2 complex. the affinity of l - gilz is greater for mdm2 (large arrow) than for p53 (small arrow). l - gilz / p53 dimer formation is prevented by inhibition of nuclear export. as a result of l - gilz binding to p53 and mdm2, ubiquitination of p53 is decreased and that of mdm2 is increased, and p53 is activated with consequent activation of p21 and p53 upregulated modulator of apoptosis (puma). long glucocorticoid - induced leucine zipper (l - gilz) interacts directly with p53 and mouse double minute 2 homolog (mdm2) in the cytosol, dissociating the p53/mdm2 complex. the affinity of l - gilz is greater for mdm2 (large arrow) than for p53 (small arrow). l - gilz / p53 dimer formation is prevented by inhibition of nuclear export. as a result of l - gilz binding to p53 and mdm2, ubiquitination of p53 is decreased and that of mdm2 is increased, and p53 is activated with consequent activation of p21 and p53 upregulated modulator of apoptosis (puma). second, what is the role of l - gilz in human cancers ? by activating p53, l - gilz suppresses the growth of xenografts in mice ; therefore, a correlation between decreased expression / function of l - gilz and hypofunction of p53 in human tumors is plausible. clearly, tumors that express wild - type p53 and whose antitumorigenic function has been switched off, for example by mdm2 overexpression, may provide more attractive targets than tumors expressing mutated p53 for potential therapeutic strategies to enhance l - gilz expression and restore p53 activity. in addition, dex upregulated l - gilz expression in a breast cancer cell line, and silencing of l - gilz reversed dex antiproliferative activity as well as expression of p53, p21, and puma. thus, l - gilz may represent a means of gc / p53 crosstalk and in this way contribute to gc therapeutic efficacy. in fact, recent demonstrations of the role of p53 in protection against sources of inflammatory stress such as reactive oxygen and nitrogen species, infectious agents, cytokines, and signaling pathways, for example the nuclear factor-b (nf-b) pathway, suggest that, through p53 activation, l - gilz may impact not only gc - responsive cancers, but also canonical gc anti - inflammatory activity. the chronic inflammatory response and stress pathways promoting carcinogenesis often recognize the same multitude of overlapping signals that are activated when tissue homeostasis is impaired by damaging stimuli and involve p53 responses to a large extent. with the finding that l - gilz activates p53, an intriguing new field of investigation has opened up that may link gcs, p53, inflammation, and cancer. these topics are already being extensively investigated, but this new common thread of l - gilz could aid in the exploration of currently unknown underlying molecular mechanisms. this study was supported by a grant from the associazione italiana per la ricerca sul cancro (airc), milan, italy (ig 10677)	a recent report from our laboratory reveals how long glucocorticoid - induced leucine zipper (l - gilz) protein binds to p53 and mouse double minute 2 homolog (mdm2), thus dissociating the p53/mdm2 complex and activating p53 with subsequent activation of downstream genes p21 and p53 upregulated modulator of apoptosis (puma). p53 activation appears to be the mechanism by which both basal and glucocorticoid (gc)-induced l - gilz inhibits proliferation and induces antioncogenic activity in human cancer.
in the post - genomic era, with the advent of rapid sequencing techniques, reliable and efficient functional annotation methods are needed. routinely, a translated protein sequence is aligned towards a data base of already annotated sequences and by this it is endowed with different features depending on the level of sequence identity (si). this similarity search is the basis for transfer of annotation by homology. the uniprot knowledgebase (uniprotkb ; http://www.uniprotkb.org/) is presently our major resource of information of protein sequences and of corresponding functions and structures, when available. it provides links also to other resources / data bases, allowing a comprehensive knowledge of experimental and computational characteristics of known / putative proteins and genes. however, only 4.4% of the all protein universe that presently (uniprotkb release 2011_03 ; 8 march 2011) includes some 14 million of sequences has evidence at the protein and at the transcript level. with this scenario, inference of function and structure among related sequences requires the definition of rules to increase the reliability of annotation this is routinely obtained with clustering methods by which sequences are included into sets of similarity. examples of hierarchical clustering include systers (1), picasso (2) and iproclass (3). clustr (4,5) and protonet (6,7) are the only web servers that comprise the large number of sequences made available by fully sequenced genomes and the entire uniprotkb. both clustr and protonet cluster sequences according to different levels of si, as set by different e - value thresholds, and with different hierarchical algorithms. alternatively, non - hierarchical clustering partitions a sequence data set into disjoint clusters (8,9). however, neither hierarchical nor non - hierarchical methods consider explicitly proteins containing multiple domains or proteins that sharing common domains do not necessarily have the same function. proteins with different combinations of shared domains can have different molecular and biological functions, as recently re - discussed (10). in order to address these problems, we developed bar (11), an annotation procedure that relies on a non - hierarchical clustering method and a large - scale genome comparison where pairs of sequences are selected with very strict criteria of similarity and overlapping of the alignment as described in the next section. we provided statistical validation that bar allows reliable functional and structural annotation in addition to that given by commonly used databases (11). here, we introduce bar, an updated and extended version of bar that includes : (i) a 5-fold increase in sequences ; (ii) go terms from the three main roots (molecular function, biological process and cellular localization ; http://www.geneontology.org/) ; (iii) pfam domains (http://pfam.sanger.ac.uk/) ; (iv) known ligands and (v) for clusters containing pdb structure / s, a cluster hmm model and the corresponding alignment of the target sequence to the optimal template in the cluster for computing its 3d structure. bar is constructed by performing an all - against - all pairwise alignment of all protein sequences (collected from the entire uniprotkb 05_2010, with the exclusion of fragments (9 399 063 sequences), and from the proteome of complete sequenced genomes available on the same date at the national center for biotechnology information (ncbi) [www.ncbi.nlm.nih.gov/genomes/lproks.cgi (prokaryotes) ; www.ncbi.nlm.nih.gov/genomes/leuks.cgi (eukaryotes) ] and at ensembl (http://www.ensembl.org/info/data/ftp/index.html) for a total of 988 complete proteomes (the list of the species is available at bar+ web site). for the sake of comparison, we also used the entire swissprot 03_2011 (8 march). similarly to bar (11), bar is also a non - hierarchical clustering method relying on a comparative large - scale genome analysis. the method relies on a non - hierarchical clustering procedure characterized by a stringent metric that ensures a reliable transfer of features within clusters. in this new version, the method takes advantage of a larger scale pairwise sequence comparison than bar, including 13 495 736 protein sequences. alignment is performed with blast (12) in a grid environment (11). from this we compute for each pair both the si and the coverage (cov) defined as the ratio of the length of the intersection of the aligned regions on the two sequences and the overall length of the alignment (namely the sum of the lengths of the two sequences minus the intersection length). each protein is then taken as a node and a graph is built allowing links among nodes only when the following similarity constraints are found among two proteins : their si is 40% and cov is 90%. by this, clusters are simply the connected components of the graph (11). seventy percent of the whole data set (9 401 223 sequences) falls into 913 962 clusters. the number of sequence in the clusters ranges from two up to 87 893 in the most populated (molecular function : abc transporter). given our stringent criteria, 87% of the clusters contain sequences whose standard deviation (sd) of the protein length is 5 residues. the remaining sequences (30% of the total) originate singletons (containing just one sequence). well annotated sequences are characterized by functional and structural annotations derived from uniprotkb entries (figure 1). these include go, pfam, pdb and scop (http://scop.mrc-lmb.cam.ac.uk/scop/) (when available). to assess whether go and pfam terms are significant in a cluster, we compute p - values and given the multiplicity of the terms, we applied the bonferroni correction (11). we evaluated the cumulative distribution of bonferroni corrected p - values by adopting a bootstrapping procedure. from this we set the threshold p - value at 0.01 in order to discriminate among random and significant (cluster associated) features (11). validated features (significant for the cluster) are those endowed with p 0.01. according to our procedure when hypothetical and or putative proteins fall into an annotated and validated cluster, they can safely inherit go terms and pfam domain / s even in the case of very low si with the most annotated proteins. these sequences can therefore be labelled as distantly related homologues and inherit function and structure (when available) in a validated manner. we previously discussed that this procedure can increase the level of annotation of uniprotkb (11). here we increase the level of structural and functional annotations of cluster - included sequences by 54% (figure 2a). they can anyway carry along information (figure 2b), provided that each singleton is endowed with pdb and/or pfam and/or go annotation. our method collects sequences from the protein universe (uniprotkb) including also some 988 genomes. by this, all the features [pdb (scop classification) (red circles), go terms (including molecular function, biological process and cellular localization) and pfam models (blue circles) are also included. an extensive blast alignment is performed of all the 13 495 736 sequences in a grid environment. the sequence similarity network is built by connecting two sequences only if their si is 40% with an overlapping cov 90%. about 913 762 clusters are obtained by splitting of the connected components. by this, any cluster may contain from 2 up to 87 893 sequences (one cluster containing abc transporters from prokaryotes, eukaryotes and archaea). when clusters are endowed with pdb template / s, a cluster - hmm is generated by considering all the sequences that have an identity 40% and a cov 90% with the structure / s (pink subset). the cluster - hmm can be used to align all the other sequences in the cluster to template / s. figure 2.different types of annotations are possible with bar. after clustering and depending on the features (structure, domains and function) annotated in the cluster, the percentage of sequences endowed with a given annotation type and inheriting validated annotation (p < 0.01) is indicated. percentage is computed with respect to 9 401 223 comprised in 913 762 clusters. inherited : sequences that inherit annotations by falling into a cluster. without validated annotation : the slice comprises sequences with no annotation and not validated annotations. our method collects sequences from the protein universe (uniprotkb) including also some 988 genomes. by this, all the features [pdb (scop classification) (red circles), go terms (including molecular function, biological process and cellular localization) and pfam models (blue circles) are also included. an extensive blast alignment is performed of all the 13 495 736 sequences in a grid environment. the sequence similarity network is built by connecting two sequences only if their si is 40% with an overlapping cov 90%. about 913 762 clusters are obtained by splitting of the connected components. by this, any cluster may contain from 2 up to 87 893 sequences (one cluster containing abc transporters from prokaryotes, eukaryotes and archaea). when clusters are endowed with pdb template / s, a cluster - hmm is generated by considering all the sequences that have an identity 40% and a cov 90% with the structure / s (pink subset). the cluster - hmm can be used to align all the other sequences in the cluster to template / s. different types of annotations are possible with bar. after clustering and depending on the features (structure, domains and function) annotated in the cluster, the percentage of sequences endowed with a given annotation type and inheriting validated annotation (p < 0.01) is indicated. percentage is computed with respect to 9 401 223 comprised in 913 762 clusters. inherited : sequences that inherit annotations by falling into a cluster. without validated annotation : the slice comprises sequences with no annotation and not validated annotations. in bar, when pdb templates are present within a cluster (with or without their scop classification), profile hmms are computed on the basis of sequence to structure alignment and are cluster associated (cluster - hmm) (figure 1). when different templates are present in a cluster the structural alignment among them is computed with mustang (13). multiple alignments comprising all the overlapping templates and the sequences similar to them (with si 40% and cov 90%) are computed with muscle (14) and fed to hmmer 2.3 (15) in order to train the profile - hmm. by this, a library of 10 858 hmms is made available for aligning even distantly related sequences to a given pdb template / s. the server also provides the pairwise query sequence structural computed with the viterbi decoding implemented in hmmer from the correspondent cluster - hmm and useful for further processing and/or computing the corresponding 3d structure. bar allows 35 possible fine grain types of annotations (plus no annotation) (table 1). the most complete type of annotation is the one with pdb (with and without scop annotation) and go terms and pfam domains with p 0.01 (validated) (first row in table 1). interestingly, enough 0.11% of the total sequences in our database are sufficient to annotate in a validated manner and with the most complete annotation another 21.99% sharing common clusters (8251 ; 0.90% of the total), with an annotation gain factor higher than 200. summing up (along the first row of table 1), we can conclude that validated functional annotation is possible within 10% of the clusters. eleven percent of the sequences remains without annotation and are included in 45% of the clusters. about 57% of singletons (corresponding to 17% of the total set) table 1.the fine grain types of annotation with barpdb (%) scop monoscop multiwithout pdbgo validated pfam validated clusters8251 (0.90)3613 (0.40)1461 (0.16)83 266 (9.11) sequences2 982 449 (22.10)1 408 542 (10.44)1 028 565 (7.62)2 903 431 (21.51) inherited2 967 743 (21.99)1 404 011 (10.40)1 026 154 (7.60)1 382 310 (10.24) pfam clusters8334 (0.91)3647 (0.40)1463 (0.16)85 886 (9.40) sequences2 984 057 (22.11)1 409 647 (10.45)1 028 569 (7.62)2 922 876 (21.66) inherited2 969 285 (22.00)1 405 095 (10.41)1 026 156 (7.60)1 398 603 (10.36) without pfam clusters320 (0.04)123 (0.01)256251 (0.68) sequences42 202 (0.31)15 415 (0.11)7363 (0.05)143 533 (1.06) inherited41 825 (0.31)15 303 (0.11)7331 (0.05)93 568 (0.69)go pfam validated clusters8938 (0.98)3887 (0.43)1504 (0.16)133 895 (14.65) sequences3 042 649 (22.55)1 450 437 (10.75)1 029 707 (7.63)3 311 421 (24.54) inherited3 026 916 (22.43)1 445 521 (10.71)1 027 219 (7.61)1 617 763 (11.99) pfam clusters9357 (1.02)4033 (0.44)1526 (0.17)322 937 (35.34) sequences3 045 465 (22.57)1 451 928 (10.76)1 029 755 (7.63)3 739 076 (27.71) inherited3 029 337 (22.45)1 446 890 (10.72)1 027 247 (7.61)1 852 223 (13.72) singletons2608 (0.02)1051 515 720 (11.23) without pfam clusters452 (0.05)176 (0.02)3045 539 (4.98) sequences46 311 (0.34)17 020 (0.13)7400 (0.05)330 354 (2.45) inherited45 803 (0.34)16 862 (0.12)7362 (0.05)226 500 (1.68) singletons27922129 212 (0.96)without go pfam validated clusters679 (0.07)345 (0.04)1554 314 (5.94) sequences44 172 (0.33)27 775 (0.21)654547 459 (4.06) inherited43 416 (0.32)27 410 (0.20)633221 585 (1.64) pfam clusters779 (0.09)377 (0.04)16122 236 (13.38) sequences44 582 (0.33)27 983 (0.21)656695 684 (5.15) inherited43 735 (0.32)27 592 (0.20)634301 792 (2.24) singletons20510702 834 (5.21) without pfam clusters270 (0.03)83 (0.01)5412 192 (45.11) sequences5308 (0.04)1771 (0.01)1541 494 443 (11.07) inherited5023 (0.04)1689 (0.01)149 singletons129101 743 526 (12.92)percentage is evaluated with respect to the total number of sequences in the data base (13 495 736 sequences). bold character : sequences that inherit the annotation typevalues are negligible. validated : p 0.01 (see text for details, 11). within bar clusters, 35 different types of annotations are possible : (i) + go+pfam+pdb [with or without scop (monodomain, multidomain) ] ; go and pfam are or not validated (no. of levels = 12). (ii) + pfam+pdb (with or without scop) (no. of levels = 6). (iii) + go+pdb (with or without scop) (number of levels = 6). (iv) + pfam+go (no. of levels = 4). (v) + pdb (with or without scop) (number of levels = 3). (vi) + go (no. of levels = 2). (vii) + pfam (no. of levels = 2). seventy percent of the initial set fall into clusters (913 962) and 53% in validated clusters. some 6% of the sequences are annotated without validation and the remaining 11% are not annotated (rightmost bottom cell). about 17 and 13% of the sequences are singletons with and without annotations, respectively. the fine grain types of annotation with bar percentage is evaluated with respect to the total number of sequences in the data base (13 495 736 sequences). bold character : sequences that inherit the annotation type values are negligible. validated : p 0.01 (see text for details, 11). within bar clusters, 35 different types of annotations are possible : (i) + go+pfam+pdb [with or without scop (monodomain, multidomain) ] ; go and pfam are or not validated (no. of levels = 12). (ii) + pfam+pdb (with or without scop) (no. of levels = 6). (iii) + go+pdb (with or without scop) (number of levels = 6). (iv) + pfam+go (no. of levels = 4). (v) + pdb (with or without scop) (number of levels = 3). (vi) (vii) + pfam (no. of levels = 2). seventy percent of the initial set fall into clusters (913 962) and 53% in validated clusters. some 6% of the sequences are annotated without validation and the remaining 11% are not annotated (rightmost bottom cell). about 17 and 13% of the sequences are singletons with and without annotations, respectively. when a query sequence is submitted, there are three possible outcomes (figure 3). the sequence can match a sequence already present in the cluster (or in a singleton). by this, non - annotated proteins can inherit functional and structural annotation from other proteins within the same cluster. validated annotations are inherited when clusters are endowed with validated go and pfam (p < 0.01). the query sequence may then align with any other sequence in bar with the stringent criteria of our procedure and, therefore, find a cluster from where it can safely inherit all the corresponding structural and functional features. alternatively, when the criteria are not met, all the blast matches are returned. provided that the sequence after running blast has a level of si 40% with a cov 90% to any sequence of bar, it is included into a cluster. in the above example, the cluster is well annotated and the sequence inherits all the possible annotations from the cluster including go terms (203), pdb / s, ligands, scop and pfam annotations and the cluster - hmm. furthermore in pir format alignment / alignments of the query sequence to the cluster template / s with cluster hmm is / are also provided. provided that the sequence after running blast has a level of si 40% with a cov 90% to any sequence of bar, it is included into a cluster. in the above example, the cluster is well annotated and the sequence inherits all the possible annotations from the cluster including go terms (203), pdb / s, ligands, scop and pfam annotations and the cluster - hmm. furthermore in pir format alignment / alignments of the query sequence to the cluster template / s with cluster hmm is / are also provided. this is based on the notion that indeed the bar annotation system increases its capacity only when we add information. this is achieved when proteins with evidence at the transcript and protein level (e.g. : pdb new files and/or proteins with go / pfam terms) are included in the system. for example, by comparing uniprotkb 05_2010 with swissprot 03_2011, we collected some 2445 sequences carrying information according to our criteria (evidence at protein / transcript level). by aligning this set towards bar clusters, about 8% aligns with singletons and only 0.03% of the total number of bar singletons become new clusters (with two protein sequences). another 7% fall into non - validated clusters without affecting the statistical significance of the cluster - specific annotation. we are currently planning to include other annotation resources in order to extend our annotation process with more protein domains and their interactions. d.p. is the recipient of a miur (ministero istruzione universit ricerca) fellowship supporting his ph.d. program ; miur - firb (fondo per gli investimenti della ricerca di base) 2003/libi - international laboratory for bioinformatics delivered (to r.c. funding for open access charge : fondo ordinario per le universit (ffo) 2010 delivered (to r.c. and	we introduce bar - plus (bar+), a web server for functional and structural annotation of protein sequences. bar+ is based on a large - scale genome cross comparison and a non - hierarchical clustering procedure characterized by a metric that ensures a reliable transfer of features within clusters. in this version, the method takes advantage of a large - scale pairwise sequence comparison of 13 495 736 protein chains also including 988 complete proteomes. available sequence annotation is derived from uniprotkb, go, pfam and pdb. when pdb templates are present within a cluster (with or without their scop classification), profile hidden markov models (hmms) are computed on the basis of sequence to structure alignment and are cluster - associated (cluster - hmm). therefrom, a library of 10 858 hmms is made available for aligning even distantly related sequences for structural modelling. the server also provides pairwise query sequence structural computed from the correspondent cluster - hmm. bar+ in its present version allows three main categories of annotation : pdb [with or without scop () ] and go and/or pfam ; pdb () without go and/or pfam ; go and/or pfam without pdb () and no annotation. each category can further comprise clusters where go and pfam functional annotations are or are not statistically significant. bar+ is available at http://bar.biocomp.unibo.it/bar2.0.
increases in weight and adiposity at the population level were first observed in high - income western countries. research has linked these body composition changes to the nutrition and physical activity transitions which are characterized by an increased consumption of refined and processed foods and decreased levels of physical activity and are closely associated with social and economic changes impacting urbanization, food systems, labour demands, and transportation choices [25 ]. these transitions seem to be occurring simultaneously and low- and middle - income countries are now progressing through them experiencing similar body composition changes to those that have already occurred in high - income countries [68 ]. in fact, in the last decade the prevalence of obesity has tripled in several low- and middle - income countries. as a result, obesity and its related chronic diseases are significant public health issues worldwide [10, 11 ]. in addition to the rise in childhood obesity and inactive lifestyles, secular changes in children 's fitness a strong and independent marker of chronic disease risk [12, 13]have been documented. tomkinson and colleagues calculated that the average annual decline in the aerobic fitness of 619-year - olds from five geographical regions (africa, middle and east asia, australia, europe, and north america) was 0.36% between 1958 and 2003. there is also evidence from developed countries supporting the notion that childhood obesity and fitness levels are negatively correlated. whether or not such associations are consistent in developing countries, and whether changes in body composition and fitness at different stages of the nutrition and physical activity transitions reflect those for obesity, requires further investigation. by comparing the body composition and fitness of children living in countries situated at different stages of the nutrition and physical activity transitions, global correlates of childhood obesity can be better understood. by examining the consistency of these correlates across countries, if correlates are similar from one country to the next, it is likely that comparable factors have contributed to the observed changes and that preventive efforts that work in one country may, if appropriately contextualized, be successful in another. thus, intercountry comparisons can serve to raise awareness, guide the development of preventive initiatives, and further our understanding of this public health concern. the objectives of this study were to (1) compare body composition, aerobic fitness, and muscular fitness measures in children from three countries that currently sit at different stages of the nutrition and physical activity transitions (canada - end stages, mexico - mid stages, and kenya - early stages) and (2) to examine the intercountry differences in the relationships between body composition and fitness measures. the study population consisted of school - aged children from three countries that currently sit at different stages of the nutrition and physical activity transitions (canada, mexico, and kenya). canada represents a high - income country that currently sits at the final stage of the transitions as shifts in diet and physical activity occurred decades ago and considerable efforts have been underway for the past decade or so to reverse obesity [2, 3, 6 ]. mexico represents a middle - income country that is at the mid - stages of the transitions as changes in dietary intake and physical activity have occurred, but much later than those observed in high - income countries, and only recently has the issue of obesity begun to be addressed [2, 3, 6 ]. finally, kenya represents a low - income country that is at the early stages of the transitions as shifts in diet and physical activity are only beginning to emerge [2, 3, 6 ]. canadacanadian participants consisted of a representative sample of 736 children aged 913 years who participated in the canadian health measures survey (chms) [1618 ]. the chms is a nationally representative cross - sectional survey with data collected from 15 sites across canada between march 2007 and february 2009. data collection included a combination of a personal interview (demographic information) and a visit to a mobile examination centre for the collection of physical measures, including anthropometry and fitness. canadian participants consisted of a representative sample of 736 children aged 913 years who participated in the canadian health measures survey (chms) [1618 ]. the chms is a nationally representative cross - sectional survey with data collected from 15 sites across canada between march 2007 and february 2009. data collection included a combination of a personal interview (demographic information) and a visit to a mobile examination centre for the collection of physical measures, including anthropometry and fitness. mexicothe study population consisted of a convenience sample of 193 boys and girls from four public schools located in the urban core of guadalajara, mexico. children in grades 5 and 6 (1013 years of age) from the selected schools were invited to participate. trained personnel directly measured body composition and fitness indicators. the study population consisted of a convenience sample of 193 boys and girls from four public schools located in the urban core of guadalajara, mexico. children in grades 5 and 6 (1013 years of age) from the selected schools were invited to participate. trained personnel directly measured body composition and fitness indicators. kenyaparticipants consisted of a convenience sample of 179 school children aged 913 from four schools in kenya. two of these schools were located in urban areas and two were located in rural areas. data were collected at the four schools by members of our research team in november 2008. body composition and fitness data participants consisted of a convenience sample of 179 school children aged 913 from four schools in kenya. two of these schools were located in urban areas and two were located in rural areas. data were collected at the four schools by members of our research team in november 2008. body composition and fitness data ethics approval for data collection was granted for all three study populations by respective institutional review boards. informed consent / assent was also obtained from the child participants and their parents or guardians. with the exception of the aerobic fitness measures in mexico and kenya, all body composition and fitness data were collected in each country using comparable equipment according to the canadian physical activity, fitness, and lifestyle appraisal (cpafla). height (to the nearest 0.1 cm) and weight (to the nearest 0.1 kg) were measured by trained personnel using calibrated stadiometers and scales, respectively. these measures were used to calculate body mass index (bmi, kg / m). subjects were classified into four categories (underweight, normal weight, overweight, and obese) according to the international obesity task force age- and sex - specific bmi cut - points [20, 21 ]. triceps skinfolds were measured in duplicate (or triplicate if measures varied by > 0.4 mm) to the closest 0.2 mm using harpenden skinfold calipers (baty international, uk). gulick measuring tapes were used to measure the waist circumference, to the nearest 0.1 cm, according to the world health organization and cpafla protocols (i.e., midpoint between last floating rib and top of iliac crest in the mid - axillary line). a hand dynamometer (canada : takei scientific instruments, japan ; mexico / kenya : lb9011 senoh, japan) was used to measure grip strength in kg. the combined maximum score for each hand was calculated. in the canadian population aerobic fitness was measured using the modified canadian aerobic fitness test (mcaft), during which children had to complete one or more 3-minute stepping stages (up and down steps with increasing intensity) at predetermined speeds based on their age and sex. children aged 614 years started at what is stage five for women to a maximum of three stages. participants ' heart rate was recorded after each stage, and the test was completed when it reached 85% of their age - predicted maximal heart rate (220 age). predicted maximal aerobic power (vo2max) was calculated for all participants using the pediatric - specific equation vo2max (ml / kg / min) = 3.23 (oc) 1.31 (bmi) + 1.39 (age) 49.21, where oc is the oxygen cost of stepping. other equations suggested specifically for adults using the mcaft were not used as these have not been validated on children [19, 25, 26 ]. in the mexican and kenyan populations this test involved continuous running by participants between two lines 20 metres apart in time to recorded beeps on a compact disc. the participants continued running between the two lines, turning when signalled by the recorded beeps. each minute, a sound indicated an increase in speed and the beeps became closer together. if children did not reach the line in time for each beep, the child had to run to the line, turn, and try to catch up with the pace within two more beeps. the test was stopped when the child failed to reach the line (within 2 metres) for two consecutive ends. the level at which the child ended the test was recorded, and leger 's equation was then used to calculate peak oxygen consumption (vo2max). this test is currently the most widely used aerobic fitness field test within children and adolescents and has been shown to be a reliable and valid method of estimating vo2max in this age group. all analyses were performed using sas version 9.1 (sas institute, cary, nc, usa). pearson correlations were completed between the three body composition measures within each sex and country subgroup. linear regression models were used to examine the associations between the body composition (bmi, triceps skinfold, and waist circumference), aerobic fitness, and muscular fitness variables. regression diagnostics showed that residuals of the dependent variables (bmi, triceps skinfold, and waist circumference) were normally distributed, and thus no transformations were needed. differences in the descriptive and regression analyses across countries were determined by examining whether 95% confidence intervals of the means and regression (intercepts and coefficients) overlapped. because of the complex sampling strategy, bootstrapping techniques were used on the canadian data to generate the confidence intervals [29, 30 ]. there were no differences in the mean height of girls in all three countries, but the mean height of kenyan boys was less than that of canadian and mexican children. the mean bmi, waist circumference, and skinfold values of canadian and mexican boys and girls were higher than those of their kenyan counterparts. there were no differences between canadian and mexican children for these three body composition measures with the exception of waist circumference, which was higher in mexican boys. the prevalence of obesity was highest in mexican children while the prevalence of underweight was highest in the kenyan children. however, aerobic fitness (vo2max) was different in boys across all three countries with the kenyan 's having the highest values and the canadian 's having the lowest values. in girls, table 2 shows the correlations between the three body composition measures within each sex and country subgroup. correlation coefficients were quite strong (r value range of 0.620.95), irrespective of sex and country. the correlations in kenyan boys and canadian girls tended to be weaker than in the other sex and country subgroups. bmi tended to be more strongly correlated with waist circumference than triceps skinfold, regardless of sex and country. table 3 shows the results from the age - adjusted linear regression analyses looking at the association between aerobic fitness and the three body composition measures. the table displays the slopes (beta - coefficient) of the regression lines, the model fit (r), and the predicted bmi at a vo2max of 40 and 50 ml / kg / min for each sex and country subgroup. the overall patterns of findings indicate the following : (1) aerobic fitness and body composition measures were negatively associated irrespective of country, sex, and body composition measure examined. (2) the slopes of the regression lines and predicted bmi at a low aerobic fitness (e.g., 40 ml / kg / min) tended to be greater in mexican children than in canadian and kenyan children. thus as illustrated in figure 1 for bmi, mexican children with low aerobic fitness levels had higher body composition values than did canadian and kenyan children. however, the body composition values of children in all three countries were similar in those with high aerobic fitness (e.g., 50 ml / kg / min). (3) the r values for both sexes were higher in canadian children (range 0.370.53) than in mexican children (range 0.310.37) and higher in mexican children than kenyan children (range 0.110.32). thus, aerobic fitness was more strongly associated with obesity in the most developed country (canada) and least strongly associated with obesity in the least developed country (kenya). table 4 and figure 2 show the results from the linear regression analyses looking at the associations between the muscular fitness (grip strength) and body composition measures. the overall findings indicate the following : (1) muscular fitness was not associated with any of the body composition measures in boys and girls from kenya. (2) muscular fitness was positively associated with bmi and waist circumference, but not skinfold thickness, in boys and girls from canada and mexico. these associations were weak (r range = 0.090.14) in canadian children and mexican boys and were of a modest strength (r = 0.32) in mexican girls. the results provide supporting evidence of intercountry differences in the aerobic fitness and body composition of children from countries at different stages of the nutrition and physical activity transitions. negative relationships between aerobic fitness and obesity were observed in boys and girls from all three countries ; however, these relationships were more pronounced in mexican children than in canadian and kenyan children. differences in aerobic fitness were observed across all three countries wherein kenyan children were the most fit and canadian children were the least fit. although mixed results have been reported in the literature (possibly resulting from the use of invalid self - reported physical activity questionnaires), evidence based on valid questionnaires, objective physical activity measures, and physical activity interventions suggest that aerobic fitness reflects the amount of aerobic physical activity performed in recent weeks and months [3234 ]. thus, results from the current study are consistent with each country 's current stage within the physical activity transition. these results are also supported by tomkinson and olds who compared the secular decline in the aerobic fitness of 619-year - old children from 27 countries in recent decades. their results showed that the rate of decline in high - income countries was greater than that of middle- and low - income countries (0.49% versus 0.39% per year). in the current study irrespective of country, grip strength was not related to triceps skinfold thickness ; however, grip strength was a weak positive correlate of bmi in canadian and mexican children. because weight gain is associated with increases in both lean body mass and fat mass, the positive associations observed were likely driven by a greater lean body mass in the heavier children within canada and mexico. we speculate that the positive effects that the increased lean body mass had on muscular fitness in the heavier canadian and mexican children were not reflected in higher grip strength values than in kenyan children because these effects were negated by decreases in physical activity that affected muscle quality (e.g., strength per kg of muscle). it is also possible that insufficient variability in the bmis of the kenyan sample resulted in a lack of power to detect meaningful associations. the low prevalence of overweight and obesity in the kenyan children (5.6%) examined in this study was expected given their stage of the nutrition and physical activity transitions and previously published data from that country. in particular, the 2003 kenya global school - based student health survey found that only 5.9% of 1015-year - old boys and girls were overweight or obese [8, 36 ]. although mexico sits at an earlier stage of the nutrition and physical activity transitions than canada, the prevalence of obesity in the mexican children (9.2% boys, 8.4% girls) studied here was slightly higher than in the canadian children (8.6% boys, 7.2% girls). although this observation is inconsistent with where the two countries currently sit within the nutrition and physical activity transitions, this was not unexpected as these differences are consistent with nationally representative data for the two countries. specifically, the prevalence of obesity in 519-year - old boys and girls in the 2006 mexican national health and nutrition survey was between 16.5% and 23.3% while the prevalence of obesity in 617-year - old boys and girls in the 2004 canadian community health survey was between 7.5% and 11.1%. the higher rates of obesity in mexican children may be due to a variety of factors including differences in dietary and physical activity behaviours, biological differences, and how they interact with their environments. growth stunting (very low height for age) could also be a plausible explanation for the higher obesity rates observed within the mexican population. for example, between 1988 and 2006 stunting decreased from 27% to 16% in mexican children under the age of 5 and results from the 2006 mexican national health and nutrition survey found that only 9.9% of children between the ages of 5 and 11 were stunted. furthermore, within the current study no differences were observed between the height of canadian and mexican children suggesting that, in the current study, the higher rates of obesity were not likely due to stunting. although temporality of relationships can not be addressed in this study, the relations between the aerobic fitness and body composition measures suggest that low fitness has a greater impact on the body composition of mexican children than on that of canadian and kenyan children. thus, as mexico continues to progress through the physical activity transition, wherein their physical activity and fitness levels approach those currently observed in canada, we can anticipate that the obesity levels in mexican children will rise at a faster rate than what has occurred in canada in recent decades. conversely, as kenya progresses through the physical activity transition, the increased prevalence of obesity in the population may more closely match what has occurred in canada. nonetheless, our findings suggest that it may be inaccurate to project changes in children 's body composition in developing countries based on previous trends observed in developed countries. thus, reproducing preventive physical activity initiatives that have been successful in high - income countries may have varying levels of success in lower - income countries. for example, our findings suggest that more substantial changes in physical activity and fitness would need to occur within mexican children to have the same body composition benefits observed in predominately non - hispanic white populations such as canada. dietary initiatives may also differ ; however, the differential effects of diet on the body composition of children in different countries requires further investigation. as with all studies, because the kenya and mexico testing sites were at schools that did not have a gymnasium, the aerobic fitness testing was performed outdoors where the high temperature and humidity could not be controlled. altitude also negatively impacts aerobic fitness performance, and therefore the vo2max values obtained around the city of nairobi in the kenyan children were likely underestimated (though this would only further strengthen our findings). in addition, the aerobic fitness of canadian children was assessed using the mcaft test as opposed to the 20 m shuttle run test that was used in mexico and kenya. thus, equations used to estimate vo2max were different for canadian youth resulting in possible comparability issues. furthermore, the mexican and kenyan samples were convenience samples, which limit the generalizability of the findings, particularly as it pertains to how they may have been influenced by urban / rural status. approximately 50% of the kenyan sample was from an urban area, while in the country as a whole only 22% of the population is urbanized. the entire (100%) mexican sample was from an urban area, while in the country as a whole 78% of the population is urbanized. even within a country children may sit at different stages of the nutrition and physical activity transitions depending on where they live. in kenya, for instance, children residing in urban areas are more obese and have lower physical activity and fitness levels than children residing in rural areas [7, 8 ]. while this urban / rural issue may have influenced the descriptive data, they were unlikely to have influenced the relations between the fitness and body composition measures that is, when relationships between body composition and fitness measures were assessed by rural and urban dwelling in the kenyan sample, no significant differences were observed in the intercepts and regression coefficients (data not shown). in conclusion, there appear to be differences in the fitness and body composition measures of children from countries that currently sit at different stages of the nutrition and physical activity transitions. while negative relationships between aerobic fitness and obesity were observed in children from all three countries examined in this study, these relationships were more pronounced in mexican children. this may, in part, explain why the prevalence of obesity was higher in mexican children than in their canadian counterparts even though mexico is at an earlier stage of the nutrition and physical activity transitions.	background. the physical activity transition is contributing to an increase in childhood obesity and a decrease in fitness worldwide. this study compared body composition and fitness measures in children from three countries and examined intercountry differences in the relationship between these variables. methods. participants consisted of 736 canadian, 193 mexican, and 179 kenyan children aged 913 years. body mass index (bmi), waist circumference, triceps skinfolds, aerobic fitness, and muscular fitness were measured. linear regression was used to examine associations between variables. results. the prevalence of obesity was the highest in mexican children (9.2% boys, 8.4% girls) and the lowest in kenyan children (0.9% boys, 2.8% girls). aerobic fitness (vo2max in ml / kg / min) was the highest in kenyan children (50.2 boys, 46.7 girls) and the lowest in canadian children (41.3 boys, 38.3 girls). aerobic fitness was negatively associated with body composition measures irrespective of country and sex. mexican children with low aerobic fitness had higher body composition measures than canadian and kenyan children. muscular fitness was not associated with the body composition measures in kenyan children but was a weak positive correlate of bmi and waist circumference in canadian and mexican children. conclusion. the current study provides some evidence to support the physical activity transition hypothesis.
mary s hospital, and the patient gave consent for video recording and academic use. the patient, a 47-year - old woman, had suffered from insidious onset of blepharospasm, oromandibular dystonia, and spasmodic dysphonia for 2 years before visiting our neurology outpatient clinic. the patient complained of decreased social activity during the course of her disease due to hyperkinesia of facial muscles and functional blindness secondary to blepharospasm. the patient was never exposed to neuroleptics, nor did she have a family history of dystonia or any perioral trauma. upon examination, the patient demonstrated severe blepharospasm and oromandibular dystonia, which became aggravated when the patient attempted to open her eyes or tried to speak (video 1 in the online - only data supplement). the results of neuropsychological testing for memory and frontal lobe functions were within normal limits. evaluations were normal, including magnetic resonance imaging of the brain, serum chemistries, a complete blood count, serum ceruloplasmin, serum and urine copper levels, thyroid function tests, and genetic testing for the dyt1 mutation in the torsion a gene. the patient had been treated with 50 mg quetiapine, 8 mg trihexiphenidyl, and 40 mg baclofen, but these drugs were not effective, and side effects of somnolence were observed. considering both the treatment - resistant symptoms and the severe disabilities, we decided to perform bilateral gpi dbs to control her dystonic symptoms. the initial stereotactic coordinates determined by mri - stereotactic planning were established to target the posteroventral lateral gpi. postoperative ct and mri were used to verify electrode position within the gpi and to exclude asymptomatic cerebral hemorrhage (figure 1). postoperative programming of the dbs was performed 1 day after the staged implantation of the pulse generator. all major clinical symptoms indicated significant improvement in blepharospasm and in oromandibular and orofacial dystonia (video 2 in the online - only data supplement). speech difficulties caused by spasmodic dysphonia and/or oromandibular dystonia also responded well to pallidal stimulation. no clinically relevant bradykinesia or other abnormal movement was observed in our patient, nor were depressive mood or emotional instability. the parameters of gpi dbs were as follows : a pulse width of 60 sec, frequency of 130 hz, and stimulation amplitude of 2.1 v on the right side, and a width of 90 sec, frequency of 130 hz, and amplitude of 1.9 v on the left side. the exact mechanism of the observed effect of pallidal stimulation on dystonia remains unclear, but it is possible that dbs improves dystonic symptoms by changing the plasticity in the cortical - basal ganglia circuit. thus, symptoms do not return immediately after the discontinuation of therapy, and continuous stimulation may not be needed in some of these patients. the most effective target area within the gpi for controlling this symptom by dbs remains under debate. moreover, which phenotypes among the symptoms constituting meige syndrome will respond best to dbs also remain unknown. all of these uncertainties make the publication of all previous case series and our case report important for understanding the efficacy of gpi - dbs in meige syndrome. hypothesized that in patients with meige syndrome and segmental dystonia, the gpi - dbs response may be better than in patients with purely cranio - facial dystonia. houser and waltz, in contrast, hypothesized that improvement could be predicted simply by the presence of isolated cranio - facial dystonia. recently, the important observation was made that although disease duration can be a good predictor of the outcome of pallidal stimulation in patients with primary dystonias, no particular predictive value should be assigned to age at onset, age at surgery, severity of disease, dyt1 status or the presence of phasic or tonic involuntary movements. two recent reports indicated bradykinesia as a therapy - related adverse effect of pallidal neurostimulation in meige syndrome at high voltage. cases of mania, depression, suicide and suicide ideations were observed in connection to pallidal stimulation., who reported two suicides in 16 gpi - dbs treated dystonic patients, stated that these findings illustrate that there is a prominent risk of suicide after dbs of the gpi in medication - refractory dystonia. in summary, this case report, in combination with other previous reports, suggests that bilateral gpi dbs may be an effective treatment for medically refractory meige syndrome, without significant adverse effects.	medical therapies in patients with meige syndrome, including botulinum toxin injection, have been limited because of incomplete response or adverse side effects. we evaluated a patient with meige syndrome who was successfully treated with deep brain stimulation (dbs) in the globus pallidus interna (gpi). this case report and other previous reports suggest that bilateral gpi dbs may be an effective treatment for medically refractory meige syndrome, without significant adverse effects.
recognising a specific object and saying aloud its name promptly are rather effortless for the most part. however, deficits in naming objects emerge as a frequent symptom of brain damage (bayles and tomoeda, 1983 ; bell., 2001 ; hodges., 2000 ; hodges and patterson, 2007) occurring, for instance, in at least 14% of stroke patients (e.g. nkleby., 2008 ; tatemichi., 1994). in clinical practice, object naming is widely used as a test of language functions in bedside neuropsychological examination (e.g. in moca, mmse). it is also common as a behavioural treatment approach for naming disorders, or aphasia at large, to train whole word naming to simple pictures (e.g. conroy., 2009 ; nickels, 2002). in this study, we examined the cognitive and neural relevance between object naming and other visual speech production tasks using a lesion deficit mapping approach. posit that naming an object requires at a minimum four processing steps to take place : 1) visual perception ; 2) retrieval of semantic knowledge about the object ; 3) access to the associated phonological representation ; and 4) articulation. likewise, a neuroanatomically - constrained model (ueno., 2011 ; ueno and lambon ralph, 2013) specifically highlights the interactive contribution of semantic and phonological pathways in supporting naming. disruptions to various parts of these pathways, using computational stimulation, have been shown to affect naming and other spoken language abilities. in correspondence with the computational account, an elegant vbm study by butler and collaborators (butler., 2014) examined the common neuro - cognitive components that are shared across a number of language (including object naming) and executive function tasks. also, as reported in this study, the phonological component was related to the left perisylvian regions encompassing the temporal, insula and inferior frontal cortices while the semantic component was related to the left anterior temporal area. evidence from neuropsychological reports suggests that object naming is supported by a large network of different brain regions along the sylvian fissure with the left frontal and temporal lobes being particularly critical (damasio., 2004, 1996 ; (2013) used voxel - based lesion symptom mapping to relate performance on a test of object naming to neural correlates based on the lesion maps of patients with left hemispheric stroke. their results showed an association between naming deficits and lesions to significant portions of the left temporal cortex including the superior and middle sections and underlying white matter with an extension to the inferior parietal cortex. similar patterns of extensive left perisylvian lesions were reported in studies using cortical electrical stimulation during neurosurgery (corina., 2010) and perfusion - weighted magnetic resonance imaging (deleon., 2007). in particular, deleon and colleagues (2007) identified the lesions to the superior and middle temporal gyri and the anterior temporal pole to be most predictive of the lexical semantic mapping deficits (i.e. a failure to linking concepts to phonological output) in naming. additionally, a recently growing body of literature has emphasised the role of the anterior temporal lobe (atl) in naming (e.g. domoto - reilly., 2012 ; notably patients with semantic dementia typically have prominent atl atrophy and progressive anomia (i.e. naming impairment) (bright., 2008 ; jefferies and lambon ralph, 2006 ; noppeney., 2007). according to patterson and roger (patterson., 2007 ; rogers., 2004) hub of the brain, integrating modality - specific representations (e.g. smell, shape, colour, name) from different regions to constitute domain - general concepts (see also lambon ralph, 2014 for a review). in many neuropsychological studies of object, 2007), patients have been restricted to those only with left hemispheric damage. this limited the ability to draw inferences about potential contributions of particular regions in the rest of the brain to a given function. (2006) examined the anatomical organisation of object naming using voxel - based morphometry (vbm) in patients with a range of neurodegenerative diseases. they reported a link between overall naming performance and bilateral atrophy in the superior and inferior temporal gyri, anterior fusiforms and hippocampi, in additional to some left - sided atrophy. similarly, studies using functional imaging show activations in extensive brain regions during object naming (garn., 2009 ; lger., 2002 ; okada., 2000 ; spitzer., 1998). price and colleagues (2005) conducted a meta - analysis of the functional imaging studies on object naming in healthy individuals. this meta - analysis study identified regions primarily along the occipito - temporal cortices on the left ; however, greater involvement of the right hemisphere was also noted when object naming was compared with baseline conditions controlling for perceptual processing and speech production. in the current study, we performed the whole brain correlation analysis using vbm. object naming is very similar to other speech production abilities such as reading as they both require speech response driven by visual inputs. interestingly, however, there is limited comprehensive account of how object naming is distinguished from other visual speech production tasks at the neuronal level. only a few fmri studies have directly contrasted the neural activation of object naming to single word reading (bookheimer., 1995 for example, moore and price 's (1999) study found shared mechanism in the inferior temporal cortex (among other regions) which responded more to both words and objects relative to viewing meaningless visual stimuli. compared with word reading, increased activation during object naming was observed in the anterior fusiform. the authors (moore and price, 1999) explained that the anterior part of fusiform has been linked to semantic processing, with object naming being more dependent on semantic processing than reading. functional imaging studies of other speech production tasks alone such as sentence production in picture description (e.g. grande., 2012) highlight the involvement of a large bilateral network which includes both the anterior (e.g. inferior frontal gyrus, anterior part of superior and middle temporal gyri) and posterior (e.g. temporo - parietal and occipital cortices) regions of the left hemisphere. however, there is a lack of neuropsychological data directly comparing performance on object naming with a series of visual speech production tasks using a common set of patients. the present study used performance data from a stroke sample on a clinical cognitive screen (bcos ; humphreys., 2012). the bcos assesses language abilities including object naming as well as reading and picture description (see the behavioural measures subsection and the supplementary material s1, for detailed description). all these tasks assess identification of visual stimuli and generation of spoken responses. despite the similarities, each task potentially has its specific demands. to increase the demands on recognition and semantic processing, the object naming task in bcos includes low frequency object items. in contrast, the sentence production (picture description) task is designed to assess primarily syntactic and morphological processing while demands on recognition and semantic / name retrieval of the target objects were made minimal (by using very frequent object items, e.g. book, and also by actually providing the name of the target objects alongside the picture stimulus to the participant). the sentence reading task requires the participant to read aloud a sentence containing some relatively low frequency and exception words (i.e.irregular words as described in coltheart., 2001). this task would tap the lexical and non - lexical phonological processing finally, bcos also assesses nonword reading, which can only be achieved by non - lexical phonological processing and not aided with semantic knowledge. table 1 outlines the potential cognitive language processes underlying these four visual speech production tasks. we speculate that the object naming task may have greater demands on recognition and semantic knowledge of objects relative to other tasks tested in the present study. in a large sample of sub - acute stroke patients, we examined the lesions associated with impaired object naming and then in relation to other visual speech production tasks (in order to isolate regions specific to object naming). as another approach, we also performed a principal component analysis in order to identify the shared and unique mechanisms of object naming and the other language tasks. we applied a fully - automated voxel - based correlational method to assess the relationship between the performance on the language tasks (based on the raw and pca scores) and the density of grey and white matters (based on patients ' clinical ct scans). all patients were recruited from the stroke units of 12 hospitals in the west midlands, uk, as part of the birmingham university cognitive screen trial (bucs ; http://www.bucs.bham.ac.uk). the broad inclusion criteria of the trial were that the patient should be at the sub - acute stage (< 120 days post - stroke), physically stable and well enough to maintain concentration for around an hour to complete the cognitive assessment (judged by a trained assessor of the multi - disciplinary stroke team). the sample of this present study was made up of 280 patients (141 males, average age : 70.88 years 14.06std, ranging between 26 and 93 years) selected from the bucs database of 532 cases with clinical ct scans available. as previously estimated in the patient group of the bucs trial, 41.4% had middle cerebral artery (mca) stroke, 10.4% posterior cerebral artery stroke and 13.4% due to other affected vascular territories (chechlacz., 2014a). for the present study, we excluded patients whose ct scans were of poor quality (n = 37), or if the scans showed abnormally large ventricles (n = 4). to control for the potential confounding effect due to the presence of abrupt high intensity signals, we also eliminated cases with haemorrhage (n = 42). we further excluded patients who were non - right - handed (n = 54), or who were scanned more than 120 days post - stroke (n = 1) or on the same day (within 24 h) of their stroke (n = 114). as shown, lesions of the patients cover the entire brain in the two hemispheres with maximum overlaps in the right mca territory (see supplementary material s3 for the method used to create the individual lesion maps). all patients provided written informed consent conforming to the ethics protocols approved by the uk national health service ethics committee, the local nhs trusts and the birmingham university ethics procedures. the cognitive abilities of the patients were examined using the bcos cognitive screen (humphreys., 2012) domains of daily cognitive functions : i) language, ii) attention and executive functions, iii) memory, iv) praxis and v) number processing. this is achieved by 27 paper and pencil tasks with each designed to tap into various cognitive processes under each domain (http://www.cognitionmatters.org.uk/bcos.php). the tests were designed to be aphasia and neglect friendly, to be as sensitive as possible to identify cognitive impairments (with validated age - matched cut - off scores) and to optimise time efficient test administration (i.e. the entire screen was developed to be completed within 60 min). during the study, experimenters were blind to the specific condition of the patient and the location of any lesion. on average, patients were tested 24 days post - stroke (with 65% tested within the first month after stroke). the items were chosen to cover a range of frequency according to the subjective familiarity ratings (469543 out of 700) from the mrc psycholinguistic database (wilson, 1988). in order to represent a variety of semantic categories, half of the items were living things (e.g. bat) and half non - living (e.g. spanner). among the living items there were 2 animals, 3 fruits and 2 vegetables while the non - living category consisted of 2 tools, 1 kitchen implement and 4 other household implements. in order to detect word production problems sensitive to stimulus length, half of the items had a long name (being composed of 69 letters) and half a short name (35 letters). during the task, participants were presented with each drawing of an object printed centrally on an a4 sheet of paper. a maximum of 15 s was allowed per item for the patient to give a response. in addition to object naming, we assessed performance on other bcos tests that also required speech output to visual stimuli. the tests included sentence production to a picture (to describe what a person was doing), reading a sentence containing some low frequency and exception words and reading some nonwords (for details, refer to the supplementary material s1). for each patient, computed tomography (ct) images were collected as part of the standard clinical procedures. the scans were acquired using one of these scanners : siemens sensation 16 ; ge medical system lightspeed 16 or lightspeed plus. the ct images were provided by the hospitals in digital dicom format after they had been anonymised. these images covered the whole brain with an in - plane resolution of 0.5 0.5 mm and a slice thickness of 45 mm. the in - plane resolution (along the x y plane) of a ct image was higher than that of a typical mr structural scan (1 1 mm), but the resolution along the superior inferior direction (z - axis) was poorer in ct compared to mr. ct images depict the density of the tissue and as such have a clear biological interpretation. however, changes in tissue density, especially due to ischemic stroke, may be underestimated on a ct scan, at least when the scan is conducted within the first 24 h after a stroke (mohr., 1995). therefore, in the current study, we included only patients who had their ct taken at least 24 h post - stroke. also, to account for possible changes in lesions following a stroke, the analysis models included as a covariate the interval (in days) between the stroke and the ct scan. on average the ct scans were taken 7.26 days after stroke, with 74% of cases within 1 week of the stroke. the ct images were pre - processed using spm8 (statistical parametric mapping, welcome department of cognitive neurology, london, united kingdom ; http://www.fil.ion.ucl.ac.uk/~spm). the quality of the ct scans was first assessed visually to ensure that only good quality data (e.g. free from head movement and other image artefacts) were included in the analysis. this quality check was done on the raw ct images and also on the segmented, normalised images by comparing them to the a - priori tissue templates. pre - processing started with converting the images to nifti format and normalising (ashburner and friston, 2003) them to an in - house ct template. this initial normalisation stage was primarily based on skull shape and aimed to transform the images into mni (montreal neurological institute) space to optimise the following procedures. next, the unified - segmentation algorithm implemented in spm12 (i.e. seg8 in spm8 ; ashburner and friston, 2005) was employed. in the unified model, the priors of the tissue class, from which intensities are drawn, the a - priori tissue class maps indicate the probability of finding expected signal sources at each voxel : grey matter (gm), white matter (wm), cerebrospinal fluid (csf), bone, fat and air in the brain. to account for the presence of damaged tissue due to stroke, a modified segmentation procedure similar to the approach of seghier and colleagues (2008) was adopted to include a seventh tissue class. in creating an additional prior for abnormal tissue, we assumed that in each grey or white matter voxel there is a 10% chance of it belonging to an abnormal tissue class. this 10% estimation was computed based on the lesion volume size (versus the brain size) estimated in the bucs database (for details, see chechlacz., 2012). furthermore, for the grey and white matter tissue classification, we assumed a single gaussian distribution for the underlying intensities. to account for potential inhomogeneity of the abnormal tissue we used 2 different gaussian distributions to model the intensities in this tissue class. what is more, ct images as opposed to mri do not suffer field bias due to field strength inhomogeneity ; therefore we did not correct for that in the model. finally, the segmented white and grey matter images were normalised using the parameters estimated in the unified - segmentation algorithm again and smoothed using a 12-mm full - width - at - half - maximum (fwhm) gaussian kernel to accommodate random field theory assumptions of continuity (worsley and friston, 1995). the preprocessed gm and wm maps were then used in our analyses to explore voxel by voxel the relationship between brain lesion and behavioural performance. for the additional visual speech production tasks used in the analyses, we replaced missing data with the group average. data from 2 patients were missing in sentence reading, 8 in sentence production and 8 in nonword reading. to examine the relationship between the performance on object naming and the covariates, a non - parametric spearman - rank correlation (two - tailed) analysis was carried out. principal component analysis (pca) was performed using matlab 7.9 (the mathworks, natick, ma, usa) to identify shared and unique components of object naming in relation to other language tasks. our sample consisted of 280 subjects, which is considered fairly adequate (n = 300) for pca (comrey and lee, 1992). to increase the robustness of the analysis, especially for small to moderate sample size, a subject - to - variable ratio of at least 5:1 has also been suggested (hatcher, 1994). with 280 cases and 4 variables in this study, the use of pca to rotate behavioural data which is then related to the distribution of brain lesions has been demonstrated in recent neuropsychological studies of stroke (butler., 2014, 2014b) and developmental prosopagnosia (garrido., 2009). to account for differences in the maximum scores of the language tests (object naming : 14 ; picture description : 8 ; sentence reading : 40 ; nonword reading : 6), we re - scaled the raw scores on each test linearly to range between 0 and 20. we used in the pca the re - scaled scores of the 280 participants on the four language tests and then extracted component loadings (i.e. coefficients) and eigenvalues. individual performance scores on each principal component were also derived and used in the vbm analyses. using spm8, all reported vbm analyses were performed on the scans from 280 patients to determine the neural correlates of object naming and its related cognitive components. random effects analyses were conducted within the general linear model framework and correlations between the behavioural measures and the integrity of brain tissues were computed (ashburner and friston, 2001). first, we determined the common lesions that were associated with object naming deficits in our sample using the participants ' raw scores (analysis 1). second, we identified the lesions unique for object naming by controlling in the model for other language tasks including sentence production, sentence reading and nonword reading (analysis 2). third, we examined the neural correlates of the individual scores on the principal components derived from the pca (analysis 3). this model included all principal components though we focused primarily on the lesions associated with a shared language component and a component that was unique to object naming. reduced integrity of grey and white matter was analysed separately for each model. in all analyses, the following measures were included as covariates of no interest : age, gender, years of education, interval between stroke and ct scanning, interval between stroke and cognitive testing, and measures of general cognitive state (we used tests of each participant 's overall orientation, see supplementary material s2 for details). inclusion of these covariates allowed us to control for various confounding factors that might have a potential impact on cognitive performance or the extent of lesion. we focused on results that were reliable at a fwe - corrected threshold of p < 0.05 at the cluster level, unless stated otherwise. for completeness we report in the tables all clusters with a voxel - wise threshold of z = 2.32 and an extent threshold of at least 300 voxels (the expected cluster size by chance with the above z threshold is 233 voxels). anatomical labelling was based on the anatomical automatic labelling toolbox (tzourio - mazoyer., 2002), duvernoy 's (1999) human brain atlas, the jhu white matter tractography atlas (hua., 2008) and the mri atlas of human white matter by mori (2005). on average, patients were able to name 10.02 (sd = 3.88) objects correctly. compared to the cut - off points established from the age - matched healthy controls (http://www.bcos.bham.ac.uk), 108 patients scored lower than the cut - off points and table 2 provides the descriptive data and average scores on the cognitive and language tasks. performance on object naming was significantly but weakly associated with age (rs = 0.164, p < 0.01) and years of education (rs = 0.218, p < 0.01). in addition, individuals who performed worse at object naming also had poorer overall orientation, measured in terms of their knowledge of personal information (rs = 0.548, p < 0.01) and time and space (rs = 0.328, p assessment on the time and space measure was based on multiple - choice tests and hence did not rely on speech production. not surprisingly object naming was correlated significantly with all other language measures including sentence production (rs = 0.596, p < 0.01), sentence reading (rs = 0.636, p < 0.01) and nonword reading (rs = 0.566, p < 0.01). as an attempt to dissociate processes underlying object naming and the other visual speech production tasks, we ran a pca on the re - scaled raw data of four language tests (i.e. object naming, sentence production, sentence and non - word reading). the pca revealed that the four tasks all loaded on the first component with loadings ranging between 0.4 and 0.6. as all the tasks required both visual perception and generation of a verbal response, we assume that this component represents a process relating to the conversion of visual information into phonological outputs. another component of interest accounted for 4.7% of the variability and had a dominant high loading from object naming, separating it from the other three visual speech production tasks. this suggests that there are processes that are uniquely involved in object naming but not in any other language tasks tested. table 3 shows the loadings on these two components and the correlations between the raw scores on each language test and the individual scores on each of the two principal components. there were two other components from the pca outputs : 1) one dissociating nonword reading from the other three language tasks (11.6% of the variance explained) ; and 2) one specifically dissociating sentence reading from sentence production (5.8% of the variance explained). as these two other components were not unique for object naming, they were not the central focus of the present study (although they were also included in the glm model). to further explore the presence of a unique object naming component we counted the number of patients who were classified as impaired in object naming but showed normal performance on all the other language tasks., there were 10 patients classified as impaired in all the three language tasks but retained normal functioning in object naming. this may suggest a potential double dissociation between object naming and other visual speech language tasks although it appears to be a very rare phenomenon. as lesion size is a factor potentially contributing to the severity of any deficit, each patient 's lesion volume was calculated. we then examined its relationship with the performance on the four language tasks and the rotated scores for the shared and unique naming components. lesion volume correlated weakly with object naming (rs = 0.167, p = 0.005), sentence reading (rs = 0.136, p = 0.023), sentence production (rs = 0.207, p < 0.001) and nonword reading (rs = 0.126, p = 0.035). regarding the pca components, lesion volume again correlated weakly with the general language component (rs = 0.175, p = 0.003) but not at all with the unique naming component (rs = 0.058, p = 0.337). next, we related the behavioural measures to the neuroimaging data in order to determine the structural lesion correlates of object naming and their associations with underlying cognitive processes represented by the two principal components of interest. here, we report the results of three analyses, and for each we computed separate probability maps for the grey and white matter (gm & wm, respectively) : analysis 1 correlation with only the raw score of object naming ; analysis 2 correlation with object naming after accounting for performances on the other visual speech production tasks ; and analysis 3 (a) correlation with the individual scores on the shared language component and (b) with the unique object naming component. this vbm analysis based on the raw scores of object naming alone revealed a significant positive relationship with voxels in the left fronto - temporal and medial temporal regions and also the bilateral occipital cortices. in particular, impaired performance was significantly associated with gm damage in the bilateral posterior visual cortices, the left superior temporal gyrus extending to the insula and inferior frontal gyrus, and the left fusiform (table 4, analysis 1). to identify the neural correlates specific to object naming but not to other visual speech production tasks, we included the scores of 3 other language tests (i.e. sentence production, sentence reading and non - word reading) as covariates in a separate model. the results of this analysis did not survive the cluster - level threshold of p < 0.05 with fwe correction. however, they do suggest that lesions to the bilateral anterior superior temporal and inferior frontal gyri, the left hippocampus and several regions in the cerebellum were associated uniquely with object naming (table 5, analysis 2). notably, lesions to the left fusiform and lingual gyri were not found to be associated uniquely with object naming after we controlled for the other language tasks. this vbm analysis correlated each brain voxel with the subject 's score on a principal component generated from the pca procedure. from analysis 3a (table 4), the share component was reliably correlated with lesions in the left lateral fronto - temporal and fusiform regions and the bilateral visual cortices. these were similar to the lesion pattern observed when performance on object naming was modelled alone (without controlling for any other language tasks, i.e. table 4, analysis 1 ; see also fig. 2). unique naming component (table 5, analysis 3b) mirrored the results yielded in analysis 2 (table 5) where performances on the other three language tasks were partialled out from object naming (fig. 3). vbm analysis of wm maps and object naming scores alone revealed a significant relationship between object naming deficits and reduced white matter density in an extensive region encompassing the temporal and inferior parietal lobes in the left hemisphere and the bilateral visual cortices (table 6, analysis 1). damage to wm in temporo - parietal areas is likely to disconnect the inferior fronto - occipital fasciculus, inferior longitudinal fasciculus, parts of the superior longitudinal fasciculus including the arcuate fasciculus (hua., 2008 ; lesions in the bilateral occipital cortices, on the other hand, are linked to damage of the posterior end of the inferior fronto - occipital fasciculi (hua., 2008 ; after controlling for performances on the three other visual speech production tasks, object naming correlated with wm integrity of a smaller area in the left temporal lobe and a small cluster of voxels in the cerebellum (table 7, analysis 2). however, these relationships were only weakly reliable at the cluster level. lesion to this temporal area affects most likely the temporal tail of the arcuate fasciculus. damage to the cerebellum, on the other hand, is likely to impede transfer of information along the middle cerebellar peduncle. shared component and wm density of an extensive region in the left temporal and parietal cortices and the bilateral occipital cortices (table 6, analysis 3a). there is a high degree of similarities between these results and those of analysis 1, which modelled object naming alone (fig. 4). in contrast, the white matters associated with the unique naming component (table 7, analysis 3b) greatly overlap with the outputs of analysis 2 (table 7), the one that looked at object naming after partialling out the performances on other language tasks (fig. finally, we included lesion volume as an additional covariate in all the vbm analyses and the pattern of results did not change. to examine the relationship between object naming and other visual speech production tasks, we carried out a principal component analysis across the language tasks. this component was linked to damage to the bilateral posterior occipital cortices and left - lateralised regions including the fusiform and the superior temporal gyrus (stg) extending into the insula and the inferior frontal gyrus (ifg). similarly, the white matter damage associated with this shared language component was left lateralised. these regions were also related to poor object naming when it was assessed alone without controlling for performances on other visual speech production tasks. in contrast to these analyses of common language processes, we also evaluated the processes particularly stressed in object naming by (i) including the other visual speech production tasks as regressors in the vbm analysis and (ii) examining the unique naming component that dissociates object naming from the other tasks. these analyses indicated particular involvement in object naming of the two anterior superior temporal poles (extending to ifg), as well as the hippocampus and cerebellum. pca across object naming, sentence production, and sentence and nonword reading produced a component that accounted for more than 75% of the total variance and the loadings from the four tasks on this component ranged between 0.4 and 0.6. shared component is likely to stress more visual recognition, phonological retrieval and articulation because all the tasks rely on speech response to a visual input and the nonword reading task would not require semantic processing. when analysed alone, object naming (analysis 1) was linked to the same left lateralised network as the shared component (analysis 3a). this indicates that without additional care being taken to isolate factors stressed by object naming, lesion symptom analyses of object naming tend to highlight visual and phonological processing found in a number of language tasks. the four language tasks employed in the present study all potentially demand high - level processing of visual inputs. this may be why the shared component was linked to the lateral occipital cortex and the fusiform gyrus. in agreement with our findings, these two regions have often been associated with processing of complex visual inputs such as faces, objects and words (bar., 2006, 2001 ; dien, 2009 ; grill - spector., 2001 ; herbster., 1997 ; kanwisher and yovel, 2006 ; malach., 2002 ; mccarthy., 1997), even though each type of stimulus may recruit slightly different segments of the striate and extrastriate cortices (see dehaene., 2002 ; mccandliss., 2003, but also price and devlin, 2003 ; starrfelt and gerlach, 2007). yet, it is likely that stroke which typically affects a large area in the brain impairs various types of high - level visual processing simultaneously (as in our case) due to the spatial proximity of the corresponding loci within the occipito - temporal regions. the involvement of the left inferolateral frontal gyrus (extending to the insula) and the superior temporal gyrus in shared language processes is not surprising. these results accord with a recent study showing links between these temporo - frontal areas and a phonological factor of language (butler., 2014). the ifg has long been held to play an important role in the production of meaningful speech (broca, 1861). infarction to this frontal area has been related to a number of speech impediments including apraxia of speech and expressive aphasia (for a detailed review, see caplan, 1987). besides, a body of evidence implicates also the left insular gyrus (a neighbouring brain structure of the ifg) in speech production (dronkers, 1996 ; kleist, 1934 ; mazzocchi and vignolo, 1979 ; mohr.. a recent review of the clinical and functional imaging literature suggests that the insula participates in articulatory planning and control processes (ackermann and riecker, 2010). along with stg these areas constitute part of the dorsal pathway that has been proposed to specialise in phonological processing (hickok and poeppel, 2007 ; rauschecker and scott, 2009 ; saur., 2008). the present study also observed associations between white matter lesions in the left temporal and bilateral ventral occipital lobes and impairment in a general visual - to - speech language function, represented by the shared component. putting together the previously proposed functions of various parts of the visual - to - speech network, here we suggest that impairment in the general language function requiring visual - to - speech interaction can also be the results of disconnection between the occipital and ventral temporal lobes and/or also between the occipital and inferior frontal lobes via the temporal regions. this was done by analysing the neural correlates of object naming with the other three language tasks included as regressors, and also by assessing the unique pca component that isolated object naming from the other tasks. the unique object naming component was associated with the bilateral anterior temporal lobes, the hippocampi and several cerebellar areas (analysis 3b). corroborating evidence was provided by an additional analysis (analysis 2) using object naming as the variable of interest while controlling for other visual speech production tasks. however, these unique associations did not reach family - wise significance (apart from a cerebellar area). this is probably because the occurrence of cases of deficits at object naming only in the absence of more general language impairment is rather rare. nevertheless, we note that the observation of a potential bi - anterior temporal association with object naming is in agreement with previous studies testing semantic dementia (jefferies and lambon ralph, 2006 ; lambon ralph., 2007) and temporal lobe epilepsy (hodges and patterson, 2007 ; lambon ralph., 2012 these past studies concur that the anterior temporal lobes contribute to semantic representation (see also gough., 2009 ; tranel., 1997 ; woollams, 2012) necessary for accurate object naming. putting this together with the results of left - lateralised lesions associated with the shared language component (discussed earlier), our findings are complementary to a proposed model of naming that suggests a left - localised phonological representation system which connects strongly to a bilaterally distributed conceptualisation network (lambon ralph., 2001 ; schapiro., 2013). the unique involvement of cerebellar areas and the hippocampus in object processing is less clear. recent literature has implicated the hippocampus in some forms of semantic processing (bonelli., 2011 ; holdstock., 2002 ; 2003) and the cerebellum in high - level cognitive operations (desmond and fiez, 1998 ; molinari., 1997). still, further investigation is needed to clarify the unique contribution of these neural substrates to object naming deficits following stroke. our study provides additional evidence for the use of the clinically acquired behavioural and structural imaging data in voxel - based correlation analysis (refer also to chechlacz., 2012). specifically, the behavioural data were derived from a large - scale clinical trial of cognitive testing and the ct scans were collected from clinics and hospitals as part of the standard everyday medical practice. despite being the preferred modality in clinical stroke units (e.g. karnath. (2004) reported that ct was used for 72 out of 140 stroke patients at admission), ct images are not usually used in statistical anatomical research. only recently the first high - resolution ct template (to aid normalisation of the images) was published (rorden., 2012) and image - processing algorithms were improved to make statistical analysis of large ct datasets more feasible (gillebert., 2014). symptom mapping to answer a psycholinguistic question and the findings accord with past studies based on other high resolution structural scans. ct scanning measures tissue density and provides meaningful biological signals, making them relatively easy for comparison across scanner sites. however, ct scans, similar to t1- and t2-weighted mr images, fail to detect cortical dysfunction arising from inadequate cortical perfusion within a region that is structurally intact. abnormal reduction in perfusion may contribute to cognitive deficits (for example see hillis., 2000 ; karnath., 2005 ; ticini., 2010 besides, it has been shown that lesions caused by ischemic stroke may not be immediately identified when ct scans are acquired too early (wardlaw and farrall, 2004), especially within the first 24 h post - stroke (mohr., 1995). the current study, therefore, excluded scans that were taken less than a day after a stroke. as tissue loss continues for a few weeks to several months after stroke, signals arising from lesioned tissues may vary with time. to control for that, we added the interval between the stroke and the scan acquisition as a covariate measure. behavioural and imaging measurements may not be as accurate as those acquired in a laboratory environment. this is mostly because under the time pressure in clinical settings measurements are designed to capture the most essential diagnostic information at the expense of the reliability gained through trial repetition. however, the advantage we have had here is a much larger sample size compared to a typical lesion symptom mapping study, as well as the reduced burden imposed on patients wishing to contribute to research. after all, we believe that this represents a reasonable trade - off (for more information about this clinical trial, see also bickerton., 2014). moreover, in many past studies patients were pre - selected based on specific lesion locations (e.g. baldo., 2013 ; deleon., 2007 ; looking at left hemispheric damage only) or cognitive impairment (e.g. mesulam., 2009 ; schwartz., 2009 ; focusing only on specific aphasic patients). here, we used minimal exclusion criteria, meaning that our results can be generalised to a broad population of patients with sub - acute stroke at large. as another point to note, the four tests included in our comparison all require at least visual and phonological (/motor) processing and both types of processing were captured by the shared pca component. object naming is a complex cognitive task and damage to any parts of the cortical network sustaining the underlying cognitive processes would lead to deficits at object naming. the fact that lesions are usually sampled unevenly across the brain in stroke patients (ng., 2007) may be a limitation for mass - univariate (voxel - wise) analysis since this is likely to reduce the statistical power of identifying brain function (lesion deficit) relationship contributed by less frequent lesions (see also chechlacz. yet, it is worth mentioning that the lesion coverage of our patients encompassed the entire brain. as a final note, the current study used parametric analysis, with both the brain signal and the behavioural measures represented as continuous variables. compared to having lesions manually delineated by the researchers or any human staff, the brain tissue density was automatically assessed using a unified - segmentation algorithm. also, it is blind to the cause of tissue reduction and would note any abnormal tissue change. lesion per se but there could be other conditions causing neural abnormality. be that as it may, we argue that overlooking tissue loss that is not primarily caused by a stroke insult (it would be the case in manual lesion delineation procedures) may result in misinformed function lesion mapping because any abnormal tissue change, whatever the cause, can impair cognitive functioning. firstly, we tested the relevance between object naming and other common spoken language abilities because in many bedside neuropsychological assessments object naming is often tested to indicate retained language function (e.g. in moca, mmse). our results suggest that deficit at object naming in the majority of patients can be a good predictor for more general language impairment, which is evidenced by the great extent of the shared lesions contributing to a secondly, our study provides evidence for the possibility of using data collected primarily for everyday clinical assessment to address a scientific question in the psycholinguistic context. particularly, clinical ct scans were analysed with the use of the most up - to - date statistical tools that were originally developed for handling high resolution imaging data. as discussed above, our findings correspond with the past literature based on other neuroscience techniques. finally, to date there are various treatment approaches (and their variations) for naming impairment, or aphasia at large, including explicitly training individuals in whole word naming (with or without provision of particular cues) (for review see nickels, 2002). another approach to the problem specifically directs at the level of phonologic processor through training in phoneme production and comprehension of phonological sequence knowledge (kendall., 2008). our data posit that naming deficits are very likely to occur with more generic language impairment in converting visual inputs to speech production. as a result, training tapping into more general language cognitive processes such as phoneme production and/or visual form recognition may be more beneficial. the current study used vbm in a large sample of sub - acute stroke patients to determine the common and dissociable neural substrates of object naming in relation to various language tasks that require visually - driven speech production. we showed a distinction between a large neural network commonly engaged across various language tasks (within the left temporal cortex and its surrounding areas) and a number of potentially specific brain regions (particularly the bilateral anterior temporal lobes) required to support object naming. these findings are in line with the hypothesis that object naming relies on a left - lateralised language dominant system that interacts closely with a bi - anterior temporal network. beyond this, our work also highlights the value of examining patient performance in object naming in relation to other language tasks, as is done by screens such as the bcos which provide an overall profile of cognition in patients.	we report a lesion symptom mapping analysis of visual speech production deficits in a large group (280) of stroke patients at the sub - acute stage (< 120 days post - stroke). performance on object naming was evaluated alongside three other tests of visual speech production, namely sentence production to a picture, sentence reading and nonword reading. a principal component analysis was performed on all these tests ' scores and revealed a shared component that loaded across all the visual speech production tasks and a unique component that isolated object naming from the other three tasks. regions for the shared component were observed in the left fronto - temporal cortices, fusiform gyrus and bilateral visual cortices. lesions in these regions linked to both poor object naming and impairment in general visual speech production. on the other hand, the unique naming component was potentially associated with the bilateral anterior temporal poles, hippocampus and cerebellar areas. this is in line with the models proposing that object naming relies on a left - lateralised language dominant system that interacts with a bilateral anterior temporal network. neuropsychological deficits in object naming can reflect both the increased demands specific to the task and the more general difficulties in language processing.
the major complications of colonoscopy are postcolonoscopy bleeding (0.2%) and perforation (1%). a 56-year - old man presented with a history of 6 months hypogastric pain. in order to investigate the cause, colonoscopy was performed using conscious sedation with intravenous midazolam (2.5 mg) and meperidine (25 mg). the rectal mucosa was normal, but there were multiple diverticula in sigmoid colon and also some small sessile polyps (< 1 cm) near the junction between sigmoid and descending colon. there were multiple scattered diverticula up to ascending colon and another small sessile polyp was also found in cecum, from which other biopsy sample was taken (figure 1). about 5 minutes later, the patient complained of severe colicky abdominal pain, which recurred every 5 minutes and was accompanied by diaphoresis. on physical examination, blood pressure was 110/80 mmhg, heart rate was 80/min and the patient had no fever. the abdomen seemed distended and bowel sounds were high - pitched. also, the abdomen was tympanic on percussion, but there was no tenderness or guarding. plain abdominal radiography showed multiple colonic air - fluid levels and distention of the colon, especially in the right side (figure 2). abdominal radiography after the first colonoscopy showed distention of cecum, ascending colon, and transverse colon with a transition point in descending part indicating an obstructive cause in distal colon. since there was no subdiaphragmatic free air and no free air between intestinal loops, the probability of perforation was ruled out. immediately, the patient underwent second colonoscopy, but the scope could hardly pass through sigmoid colon because of spasm and edema at the site of previous polyp biopsies. the scope was gently advanced toward cecum and simultaneously decompressed the entire colon by suctioning the entrapped air. after performing the second colonoscopy, the colicky abdominal pain was relieved and abdominal distention was significantly decreased. after 2 hours, the patient passed flatus and after 12 hours, he passed liquid stool. the next day, white blood cell count was reduced to 1010/l and the patient had no pain or diaphoresis. he tolerated liquids and then full meals, and therefore, he was discharged during the second day. in recent publications, there are only few reports of postcolonoscopy small bowel obstruction, incarcerated internal hernia, ileus, and volvulus of cecum, ileum, and sigmoid colon. postcolonoscopy small intestinal obstruction usually occurs in patients with previous history of small intestinal surgery. in patients who did not respond to conservative treatments, laparotomy had mostly shown internal hernia or entrapment of small intestinal loops in adhesions. our patient did not have previous history of intestinal surgery or any complaint referring to the probability of intestinal obstruction before colonoscopy. the occurrence of symptoms immediately after colonoscopy made coincidental causes almost impossible. the most common cause of abdominal pain and distention after colonoscopy is ileus, which is mostly caused by sympathetic over - stimulation, excessive air insufflations, and the effects of opiates used for sedation. but in our patient, exaggerated, high - pitched bowel sounds, together with radiographic findings (including distention and air - fluid levels mostly confined to cecum and ascending colon), made the diagnosis of ileus impossible and led to the diagnosis of large bowel partial obstruction. the diagnosis was supported by further demonstration of narrowing at the site of polyp biopsies, which made second colonoscopy to be very hard. in our patient, the most possible explanation for sigmoid colon obstruction was the edema and spasm at the site of polyp biopsy, which was situated near multiple diverticula. fortunately, obstruction was relieved with conservative management and did not lead to surgery. in conclusion the diagnosis should be considered in case of postcolonoscopy abdominal pain, because ignoring the pain and the progression of distention may lead to intestinal perforation or even air emboli.	postpolypectomy bleeding and perforation are the major complications of colonoscopy. this report presents a rare case of colon obstruction immediately after colonoscopy. a 56-year - old man underwent colonoscopy because of 6 months lower abdominal pain. colonoscopy revealed diverticulosis and multiple small sessile polyps in sigmoid colon. biopsy samples were taken from the polyps and the procedure was continued up to cecum. soon after the procedure, the patient complained of colicky abdominal pain accompanied by diaphoresis. in physical examination, the abdomen seemed distended and bowel sounds were high - pitched. there was no abdominal tenderness or guarding. plain and upright abdominal radiography showed multiple colonic air - fluid levels. immediately, the patient underwent second colonoscopy, but passage of scope through sigmoid colon (at the site of biopsies) was somewhat hard because of edema and spasm. the colonoscope proceeded gently up to cecum and decompressed the entire colon by suctioning the air that was entrapped in proximal parts. by second colonoscopy and further conservative treatments, the patient s condition improved without any surgical procedure and was discharged after 24 hours.
acetate consumption in resting human contributes to less than 10% of global co2 production when measured with [c]acetate. in resting, human acetate is mainly metabolized in the liver [2, 3 ]. in 2002, van hall. showed that [c]acetate turnover in active skeletal muscle was greatly increased compared with rest and represented, during exercise, a major part of whole body acetate turnover. these authors studied the kinetics of increase and decrease of [c]acetate in peripheral blood under constant infusion of acetate, measurement intervals being spaced by 30 min after initiation and later stopping of exercise. higashi., furthermore, while studying the kinetics of [c]acetate uptake in lung cancer, showed that heart uptake peaked a few minutes after injection, remained maximal over about 6 min before decreasing again during the next 20 min. we hypothesized that skeletal muscle [c]acetate uptake might be visualized by positron emission tomography (pet) in analogy to heart muscle activity. we thus designed an exercise related with the one shown in the report of van hall. but also respecting particular constraints of our patients and the kinetics of [c]acetate uptake in the heart described by higashi.. for this purpose, we used an exercise force development adequate for patients in the postsurgical situation after hip arthroplasty and started pet imaging shortly after exercise in order to remain within the window of maximal [c]acetate uptake demonstrated for heart muscle. this article gives the first results with [c]acetate muscle function pet performed in two patients after hip arthroplasty. both patients were studied with rest and exercise pet / computed tomography (ct) once after 3 weeks and again after reeducation at 3 months. patients gave their written informed consent to the study protocol that had been approved by the medical ethical committee, geneva university hospital, by swissmedic and by the federal office of public health, section of radioprotection. this allowed the analysis of muscle exercise participation on the surgical side compared with the healthy body side. patient 1 had a left - side hip arthroplasty through the lateral transglutaeal (hardinge) approach (table 1). patient 2 had a right - side hip arthroplasty through the posterior (kocher - langenbeck) approach. a short shaft prothesis was implanted in the latter patient and standard uptake values (suv) measurements could be performed at the glutaeus and obturator muscle levels outside of the ct artifact. table 1patient characteristics and surgical procedurepatient ipatient iiage (years)5958arthroplastyleft hipright hipsurgical approachlateral (transglutaeal ; hardinge)posterior (kocher - langenbeck)femoral devicelong shaftshort shaft patient characteristics and surgical procedure 1-[c]acetate ([c]acetate) of clinical grade (gcp, gmp) was prepared at the cyclotron unit of geneva university hospital from [c]carbon dioxide, produced itself on an iba 18/9 cyclotron, as described. studies were performed on the lso - based pet / ct scanner biograph 64 (siemens medical solutions, erlangen, germany). patients were requested to restrain from heavy muscle exercise for 24 h prior to pet, a light morning meal 6 h before [c]acetate pet was being permitted. patients were positioned on the camera bed and remained relaxed for 10 min. for their comfort, an articulated knee position was accepted using a knee support of about 57 cm. for the rest pet / ct, 200 mbq (5.4 mci) [c]acetate was injected i.v. a low - dose ct (120 kvp, 60 mas, 16 1.5 collimation, pitch of 0.8 and 0.5 s / rotation) was registered over two bed positions between the iliac crest and the upper leg. pet scanning, 4 min per bed position, was then performed, starting 5 min after tracer injection. for exercise pet, a new activity of 800 or 600 mbq [c]acetate was then injected i.v. in the first and second patient, respectively. immediately after injection patients performed 20 isometric bilateral abductor exercises of 10 s followed by a relaxation time of 3 s each with sphygmomanometer - controlled pressure limited to 100 mmhg against the ankles using a physical constraint. a second low - dose ct was again registered followed directly by pet scanning of 3 min per bed position. following fourier rebinning and model - based scatter correction, pet images were reconstructed using two - dimensional iterative normalized attenuation - weighted ordered subsets expectation maximization and ct - based attenuation correction. mean standard uptake values (suvmean) over three non - consecutive slices were calculated within regions of interest (rois) drawn over individual muscles as defined by ct, using standard formula. 3 were compared using the student - newman - keuls multigroup comparison at different significance levels (unistat 5.6, statistical package for windows, unistat ltd, london). p 0.5). at 3 months, increase in activity of muscles under exercise (suvmean 1.52 0.09) was restored being 1.7-fold above the rest suvmean (0.88 0.18), (p 0.8). at 3 weeks after surgery, the four exercise pet / ct showed, on the healthy body side in both patients, a strong increase of [c]acetate uptake in the abductor muscles as compared with rest, notably the small, median, and the external fibers of the large glutaeus muscle (fig. 1, patient 2). a similar increase in uptake under exercise was also observed in the iliac and psoas (fig. 1[c]acetate pet / ct 3 weeks after right hip arthroplasty through the posterior kocher langerhans approach. imaging at the level of the iliac and psoas (a) and obturator muscles (b) is shown with the upper row showing pet at rest and the lower row pet after exercise. each line shows, from left to right,, pet / ct superposition and the low - dose ct alone. note that the three glutaeal muscles (a) (the small, the median, and the large glutaeus muscle with the external fibers) responded with strong increase in [c]acetate uptake during the abductor exercise (lower row) compared with rest (upper row). a strong increase in [c]acetate uptake under exercise was also observed on the healthy body side in the iliac and psoas (a) and internal obturator (b) muscles, while on the surgical side these latter muscles (arrows) show similar activity under rest and exercise at 3 weeks. the postsurgical [c]acetate pet hyperactivity extending into the external obturator muscle on the right side, directly related to the surgical approach, remained stable at rest, and exercise. [c]acetate pet / ct 3 weeks after right hip arthroplasty through the posterior kocher langerhans approach. imaging at the level of the iliac and psoas (a) and obturator muscles (b) is shown with the upper row showing pet at rest and the lower row pet after exercise. each line shows, from left to right,, pet / ct superposition and the low - dose ct alone. note that the three glutaeal muscles (a) (the small, the median, and the large glutaeus muscle with the external fibers) responded with strong increase in [c]acetate uptake during the abductor exercise (lower row) compared with rest (upper row). a strong increase in [c]acetate uptake under exercise was also observed on the healthy body side in the iliac and psoas (a) and internal obturator (b) muscles, while on the surgical side these latter muscles (arrows) show similar activity under rest and exercise at 3 weeks. the postsurgical [c]acetate pet hyperactivity extending into the external obturator muscle on the right side, directly related to the surgical approach, remained stable at rest, and exercise. on the arthroplasty side, most muscles showed in symmetry a similar exercise uptake increase. rest activity was slightly higher on the surgical side compared with the contralateral side. clearly, three muscles on the arthroplasty side of the patient having undergone the posterior kocher langenbeck arthroplasty approach (fig. 1), namely the iliac and psoas muscles (fig. 1a) and the internal obturator muscle (fig. 1b) did not show any relevant increase in uptake under exercise, in contrast to the increase in these same muscles on the contralateral healthy body side. in the other patient, having undergone a lateral hardinge transgluteal arthroplasty approach, the small glutaeus muscle and the external fibers of the large glutaeus muscle showed a similar increase in [c]acetate uptake under exercise as the contralateral muscles. however the median glutaeus muscle showed only a marginal increase in uptake under exercise at 3 weeks (result not shown) compared with rest. the rest activity of the median gluteaus muscle at 3 weeks was markedly above the rest activity in the adjacent and contralateral muscles. these same muscle groups were again reevaluated using an identical study protocol 3 months after surgery, both patients having completed their reeducation program. at the second pet evaluation, a similar increase in [c]acetate uptake as of 3 weeks was again observed in all muscles that participated previously in the exercise (fig. 2). furthermore, the four previously inactive muscles on the surgery side had clearly regained activity by showing an increase in [c]acetate uptake under exercise very similar to all other active muscles. 2pet / ct imaging at 3 months post surgery of the same patient as shown in fig. 1. note that imaging on the level of the iliac and psoas muscles (a) and internal obturator (b) shows the almost complete recovery of the previously stunned iliac and psoas and internal obturator muscles (arrows) on the surgery side. exercise (lower row) increase in [c]acetate uptake on the surgery side is now similar to that on the healthy body side also for these muscles. the three glutaeal muscles (a) responded again well at 3 months to the abductor exercise. note that the postsurgical hyperactivity, reaching into the external obturator muscle, is still well visualized at 3 months post surgery but was no obstacle to good participation in the exercise of the nearby internal obturator muscle. pet / ct imaging at 3 months post surgery of the same patient as shown in fig. 1. note that imaging on the level of the iliac and psoas muscles (a) and internal obturator (b) shows the almost complete recovery of the previously stunned iliac and psoas and internal obturator muscles (arrows) on the surgery side. exercise (lower row) increase in [c]acetate uptake on the surgery side is now similar to that on the healthy body side also for these muscles. the three glutaeal muscles (a) responded again well at 3 months to the abductor exercise. note that the postsurgical hyperactivity, reaching into the external obturator muscle, is still well visualized at 3 months post surgery but was no obstacle to good participation in the exercise of the nearby internal obturator muscle. in both patients, a marked hyperactivity related with the surgical intervention was observed at 3 weeks and was only partially resolved at 3 months. pet showed also a postsurgical collection in the first patient adjacent to the large gluteus muscle at the level of the greater trochanter. the observation of a global prostate hyperactivity observed in one of the two patients was also reported to his physician. in the four exercise pet / cts, the average suvmean of [c]acetate activity significantly increased above that of rest in the healthy abductor muscles and in the ilio - psoas and the internal obturator muscles contralateral to surgery. at 3 weeks post surgery, the increase was 2.9-fold above rest activity (p 0.5). at 3 months, increase in activity of muscles under exercise (suvmean 1.52 0.09) was restored being 1.7-fold above the rest suvmean (0.88 0.18), (p this latter increase in activity of previously injured muscles was notably identical to the measured increase of non - injured muscles on the surgery side at 3 months. for comparison, the visually inactive obturator external muscle was measured both at initial pet and after 3 months and showed on all examinations a very similar activity at rest and after exercise with only a marginal increase under exercise (group d, fig. 3, p > 0.8). imaging with [f]fdg pet showed the functioning of skeletal muscles by performing specific muscle exercises after injection of patients evaluated for suspected neoplasic disease. it was shown that [f]fdg uptake increased specifically in exercising muscles compared with resting muscle groups. aiming to reduce radiation exposure in muscle function pet and giving preference, notably in the postsurgical situation, to a double pet study at rest and after exercise our first results clearly show a significant increase in [c]acetate uptake under exercise compared with rest in healthy abductor muscles. this latter observation may be related to the previous observation that [c]acetate extraction from blood was also increased in the resting leg which should not actively participate in the exercise. furthermore, allowing a minor knee articulation position for our patients aimed at permitting complete muscle relaxation, the intended abductor exercise also required some corrective exercise of the inner rotation muscles in order to counteract an external rotation tendency under the exercise pressure to both ankles. at 3 months, all active muscles on the healthy body side were also symmetrically active on the surgery side, indicating the absence of any incidental single - sided muscle acetate uptake not related to the symmetric exercise. however, at 3 weeks post surgery, four muscles directly related to the surgical procedure showed absence of exercise participation as visualized by absence of significant increase in [c]acetate uptake. whether the absence of muscle function observed at 3 weeks post surgery was the product of a direct muscle or an activation failure, such as seen in painful osteoarthritic knees, remains an open question. the regained exercise participation of these four muscles at 3 months post surgery clearly shows that muscle function impairment was temporary, unrelated to any definitive nerve damage. a nervous lesion seems unlikely since the reinervation time would be longer than the 3 months, after which time, recovered muscle function could be demonstrated. [c]acetate pet of heart muscle has been described by different authors [1113 ]. [c]acetate undergoes a high first - pass extraction in the heart muscle and enters a first and second tricarboxylic acid (tca) cycle with production of different intermediate metabolites before being converted into [c]co2 in the second tca cycle. residence time of [c]acetate in the heart is short [5, 14 ] due to metabolism of [c]acetate into [c]co2. the kinetics of [c]acetate clearance from heart muscle, as measured by dynamic pet, has been proposed as a means to estimate myocardial oxygen consumption [1517 ] and has also been shown to be related to ventricular efficiency. non - oxidative reactions in the tca cycle also produce different metabolites and oxidative production of [c]co2 has been described to be relatively slow in resting muscle. with exercise, however, oxidative production of co2 from 1-[c]acetate in skeletal muscle would be significantly more rapid than at rest. in exercising volunteers, [c]co2 in breath decreased by 50% 20 min after infusion stop of 1-[c]acetate, suggestive of a relatively short - term metabolic consumption of acetate. our pet imaging time from 5 to13 min and 5 to 11 min postinjection in rest and exercise, respectively, was designed to match the frame of maximal [c]acetate uptake in heart muscle. we thus believe that we observed a real increase in [c]acetate muscle uptake under exercise. however, in analogy to heart muscle measurements, dynamic imaging of uptake and particularly elimination phase of [c]acetate in resting and exercising muscle may be required to prove an increase of [c]acetate consumption rate by exercising muscle. different aspects of [c]acetate muscle metabolism remained unclear in the preparation of this pilot study. thus, under different study protocols, [c]acetate had been infused in volunteers at micromolar concentration and it was shown that acetate consumption rose over longer periods of time [24 ] and did not reach its peak after 3 h, suggesting the need for an activation time. secondly, under muscle exercise, venous blood draws were only measured at 30-min intervals notably after exercise initiation. after the 30-min interval, uptake under exercise had increased to a maximum. in apparent contrast with the observations of acetate turnover in skeletal muscles, heart muscle [c]acetate has been shown to increase rapidly after injection and decrease thereafter to almost background level in between 30 min after injection. we opted for a limited strength isotonic, isometric bilateral abductor exercise of 4.3-min - overall duration. regarding the short exercise that was thus performed, reliable [c or c]acetate physiological observations of skeletal muscle activity were not available. furthermore, the maximal exercise pressure had to be limited in order not to risk loosening of the freshly implanted hip prothesis. bearing in mind the mentioned limitations, the positive outcome of this study appears remarkable. we were able to observe a convincing and reproducible increase in [c]acetate uptake in the major hip abductor muscles after exercise as compared with rest. although [c]acetate consumption in the active leg had been increased about sixfold as compared with rest, the approximate doubling of uptake in [c]acetate pet under exercise compared with rest in our hip arthroplasty patients was highly significant and seems well compatible with an exercise limited in time and force development. our data are well compatible with observations of skeletal muscle exercise [f]fdg pet [10, 21 ]. a patchy exercise pet [c]acetate muscle hyperactivity was observed in our patients. this may be due to in - homogenous blood supply which is known to exist under isometric muscle exercise [22, 23 ]. [c]acetate dual muscle pet may thus prove to be an interesting new non - invasive tool to directly demonstrate active muscle function and its occasional impairment after arthroplasty. it may represent an objective method to validate the introduction of new techniques [24, 25 ] in orthopedic surgery or for studying postsurgical long - term impairment of muscle function and its repair. it may also be an interesting complement to the electro - myogram or mri and allow an in - depth evaluation of activation failures which could occasionally also be of central nature. [c]acetate dual skeletal muscle pet under rest and exercise was successfully performed in a single session on four occasions in two patients. the patients having undergone hip arthroplasty by two different approaches had shown impaired muscle activity to the different muscle groups concerned which are closely related with the surgical procedure. good recovery of the transiently stunned muscle groups was then observed in both patients at 3 months after completion of their reeducation program. taken together, these data indicate a potential usefulness of [c]acetate muscle function pet in arthroplasty patients to study different surgical approaches in relation to potential transient or occasional persistently impaired muscle functions. these observations clearly merit further evaluation.	purposebased on skeletal muscle acetate physiology we aimed studying muscle function after hip arthroplasty with [11c]acetate pet.procedurestwo male patients were investigated 3 and 12 weeks after hip arthroplasty with muscle [11c]acetate pet / ct performed at rest and exercise. median muscle suvmean were calculated on three non - consecutive transverse pet slices.resultsthe four exercise pet / ct showed, compared with rest, consistent increase in [11c]acetate uptake in active muscles contralateral to surgery. on the arthroplasty side most muscles showed symmetric activity increase under exercise both at 3 and 12 weeks after surgery, but four muscles showed only minor activity increase at 3 weeks. at 3 months, functional recovery of the latter four muscles was observed.conclusionconsistent increase in [11c]acetate uptake in healthy muscles under exercise compared with rest was observed by pet / ct. transiently impaired muscle function 3 weeks after surgery recovered at 3 months. these first observations merit further investigation.
	the inhibition of differentiation and persistence of proliferation in cell transformation is probably not only caused by the mutation of single genes. an additional mechanism of transcriptional control, not only of single genes but of gene programs, is possibly the alteration of the topoisomerases. these enzymes regulate the conformation of dna by twisting and unwinding the double strands. as has been shown previously, only the genes located in relaxed dna areas are transcribed and, therefore, the topoisomerases can be described as a gene regulation device. we present the hypothesis that topoisomerase ii action is not only altered in, but also necessary for, hl-60 granulocytic cell differentiation. thus, alteration of topoisomerases may well be a molecular mechanism of cellular differentiation.imagesfigure 1.figure 2.
ionizing radiation (ir) is a well - known carcinogen for various human tissues and a complete carcinogen that is able to both initiate and promote tumor progression 1,2,3. the mechanism for this promotion is poorly understood, but studies of mouse thymic lymphomas provide some hints 2,4,5,6,7,8. indeed, studies of radiation - induced mouse thymic lymphomas, one of the classic models in radiation carcinogenesis, demonstrated that multi - steps and many factors, like ras, pten and fas, were involved in radiation - induced carcinogenesis 4,7. our previous studies showed that erk1/2, stat3 and shp-2 are also involved in radiation induced thymic lymphoma formation in balb / c mice 9. however, an easy and effective method to protect mice from radiation - induced thymic lymphoma is still not well known 1,10. hydrogen (h2), the most abundant chemical element in the universe (constituting approximately 75% of the universe 's elemental mass), is seldom regarded as an important agent in medical usage, especially as a therapeutic gas. however, many recent studies by our lab and other labs provided evidence that h2 gas has powerful therapeutic and preventive effects for many diseases 11,12,13. found that molecular h2 could selectively reduce cytotoxic reactive oxygen species, such as hydroxyl radicals in vitro and exert therapeutic antioxidant activity in a rat middle cerebral artery occlusion model in vivo 11. since hydroxyl is very strong oxidants that react indiscriminately with nucleic acids, lipids and proteins resulting in dna fragmentation, lipid peroxidation and protein inactivation, they are also the main mediators of radiation damage 12. we hypothesized and showed by experimental studies that h2 treatment could protect cultured cells and mice from radiation damage 12,13,14. in those studies, we used a single high dose model to find that h2 is a novel protective gas on radiation induced injuries. importantly, those previous studies also showed that h2-rich saline / water is safe, easy to administer and cost - effective 13,14. in this study, we used a split dose radiation - induced thymic lymphoma model in balb / c mice to test the potential role of h2 on radiation induced carcinogenesis in a method very similar to our previously studies 9,13,14. radiation induced thymic lymphoma model was described by many groups and our previous studies 4,5,9. in detail, male wild - type balb / c mice, 5 - 6 weeks of age, were purchased from chinese academy of science (shanghai, china) and a co irradiator was introduced for total - body ionizing irradiation as described in our previous work 9,12,13,14,15. four weekly sub - lethal doses of 1.75 gy gamma - ray irradiation were delivered to 5 - 6 week old balb / c mice at a dose rate of 0.58gy / min as described previously 9. only two groups were used in this study : the h2-rich saline group (h2 (+) group) or normal saline control (h2 (-) group) as described previously 13,14. themice from these two groups were intraperitonealy injected with h2-rich saline (h2 (+) group) or normal saline (h2 (-) group) 5 minutes before each irradiation respectively as we described detailed in our previous work 13,14. we found that h2 treatment significantly increased the survival rate of mice 30 weeks ' after split dose radiation (figure 1a, p<0.05).this datum is consistent with our previous studies that h2 treatment could protect cultured cells and mice from radiation damage 12,13,14. however, the radiation - induced thymic lymphoma rate in the h2 (+) group was significantly lower than in the control group (figure 1b p<0.05) and h2 treatment significantly increased the latency of lymphoma development after the split dose irradiation (figure 1c). these data indicated that h2 protects mice from radiation induced thymic lymphoma in balb / c mice. the detrimental effects of ir on biological tissues can be mediated via increased production of free radicals and reactive oxygen species (ros) and the ros system have been found to play important role in the induction of cancers 1,8,16. to explore the potential role of ros in h2 induced protection of radiation induced carcinogenesis, we used different methods to detect changes in intracellular and extracellular ros levels in h2 treated mice and control mice 4h after the last irradiation 13. intracellular ros levels in peripheral blood mononuclear cells (pbmc) from irradiated and control mice were assessed using facs analysis with dcfh - da (27di - chlorofluorescein diacetate), which converts to highly fluorescent dcf in the presence of intracellular ros. as shown in figure 2a, ros levels were much lower in the irradiated h2 group than in the irradiated control mice. serum sod (superoxide dismutase) and total gsh (glutathione) concentrations at 4h after the last irradiation in the h2 group were significantly higher than that of the control group, while mda (malondialdehyde) concentrations in the h2 group were significantly lower than that of the control group (figure 2b, 2c and 2d). these results indicate that the h2 pre - treated groups showed an increased antioxidant status, consistent with our previous studies that showed that h2 could reduce radiation - induced free radical damage to dna 13,14. radiation therapy is now a routine treatment for certain types of cancer and over 20 percent of cancer patients will require radiation therapy during the treatments of their disease 16,17. radiation itself can induce many types of cancers, especially leukemia and lymphomas, but few simple protective methods have been found. to the best of our knowledge, this may be the first report describing treatment with h2, which reduced the risk of radiation - induced carcinogenesis in the balb / c mouse model. while the therapeutic effectiveness of h2 treatment on radiation carcinogenesis needs further study, this work provides some novel experimental evidence for the use of h2 in radiation therapy. since it is safe, easy to administer and cost - effective, it could not only protect against radiation induced death 12,13,14, but also attenuate the rate of radiation induced carcinogenesis. in conclusion, our data indicates that h2 protects mice from radiation induced thymic lymphoma in balb / c mice.	ionizing radiation (ir) is a well - known carcinogen, however the mechanism of radiation induced thymic lymphoma is not well known. moreover, an easy and effective method to protect mice from radiation induced thymic lymphoma is still unknown. hydrogen, or h2, is seldom regarded as an important agent in medical usage, especially as a therapeutic gas. here in this study, we found that h2 protects mice from radiation induced thymic lymphoma in balb / c mice.
atherosclerotic disease is a main cause of severe cardiovascular events, such as myocardial infarction and stroke. as the prevalence of atherosclerosis increases globally due to aging population, identifying atherosclerotic plaques will be of utmost importance for early diagnosis and intervention, which will substantially decrease the health care burden.1 evidence is available indicating that subclinical atherosclerosis in carotid artery is associated with the presence and severity of coronary artery disease (cad) and with an increased 10-year risk of cardiovascular events.25 the association between thoracic aortic plaques and cad has been often reported by using transesophageal echocardiography.6,7 furthermore, researchers found that the extent of thoracic aortic atheroma burden was independently associated with increased long - term mortality in patients following cardiothoracic surgery.8 although autopsy studies reported plaques in the abdominal aorta to be severe in patients with cardiac catastrophe,9 there are limited studies showing the association between cad and abdominal aortic plaques. only a small number of observational studies reported that the prevalence and extents of plaques in abdominal aortas were greater in patients with cad than in those without cad by using cardiovascular magnetic resonance (cmr). but they reported that plaques in abdominal aorta were not independent factors for cad.911 besides, due to lack of large - scale data, the results of these studies may not be convictive. although cmr was a useful tool for evaluating atherosclerotic plaques in abdominal aortas,12 it is neither cost efficient nor suitable for population - based studies. the 2014 esc guidelines recommend that population screening for abdominal aortic aneurysm (aaa) should be performed by abdominal aorta ultrasound in all men > 65 years of age.13 abdominal aorta ultrasound has become a useful tool to not only screen aaa but also evaluate atherosclerotic plaques, which is noninvasive, easy to assess, cost efficient, and could be routinely used in population - based study.1 however, the significance of abdominal aortic plaques detection by ultrasound imaging is not yet definitive. the purpose of the present study was to determine the prevalence of abdominal aortic plaques by ultrasound imaging and to explore its association with cad in patients undergoing coronary angiography. between october 2014 and june 2015, a prospective study was conducted in the department of cardiology at guangdong general hospital, guangzhou, people s republic of china. abdominal aortic ultrasound is a routine examination in patients undergoing coronary angiography in our hospital. as this was an observational study, written informed consent was not required according to the policy of the ethics committees of guangdong general hospital. ultrasound scanning of the abdominal aortas was performed in 1,667 consecutive patients undergoing coronary angiography for suspected or known cad, or prior to valve surgery. excluded were patients with aortic diseases, congenital heart disease, infectious diseases, autoimmune diseases, or neoplasma. obstructive cad was defined as the existence of astenosis > 50% of the lumen diameter of at least one major coronary vessel. the severity of the disease referred to the number of identified stenosed vessels (lumen diameter 3 mm or mobile or ulcerated plaque. grades iii and iv were considered to represent abdominal aortic plaque.15 the risk factor variables evaluated in the present study included age, sex, smoking, hypertension, diabetes mellitus, chronic kidney disease, triglyceride, total cholesterol, low density lipoprotein - cholesterol (ldl - c), and high density lipoprotein - cholesterol (hdl - c). hypertension was defined as present if there was any history of high blood pressure or if the blood pressure measured twice in the hospital exceeded 140 mmhg (systolic) or 90 mmhg (diastolic) or in case of current use of antihypertensive medications. diabetes mellitus was defined as present if the patient had a history of diabetes or if the fasting plasma glucose exceeded 7.0 or 11.1 mmol / l 2 hours after a meal or in case of current use of diabetic medications. chronic kidney disease was defined as present if the estimated glomerular filtration rate (egfr) was less than 60 ml / min/1.73 m. the egfr (expressed as ml / min/1.73 m) was computed using a prediction formula derived from the modification of diet in renal disease study:16 egfr = 186 (serum creatinine) (age) 0.742 (if female). blood samples measured by standard laboratory methods were taken in a fasting state on the morning of the day when angiography was performed. quantitative data are presented as mean standard deviation (sd) and qualitative data are presented as frequencies. differences between two groups were evaluated by the unpaired t - test for parametric variables and by the chi - square test for categorical variables. a multiple logistic regression analysis was used to elucidate the associations between abdominal aortic plaque and cad. an ordinal logistic regression analysis was used to elucidate the associations between abdominal aortic plaque and the severity of cad all the statistical analyses were made with the spss for windows software, version 20.0 (ibm corporation, armonk, ny, usa). between october 2014 and june 2015, a prospective study was conducted in the department of cardiology at guangdong general hospital, guangzhou, people s republic of china. abdominal aortic ultrasound is a routine examination in patients undergoing coronary angiography in our hospital. as this was an observational study, written informed consent was not required according to the policy of the ethics committees of guangdong general hospital. ultrasound scanning of the abdominal aortas was performed in 1,667 consecutive patients undergoing coronary angiography for suspected or known cad, or prior to valve surgery. excluded were patients with aortic diseases, congenital heart disease, infectious diseases, autoimmune diseases, or neoplasma. obstructive cad was defined as the existence of astenosis > 50% of the lumen diameter of at least one major coronary vessel. the severity of the disease referred to the number of identified stenosed vessels (lumen diameter 3 mm or mobile or ulcerated plaque. the risk factor variables evaluated in the present study included age, sex, smoking, hypertension, diabetes mellitus, chronic kidney disease, triglyceride, total cholesterol, low density lipoprotein - cholesterol (ldl - c), and high density lipoprotein - cholesterol (hdl - c). hypertension was defined as present if there was any history of high blood pressure or if the blood pressure measured twice in the hospital exceeded 140 mmhg (systolic) or 90 mmhg (diastolic) or in case of current use of antihypertensive medications. diabetes mellitus was defined as present if the patient had a history of diabetes or if the fasting plasma glucose exceeded 7.0 or 11.1 mmol / l 2 hours after a meal or in case of current use of diabetic medications. chronic kidney disease was defined as present if the estimated glomerular filtration rate (egfr) was less than 60 ml / min/1.73 m. the egfr (expressed as ml / min/1.73 m) was computed using a prediction formula derived from the modification of diet in renal disease study:16 egfr = 186 (serum creatinine) (age) 0.742 (if female). blood samples measured by standard laboratory methods were taken in a fasting state on the morning of the day when angiography was performed. quantitative data are presented as mean standard deviation (sd) and qualitative data are presented as frequencies. differences between two groups were evaluated by the unpaired t - test for parametric variables and by the chi - square test for categorical variables. a multiple logistic regression analysis was used to elucidate the associations between abdominal aortic plaque and cad. an ordinal logistic regression analysis was used to elucidate the associations between abdominal aortic plaque and the severity of cad all the statistical analyses were made with the spss for windows software, version 20.0 (ibm corporation, armonk, ny, usa). of the 1,667 study patients (male, 68.9% ; mean age, 6311 years) undergoing coronary angiography, 1,268 had cad (> 50% stenosis). compared with 399 patients without cad, 1,268 patients with cad had higher prevalence of abdominal aortic plaque (37.3% vs 17% ; p 50% stenosis). compared with 399 patients without cad, 1,268 patients with cad had higher prevalence of abdominal aortic plaque (37.3% vs 17% ; p 65 years of age (class i, level a).13 our study found that abdominal aorta ultrasound can not only detect aaa but also abdominal aortic plaque, which was an independent factor associated with the presence and severity of cad. researchers found that the extent of thoracic aortic atheroma burden was independently associated with increased long - term mortality in patients following cardiothoracic surgery.8 this is an attractive hypothesis that this relationship is not limited to patients after surgery, but describes general atherosclerotic disease patterns of the aorta, with impact for prevention of cardiovascular events. identifying abdominal aortic plaques will be of utmost importance for early diagnosis and intervention of cad, which will reduce severe cardiovascular events in turn. first, our study population was chinese patients undergoing angiography, who are generally considered to be a highly selected population at high risk for cad. because of this selection bias, our results may not be applicable to the general or other ethnic populations. therefore second, atherosclerosis varies in severity depending on multiple features that contribute to plaque progression and stability. plaque instability has been confirmed as a main cause of acute coronary syndrome.23 due to the technical limitations of ultrasound imaging, we can not assess the stability of abdominal aortic plaque and its association with coronary plaque instability. recently, researchers have found that targeted fluorescence activatable cell - penetrating peptides (acpps) probes distinguished disrupted plaques from stable plaques with high sensitivity and specificity in an animal model. the combination of anatomic, magnetic resonance imaging derived predictors for disruption and acpp uptake can further improve the power of identifying high - risk plaques.24 with the recent development in technology, it is possible and necessary to further assess the stability of abdominal aortic plaques and their association with cad. third, ultrasound imaging was used to evaluate abdominal aortic plaques, but coronary angiography was used to evaluate coronary atherosclerosis, which can not visualize plaques, and it only shows lumen characteristics. our current study can not elucidate the associations of abdominal aortic plaques with coronary artery plaques. fourth, atherosclerosis is a systemic disease affecting not only the coronary and carotid arteries but also the thoracic and abdominal aorta and the lower extremities. in conclusion, the prevalence of abdominal aortic plaques was higher in patients with cad than those without cad. abdominal aortic plaque was an independent factor found to be associated with the presence and severity of cad.	objectivecurrently, the association between abdominal aortic plaques and coronary artery disease (cad) has not yet been clarified clearly. the purpose of this study was to determine the prevalence of abdominal aortic plaques by ultrasound imaging and to explore its association with cad in patients undergoing coronary angiography.methodsbetween october 2014 and june 2015, a prospective study was conducted in the department of cardiology at guangdong general hospital, guangzhou, people s republic of china. ultrasound scanning of the abdominal aortas was performed in 1,667 consecutive patients undergoing coronary angiography. clinical characteristics and coronary profile were collected from the patients.resultsof the 1,667 study patients (male, 68.9% ; mean age, 6311 years) undergoing coronary angiography, 1,268 had cad. compared with 399 patients without cad, 1,268 patients with cad had higher prevalence of abdominal aortic plaques (37.3% vs 17%, p<0.001). in multivariate analysis, abdominal aortic plaques served as independent factors associated with the presence of cad (odds ratio = 2.08 ; 95% confidence interval = 1.502.90 ; p<0.001). of the 1,268 patients with cad, the prevalence of abdominal aortic plaques was 27.0% (98/363) in patients with one - vessel disease, 35.0% (107/306) in patients with two - vessel disease, and 44.7% (268/599) in patients with three - vessel disease. stepwise increases in the prevalence of abdominal aortic plaque was found depending on the number of stenotic coronary vessels (p<0.001 ; p - value for trend < 0.001). in an ordinal logistic regression model, abdominal aortic plaques served as independent factors associated with the severity of cad according to the number of stenotic coronary vessels (p<0.001).conclusionthe prevalence of abdominal aortic plaques was higher in patients with cad than in those without cad. abdominal aortic plaque was an independent factor associated with the presence and severity of cad.
panax notoginseng saponins (pns) constitute the principal ingredient extracted from the traditional herb medicine, p. notoginseng (burk.) fh chen (chinese sanqi), and the major effective components are ; notoginsenoside r1, ginsenoside rg1, ginsenoside rb1, ginsenoside rd, and ginsenoside re.1 pns has extensive effects on the cardiovascular system, along with other pharmacological effects, including antifatigue, immunological, anticancer, antioxidant, hepatoprotective, and renoprotective functions.2 pns preparations are used widely by the general public, especially in the people s republic of china. traditional medicine is very much welcomed by the chinese public, especially the elderly, who believe that traditional chinese medicine is natural and has no adverse effects. however, in recent years, we have observed many drug eruptions and other adverse reactions induced by a variety of traditional chinese herbs and patented drugs. these adverse drug reactions, drug eruption in particular, have also been observed with pns, but have been largely ignored. here we report on four patients who developed drug eruption due to injection of pns. case 1 was a 50-year - old woman who presented with a large area of erythema and hundreds of tiny nonfollicular pustules, and a 2-week history of itching and pain from head to foot (figure 1a). she also had a fever of more than 38c. on the day before the appearance of the skin rash, the patient had received her first injection of pns (400 mg, intended for once - daily administration), and another injection of sulfotanshinone sodium (40 mg intended for once - daily administration, and used several times previously) as a treatment for dizziness. histopathological examination of a biopsy from a pustule was consistent with a diagnosis of acute generalized exanthematous pustulosis (agep, figure 1b). we treated this patient with once - daily intravenous infusion of methylprednisolone 80 mg, and the skin rash gradually improved. a single dose of intravenous methotrexate 7.5 mg was also administered. the patient s condition improved further with gradually tapering doses of methylprednisolone (figure 1c) during a hospital stay of 20 days. case 2 (figure 2a and b) was an 89-year - old man who presented with a 1-week history of generalized erythema, tiny pustules, and pruritus. one week before appearance of the rash, he had received his first injection of pns (500 mg, intended for once - daily administration) and an injection of oxiracetam (4 g, intended for once - daily administration and received several times previously) for treatment of a cerebral infarct. we treated the patient with once - daily intravenous infusion of methylprednisolone 80 mg for 3 days. a new rash appeared on his face, so we changed the dosage of methylprednisolone to 40 mg twice daily for 4 days., new rashes appeared on several occasions during his 23-day hospital stay. a single intravenous dose of methotrexate 7.5 mg was also administered. case 3 was a 62-year - old woman with a 5-day history of generalized erythema and papules with itching. she had a fever of 38.7c. on the day before appearance of the skin rash, the patient had received her first injection of pns (800 mg, intended for once - daily administration) and an injection of oxiracetam (6 g, intended for once - daily administration and received several times previously) for treatment of dizziness. this patient was treated with an intravenous infusion of methylprednisolone, 120 mg once daily. four days later, many tiny pustules appeared on the trunk and lower limbs (figure 3a) but disappeared on the same evening. we decreased the dose of methylprednisolone to 80 mg once daily on the following day because of her improving condition ; however, the severe rash and pruritus returned, so we increased her methylprednisolone to 120 mg once daily. case 4 was a 67-year - old woman with a 2-day history of generalized erythema and pruritus, who had a fever of 37.8c. one week before appearance of the rash, the patient had received an injection of pns (500 mg, intended for once - daily administration) for multiple lacunar infarction. three days later, many tiny pustules appeared on the upper limbs (figure 3b), which disappeared on the following day. after effective treatment, all four patients were discharged from hospital, the rash disappeared, and the circulating neutrophil count and liver function returned to the normal range. the pns injections used by these patients were manufactured by different local manufacturers and were all produced as per the good manufacturing practice standards. for each patient, the duration of continuous use of pns before the appearance of the skin rash was less than 1 week. no other short - term special medication, diet, or exposure to environmental factors before the appearance of the rash was identified. gallelli reported generalized urticaria in a young woman treated with clomipramine and after ingestion of codfish, indicating a probable causal relationship between the drug - food interaction and the skin rash. no specific dietary changes were associated with use of the drug and appearance of the skin rash, so a drug - food interaction was essentially excluded. some of the patients had been using other long - term medications, such as antihypertensive and antidiabetic drugs for several years, which we thought were unrelated to the skin rash. for these reasons, we diagnosed the skin lesions in these patients as drug eruption caused by pns injection. by using the world health organization s uppsala monitoring centre for standardized case causality assessment, certain, but probable / likely, because all four patients refused rechallenge.4 given the normal dose of pns injected, the clinical manifestations of the rash, and effective treatment with methylprednisolone, the reaction was considered as a type of allergic drug eruption in all four patients. these four patients with drug eruption, comprising one male and three females, all aged over 50 years, suddenly developed skin lesions with pruritus from head to foot, and then accepted hospitalization. after observation and treatment, we found that these patients had some interesting clinical features : a short incubation period (17 days, average 4 days) ; pustular skin lesions, agep, or transient local pustules ; requirement for high - dose glucocorticoid therapy, comprising methylprednisolone given as an intravenous infusion of at least 80 mg once a day ; long hospital stays (2023 days, average 22 days) ; a significantly increased count and proportion of neutrophils in blood ; and a good outcome. we also observed that new rashes continued to appear during tapering of methylprednisolone in two cases, so we believe that decrements in the glucocorticoid dose should be very slow during treatment and an immunosuppressant, such as methotrexate may be needed. agep is a rare drug eruption presenting with acute extensive formation of nonfollicular sterile pustules on an erythematous and edematous base, and is typically accompanied by fever and leukocytosis. only 18% of agep are not due to antibiotics.5 schmid reported that drug - specific t - cells played an important role in the pathogenesis of agep, showing that secretion of interleukin-8 by t - cells and keratinocytes attracted neutrophils that filled the vesicles and transformed them into pustules. whether the mechanism of this pns - induced skin reaction is related to drug - specific t - cells and interleukin-8 needs further investigation. agep or pustular drug eruption can be induced by drugs other than antibiotics, including hydroxychloroquine,5,7 sorafenib,8,9 acetazolamide,10 gliclazide,11 recombinant interleukin-2,12 and ibuprofen.13 however, there are no reports in the literature on pustular drug eruption due to traditional chinese medicine, including pns. the pustular skin rash induced by streptococcal infection should be distinguished from pustular drug eruption. patrizi reported a case of diffuse acute sterile pustular eruption after streptococcal infection. the pustular eruption resolved after an 8-day course of antibiotic therapy. in the present cases, no elevated antistreptolysin titer were found, and high - dose glucocorticoid therapy was required. to our knowledge, this is the first report of the clinical features of pns - induced drug eruption. although there have been only four cases, we have observed the following common characteristics : pustules, fever, an elevated circulating neutrophil count, need for high - dose glucocorticoid therapy, and a long treatment duration, which would provide a useful reference and warning for clinicians.	panax notoginseng saponins (pns) are a patented product in the people s republic of china, and have extensive effects on the cardiovascular system. here we report on four elderly patients (one male and three female) with drug eruption induced by pns injection. all developed a sudden skin rash with pruritus from head to foot, and subsequently accepted hospitalization. in each case, pns had been used for less than 1 week before appearance of the rash. no specific short - term medications or changes in diet or exposure to environmental factors immediately prior to appearance of the rash were identified. these four patients had some interesting features in common, ie, pustules, fever, and elevated circulating neutrophil counts, which required high - dose, long - term glucocorticoid therapy. to our knowledge, this is the first report of pustular drug eruption induced by pns and provides a useful reference and warning for clinicians.
most of the clinical experiences to date on the use of hypertonic saline have come from trauma patients in the prehospital setting or en route to the emergency room.13 the effects of hyperosmotic - hyperoncotic solutions (hhs) in rapidly restoring hemodynanic stability, boosting cardiac function, and possibly improving outcome, along with a demonstrated safety in trauma population, provide the basis for the growing interest in their perioperative use over the last two decades. the majority of studies were carried out in elderly cardiac surgery patients who were at an increased risk for intraoperative cardiac complications. the main goals of using hhs during the perioperative period are to prevent hypovolemia and to assure adequate tissue oxygenation while avoiding fluid overload. in this review, we included 30 studies433 of human subjects treated with 7.27.5% nacl alone (hypertonic saline - hs), nacl with dextran (hsd) or nacl with hetastarch (hss). in 15 cases, seven cases involved patients undergoing aortic aneurysm repair (table 2), two cases involved providing tested hypertonic saline in neurosurgical patients (table 3), two cases involved patients undergoing abdominal hysterectomy (table 4), and four cases involved patients who underwent minor operations (table 5). seven studies3440 tested the hypertonic saline using tonicity levels other than those examined previously (ranging from 1.8% to 5.85% nacl) and were among the first reported clinical trials on the perioperative use of hs.34 two studies41,42 had no control groups, and these studies were thus excluded from this review. with the exception of two studies (involving patients undergoing cardiac valve surgery) from the group of cardiac surgeries,22,31 all clinical experiences were derived from coronary artery bypass grafting (cabg) patients. amongst all patients undergoing cardiac surgery, the most relevant finding was the substantial decrease in positive fluid balance. by titrating volume infusion against pulmonary capillary wedge pressure (pcwp), oliveira.16 while studying the effect of hsd in the hemodynamic status of cardiac patients, demonstrated an enormous positive fluid balance in the control group compared to the hsd group. hsd was being tested in regards to it s effect in the hemodynamic status and volume of fluid administrated. the ringer s solution group required almost 4000 ml more positive fluid balance than the control group to arrive at the same hemodynamic status. the tollofsrud study20 also reported a relevant 1200 ml cumulative spare volume in hhs patients using a 4 ml / kg of body weight fixed dose. even when the hhs - treated group was compared to the control group, which used colloid solutions, administering hypertonic solution was deemed to be advantageous.6,7 furthermore, preoperative hemodilution with hhs in patients with normal left ventricular ejection fraction who underwent cabg was recently reported to decrease the need for perioperative fluid independent of the type of mixed colloid used.30 recently, in a study involving 50 patients undergoing cardiac valve surgery, bueno and colleagues31 administered 4 ml / kg of hhs before cardiopulmonary bypass with a fluid balance of near zero during the first 48 hours as compared to a large positive balance in the ringer s group. finally, three studies by jarvela and colleagues25,26,28 stated that administering a single dose of 4 ml / kg of 7.5% nacl over 30 minutes to cabg patients during the postoperative rewarming phase resulted in intense diuretic effects and decreased fluid retention. excess body fluids that had accumulated during cardiopulmonary bypass and cardiac surgery were excreted because of the increased diuresis in these studies. in one study,28 weight gain in the ns - 0.9% saline control group was significantly greater than that in the hs group during the first postoperative day (1.9 1.4 kg, median, 2.1 kg and 0.8 1.5 kg, median, 0.8 kg, respectively ; p=0.005). cardiac index (ci) was another hemodynamic endpoint that was favourably altered by hypertonic saline in the majority of the studies and was independent of whether the solution infusion was titrated. studies that titrated the same infusion as controls showed a substantial increase in left ventricular filling pressure.7,16,20 in two studies8,31 that used a fixed dose, the beneficial effect of increasing the ci lasted well into the 48h after surgery. however, by administering a single 250 ml postoperative dose of hhs and hsd, sirieix.22 verified a transient increase in ci that returned to baseline values within 3 hours despite significant increases in left ventricular preload and left ventricular ejection fraction in their patients. also, the hemodynamic effects of hs lasted only about 1 hour in the jarvela study.25 however, the preoperative hemodilution with hsd and hss in the molter study30 significantly augmented ci. another important effect of administrating hypertonic solutions throughout these studies was the reduction in systemic vascular resistance. three studies8,22,31 reported a reduction in pulmonary vascular resistance after infusion of hss or hsd. regarding mean arterial pressure (map), most authors observed either a transient increase or no significant difference in map compared to controls when hypertonic solutions were infused. nevertheless, two studies required further attention. in the prien study,11 a transient drop in map was documented after infusion of a fixed dose of 250 ml hss over 15 minutes. the 20% decrease in map from baseline was seen in the first 5 minutes of the infusion and was followed by hypervolemic left ventricular failure in the majority of these patients. by applying the same rate of infusion, sirieix and colleagues22 managed the severe hypotension that developed after hypertonic solution administration in three of 16 patients. however, they observed significant augmentation of map following hypertonic solution infusion in the majority of their patients compared to those given a control solution. these patients also reported significant increase in central venous pressure (cvp) following infusion of a hypertonic solution. there was a statistically significant drop in pao2 in the control groups, while pao2 remained more constant in hss treated patients.8 bueno.31 reported that patients who underwent cardiopulmonary bypass maintained a significantly higher pao2/fio2 ratio after hsd infusion compared to those given ringer s solution. molter.30 described a relevant higher o2 delivery index in the hypertonic solution treated group. furthermore, two additional studies demonstrated earlier extubation times after hypertonic saline administration.19,31 hypernatremia was a common finding after using hhs in cardiac surgery. however, no neurological symptoms were associated with the increased sodium load, even when serum sodium reached concentrations of over 160 mmol / l.31 since the first reported study in 1987 on the use of hypertonic saline (7.27.5% nacl) in patients who underwent surgical repair of abdominal aortic aneurysm, a total of 91 such patients have been treated with hypertonic solutions to date. most of the studies gave hs mixed with hydroxyetilhyl starch and had he s as the control solution (table 2). the most striking result was the reduced need for fluid intake in patients receiving hhs. first, auler.4 assessed hs in three abdominal aortic aneurysm aaa patients and found a decreased need for perioperative fluid despite a slightly greater blood loss in this group in relation to the ns group. more remarkable results were seen in two other studies. by preoperatively titrating a bolus of 50 ml of hss to the endpoint of the highest ci at lowest pcwp in high risk patients, ellinger and colleagues14 demonstrated an average four - fold decrease in the need for fluid in these patients as compared to those in the control group, who were given he s. in the christ study,17 a fixed dose of 250 ml hhs was applied during aortic clamping for over 20 minutes and resulted in a significantly reduced fluid balance compared to the hes - treated control group (2471 949 ml versus 3387 1248 ml). additional ringer s solution was administrated in both groups to achieve defined pcwp. recently, another study that titrated dose against the best pcwp / ci ratio infused the hss solution after aortic clamping and reported less fluid retention compared to those given an he s solution (mean 162 111 ml versus mean 265 108 ml).23 other important effects of hhs on hemodynamic variables were also reported. in the ellinger study,14 the ci nearly doubled in patients treated with hss in a titrated fashion compared to that in patients treated with he s. other studies have also demonstrated increased cis.4,17 in addition, a decrease in systemic vascular resistance index was reported immediately after the infusion of hs4 and after administration of hhs during aortic cross - clamping.17 auler.4 also stated that pulmonary vascular resistance soon decreased after hs administration. no hypotension developed in patients receiving hhs, regardless of whether the infusion was titrated;14,17 the sodium load increased, but no related symptoms were reported.17 one anaphylactoid reaction occurred and was attributed to he s use.17 one patient died in the ragaller study,23 though it was unclear which group the patient belonged to. three more patients developed serious complications in that study, but they were reported to be unrelated to fluid therapy. only two intraoperative studies involving neurosurgical patients were assessed for this review. while two other studies have been published, these two were excluded due to both a lack of controls41 and the utilization of 3% nacl solution rather than 7.27.5% nacl solution.39 hemodynamic parameters did not change when hs was used as opposed to 20% mannitol in the two excluded studies. gemma el al18 found no difference in brain bulk and cerebrospinal fluid (csf) during elective procedures between infusion of a fixed dose of 2.5 ml / kg hs and administration of 2.5 ml / kg of mannitol. erard and colleagues29 reported that a single infusion of solutions of equal osmolarity, nacl 7.5% or mannitol 20%, induced a similar osmolar variation over time in both hypertonic groups. recently, kolsen - petersen and colleagues32,33 published two studies on the use of hs in abdominal surgery. based on previous studies in human blood cell cultures4345 and different animals models43,46 that reported that hypertonicity is permissive for lymphocyte proliferation43 and restrictive for neutrophil function,45,4650 the researchers observed the immunological effects of a fixed dose of hs on postoperative women who underwent abdominal hysterectomy. their first randomized double - blind study32 involved 62 women who received either 4 ml / kg of 7.5% or 32 ml / kg of 0.9% nacl over 20 min. the study found that an infusion of hs saline did not alter the postoperative immune response after abdominal hysterectomy. in the subsequent clinical trial also by kolsen - peterson el al.,33 modest changes from hs infusion were observed when compared to normal saline treated patients. these changes included a temporary increase in the number of b cells in the peripheral blood (p<0.01), an augmentation in the concentration of plasma elastase (p<0.05), and a decrease in the number of circulating neutrophils (p<0.001). in a 1995 study demonstrating the inotropic effects of hs in anesthetised patients without cardiovascular disease, goertz.15 concluded that the apparent improvement of left ventricular systolic function in response to hypertonic saline / hetastarch was caused mainly by the combined effects of increased left ventricular preload and reduced left ventricular afterload. two of these studies, conducted by jarvela and colleagues,24,27 found no significant differences regarding hemodynamic parameters and sodium load when the hs - infused group was compared to the control group. however, in a separate study by durasnel.,21 infusion of 100 ml of hs for over 15 minutes prior to spinal anaesthesia effectively prevented the occurrence of hypotension when directly compared to patients infused with normal saline. hypotension occurred in two of 24 patients in the hs group and eight of 24 in the ns group (p<0.05). the improved fluid balance seen in hhs - treated patients seems to be the result of various mechanisms. first, after the stress and shock of surgery, cells may become edematous.51 as a result, hhs rapidly shifts fluid from the intracellular compartment to the intravascular space with consequent plasma expansion.52 hss increases renal blood flow and glomerular filtration rate, induces renal vasodilatation,53 and decreases plasma aldosterone levels, all resulting in increased urine output.54 an immediate increase in urine output following hhs administration was observed in several cardiac studies.25,26,28,31 in the setting of cpb, extracorporeal circulation is particularly related to the release of cytokines, vasoactive mediators, hormones and autacoids that may cause endothelium damage, increased vascular permeability, tissue oedema, and vasoconstriction. thus, fluid accumulation is an expected event during cpb due to increased extracellular fluid and intracellular oedema. in addition to the fluid shift from erythrocytes and endothelial cells, hhs may attenuate the inflammatory response, leading to less capillary leakage. in five studies,68,16,31 the use of hypertonic solution before cpb may have led to a better inflammatory profile and accounted for the favourable fluid balance. moreover, hypertonic infusions resulted in increased plasma protein55 and decreased protein loss from the intravascular space, which may further have positives effects on fluid balance. however, while volume expansion and reduced afterload have been accepted as an explanation for the increased ci, the direct effects of hypertonic solutions on cardiac contractility remain controversial. most recent results indicated that at least a slight effect may exist.5658 the remarkable increase in ci seen in the ellinger study,14 where hss was titrated to the highest ci at lowest pcwp and suggested that hypertonic saline may improve cardiac performance. only one study presented data conflicting with this hypothesis : transient hypervolomic left ventricular heart failure was induced by a 15-minute infusion of hhs in the prien study.11 this rapid hhs infusion rate could be accountable for the transient fluid overload seen in their patients. moreover, severe hypotension occurred after a similar rate of administration in the siriex study.22 it is thus generally believed that the degree of hypotension is dependent upon the rate of infusion.59 therefore, a longer rate of infusion appears to be appropriate for intraoperative purposes. numerous reports of reductions in systemic vascular resistance index as well as a few reports of decreased pulmonary vascular resistance following hhs administration were all consistent with the existing established model.6062 the most transient hemodynamic effects seen in the javerla studies2527 could be explained by the strong diuretic effects observed immediately after infusion of hs. the decrease in volume overload along with the improved microcirculation and the decreased interstitial oedema may be responsible for the more efficient pulmonary function observed in several studies.8,20,31 large volume resuscitation with crystalloids worsened lung oedema and prolonged the need for mechanical ventilation.6365 christ.17 has suggested that less fluid excess accompanied by decreased oedema formation is particularly beneficial for high risk patients. three studies have shown that a titrated dose was the preferred method of administrating hypertonic solution in a perioperative setting. the prien study11 reported a case of left ventricular fluid overload caused by a fixed dose of 34 ml / kg over 15 minutes. ellinger.14 titrated the infusion in patients undergoing aortic aneurysm surgery and found that a fixed dosage of 4 ml / kg was too high for at - risk cardiac patients with slight hypovolemia. furthermore, severe hypotension developed after the infusion of a fixed 250 ml dose given over 15 minutes in patients who underwent cardiac valve repair in the sirieix study.22 titrated infusion gained further support from animal studies. pascual.55 administered titrated doses of hsd in a swine model of intra - operative hemorrhagia and found greater volume expansion effect of the solution in hypovolemic conditions. furthermore, nguyen.66 reported the advantages of titrated resuscitation in haemorrhaged adult sheep. thus, the use of titrated hhs infusion in this population seems to be more appropriate. however, it is clear that certain limitations exist in isolated studies of aortic aneurysm repair. none of the studies involved more than 20 patients in the tested groups, which limited the power of the statistical analysis. auler.4 performed a non - randomised study and included patients with thoracoabdominal and abdominal aneurysm in the experimental group. furthermore, the studies were not blind. albrecht and colleagues13 examined patients before surgery and during the induction of anaesthesia. in the ragaller study,23 the staff was not blinded, which increased the possibility of bias once the parameters were defined. consistent with the 2002 cochrane group review67 on the use of intravenous fluids (non - blood fluids) for abdominal aortic surgery, we also found no overwhelming evidence to support or reject the preferential use of any particular type of fluid. for neurosurgical patients, however, the current shortage of studies precluded drawing any conclusions. the lower utilization of hs in neurosurgery may be the result of a lack of studies as well as study parameters on the safety and beneficial effects of using hs.18,29 despite the similarity of the hs dose used by three studies for preloading before spinal anaesthesia,21,24,27 only one group reported that infusion of the solution was capable of preventing hypotension.21 these studies used low hs doses, while most clinical trials that reported positive effects from hs used higher doses. the discrepancy between the modest findings reported by kolsen - petersen.32,33 and the results from previous studies on the immunomodulatory properties of hs may be due to the small number of patients and the characteristics of the studied population. kolsen - petersen. studied healthy normovolemic women who underwent a standardized surgical procedure. the immunologic response to trauma depended heavily on the amount of tissue injury68 and blood loss.69 ischemic reperfusion injury seen in abdominal aortic aneurysm repair is another important cause of inflammatory response.70 however, as mentioned earlier, cpb during cardiac surgery was also associated with further inflammation. based on these results, patients undergoing aortic aneurysm repair (particularly ruptured aortic aneurysm repair) appear to be the population that experiences the most beneficial effects from hypertonic solutions. the use of colloids along with hypertonic saline prolongs volume expansion and may also positively influence cardiac function. however, colloids are also linked to a small but significant number of complications, particularly anaphylactic reactions and changes in coagulation function. an anaphylactic reaction in one patient was attributed to he s use in the christ study.17 in two other studies,16,20 drainages from the chest were slightly greater in the hsd groups, but the difference was not statistically significant. our analysis of 30 published clinical studies on the perioperative use of hypertonic saline (7.27.5% nacl) solutions has shown that administration of the hypertonic solution resulted in beneficial hemodynamic effects, particularly during cardiac surgery. the previously observed reductions in positive fluid balance, increases in ci and decreases in systemic vascular resistance further supported our recommendation of hs use during surgery. furthermore, our study further indicated that dose administration should be titrated and that the rate of infusion should be slow. this review also demonstrated the urgent need for well - designed randomized clinical trials, particularly in the setting of aortic aneurysm repair, where hs has shown many beneficial effects.	hyperosmotic - hyperoncotic solutions have been widely used during prehospital care of trauma patients and have shown positive hemodynamic effects. recently, there has been a growing interest in intra - operative use of hypertonic solutions. we reviewed 30 clinical studies on the use of hypertonic saline solutions during surgeries, with the majority being cardiac surgeries. reduced positive fluid balance, increased cardiac index, and decreased systemic vascular resistance were the main beneficial effects of using hypertonic solutions in this population. well - designed clinical trials are highly needed, particularly in aortic aneurysm repair surgeries, where hypertonic solutions have shown many beneficial effects. examining the immunomodulatory effects of hypertonic solutions should also be a priority in future studies.
bicuspid aortic valve (bav) is the most common congenital heart disease with an estimated prevalence of 0.5 - 2%.1)2)3) bav is more commonly observed in male with approximate predominance of 3:1. although bav is a congenital anomaly, development of its complications in adulthood is a characteristic feature of the natural history of this disease entity. moreover, morphological or functional abnormality associated with bav is not confined to the aortic valve and development of complications in the adjacent aorta and other anatomical cardiac structures are not uncommon.4)5) thus, thorough understanding of the complex clinical features of patients with bav is not easy and there are many unresolved issues. bav is typically made of 2 unequal - sized cusps with fusion of one commissure, which results in a central raphe or ridge in the larger cusp (fig. 1). the phenotype or anatomical patterns of bav can vary according to which commissures have fused. traditionally, classification based on fused two cusps and the orientation of the raphe has been used:6)7) for example, fusion of the right and left coronary cusps is classified as bav - rl type (fig. thus, bav - rn (bav with fusion of the right and non - coronary cusps), and bav - ln (bav with fusion of the left and non - coronary cusps) based on the fused two cusps were suggested by some investigators. although this classification seems logical, demonstration of fused 2 cusps is not easy or practical and sometimes the bicuspid cusps are symmetrical without raphe (" pure " bav), which makes demonstration of fused 2 cusps impossible. for this circumstance, the traditional approach was to use the orientation of the free edge of the cusps defined either anterior - posterior (bav - ap) or lateral (bav - la) (fig. to overcome potential problems using two different criteria for bav phenotypic classification (the orientation of the raphe and the free edge), a new classification has been suggested using the orientation of bav only. in this classification,8) two bav phenotypes - fusion of the right and left coronary cusps (bav - ap) and fusion of the right or left coronary cusp and non - coronary cusp (bav - rl)-were used (fig. the position of both coronary artery ostia is also different according to the bav phenotype (bav - ap vs. bav - rl), which is helpful for patients with relatively poor echocardiographic window. the other notable finding is that a recent animal experiment provided strong evidence that bav - ap and bav - rl types are distinct etiologic entities with the defective development of different embryological structures at different embryological time.9) the theoretical advantages of the new classification need to be tested by clinical outcome studies. considering that a consistent description of bav phenotype is absolutely necessary for assessment of its potential clinical or prognostic implication, a uniform classification scheme should be determined based on long - term follow - up data. bav phenotypic classification is attractive as it may provide new and valuable data regarding risk stratification of bav patients according to cusp morphology. of particular interest is the potential utility of this information, combined with knowledge of family history and hemodynamics, to provide a better understanding of patient prognosis. for that purpose, it is no doubt that transthoracic echocardiography plays a pivotal role for diagnosis of bav.5)6) however, not infrequently, other imaging modalities are necessary for correct diagnosis and classification of bav. sometimes, transesophageal echocardiography with better resolution is necessary for accurate diagnosis of bav (fig. special care is needed to identify the cusps separation and the site of cusp fusion. both systolic and diastolic images are necessary for this purpose, as bav might show a false tricuspid appearance in diastole based on the presence of the raphe. frequently, the two commissures can be visualized only during systole with a clear separation of the cusps and thus it is imperative to pay particular attention to the opening motion of the aortic cusps (fig. advanced imaging techniques such as computed tomography or cardiac magnetic resonance imaging would be the best option (fig. not all patients with bav develop significant valvular dysfunction. moreover, valvular dysfunction in bav patients does show wide clinical spectrum in terms of patterns of valvular dysfunction ; some patients show significant aortic stenosis, whereas others may show a prolapsing aortic cusp resulting in significant valvular regurgitation without stenotic component. in young children and adolescents, bav - rl type is reported to be associated with a higher risk of significant valvular dysfunction and more rapid development of aortic stenosis or aortic regurgitation with shorter time of intervention,10)11) which was not confirmed in adult patients with bav.6)7) in a recent clinical study using high quality multidetector computed tomography, moderate - to - severe aortic stenosis predominated in patients with bav - rl type, whereas moderate - to - severe aortic regurgitation in those with bav - ap type;8) this potential association can be an interesting hypothesis which should be tested. all these studies suffer from selection bias characterized by relatively small numbers of patients with limited inclusion criteria such as referral for consideration of open heart surgery. thus, potential association between specific bav phenotype and types of valvular dysfunction still remains elusive and to be a hypothesis, which definitely needs further investigation. non - valvular cardiovascular conditions associated with bav include coarctation of aorta, turner syndrome, coronary artery anomalies, sinus of valsalva aneurysm, aortic aneurysm, aortic dissection, supravalvular aortic stenosis, patent ductus arteriosus, ventricular septal defect, shone 's complex, and familial thoracic aortic aneurysm / dissection syndromes.12) among them, aortic dilatation with development of aortic dissection is the most important and challenging issue in clinical practice. common association of aortic dilatation with significant valvular dysfunction (aortic stenosis or regurgitation) in patients with bav remains an important mechanism of development of aortic dissection.13)14) however, it is very interesting to observe that development of aortic enlargement or dissection is not correlated with severity of valvular dysfunction (fig. 5) and can occur in bav patients without valvular dysfunction or even several years after surgical correction of valvular dysfunction (fig. 6).15)16)17)18)19) these findings support inherent primary abnormalities of the aortic media, more specifically cystic medial degeneration, a well - known risk factor associated with aortic aneurysm or dissection. in large family studies, the prevalence of bavs in first degree relatives of an individual with a bav has been reported to be 9% with autosomal dominance pattern20)21) and bav associated with ascending aortic aneurysm may also be familial. moreover, recent advances in molecular biology have demonstrated potential association with specific gene abnormalities, such as notch1 (notch homolog 1, translocation - associated) or transforming growth factor beta receptor.22)23)24) all these findings support a genetically triggered intrinsic defect involving the aortic media. however, in a recent cross - sectional analysis of 595 patients with bav, age and severity of valvular dysfunction were proven to be two independent factors associated with aortic diameter. 14) additionally, isolated aortic valve replacement for bav patients with significant valvular dysfunction showed significantly lower annual aortic dilatation rate.14)25) these observations suggest significant impact of chronic hemodynamic burden due to valvular dysfunction and protective effect of surgical intervention. further prospective investigations are necessary to overcome potential selection biases in the current clinical studies and to confirm factors associated with aortic dilatation in bav patients. another characteristic finding of bav aortopathy is that there are marked individual variations in terms of the site of aortic enlargement. echocardiography has been the primary diagnostic tool for evaluation of bav patients, which can not evaluate the whole ascending and descending aorta. by using computer - assisted cluster analysis of clinical data of new imaging modalities, such as computed tomography or magnetic resonance imaging, various patterns of bav aortopathy 7).8)26) aortic dilatation may confine to the aortic root, but, not infrequently, the aortic enlargement involves the tubular portion of the ascending aorta or entire ascending aorta up to the transverse aortic arch. standardization of the classification of bicuspid aortopathy phenotypes is necessary for further investigation to test whether there would be potential association between bav phenotype or type of valvular dysfunction with bav aortopathy phenotypes.27) an interesting observation is that type of valvular dysfunction may explain different patterns of aortic dilatation in bav patients : aortic regurgitation severity was found to be associated with aortic sinus diameter, whereas aortic stenosis severity with the tubular diameter.14)18) the association between bav - rl type and aortic enlargement has been also reported and type 3 aortopathy, the most severe form of bav aortopathy, was the most common phenotype in bav - rl patients.8) as bav - rl type showed higher frequency of moderate - to - severe aortic stenosis with a higher maximal transvalvular gradient or velocity, it may suggest that the hemodynamic burden contributes significantly to the development of bicuspid aortopathy. however, because progressive aortic dilatation occurs in many patients with normal valvular function, the hemodynamic burden caused by valvular dysfunction can not entirely explain the full spectrum of bicuspid aortopathy. one interesting point is that the direction of helical flow differs according to bav phenotype, with right - handed helical flow as the predominant pattern in bav - ap type, and left - handed helical flow in bav - rl type.28)29) thus, variations in segmental aortic dilation that correlate with specific changes in valve morphology may be related to differences in eccentric flow jets that lead to a differential distribution of wall stress. longitudinal follow - up is needed to determine whether an abnormal helical flow pattern correlates with bav aortopathy phenotype. for this purpose, accurate classification of bav although bav is characterized by a relatively long latent period before clinical manifestations in adulthood and diverse patterns of manifestations including valvular dysfunction (stenosis or regurgitation), aortopathy (aortic dissection), and acquired complications (infective endocarditis), the late outcomes in children with bav, but without valve dysfunction, have not been well studied.4) moreover, the earlier studies were from the era of cardiac catheterization. there are two large recent series to better define the un - operated natural history of bav patients in the modern era.30)31) the report from the mayo clinic included 212 bav patients without valvular dysfunction (mean age 32 20 years) and the follow - up duration were 15 6 years.30) overall survival was excellent with the 20-year survival rate of 90 3% and nobody died of cardiac diseases. the probability of aortic valve or aorta surgery was 27 4% and no one underwent aortic dissection surgery. toronto general hospital group included 642 patients with a spectrum of valve dysfunction (mean age 35 16 years) and the follow - up duration was 9 5 years. the 10 year survival rate was 96 1% and the probability of aortic valve or aorta surgery was 22 2%. the incidence of aortic dissection and infective endocarditis were 2 1% and 2%, respectively.31) these data support the notion held by many that eventually most patients with bav would require some form of intervention. however, importantly, in both of these series, fatal events were rare and development of aortic dissection, the most serious clinical manifestation of bav, does not occur frequently contrary to the previous expectation. both studies confirmed that age and valvular dysfunction are independent risk factors associated with development of adverse clinical events in bav patients. thus, the risk of bav patients should not be over - emphasized and the clinicians should encourage the patients and their family members to accept that the over - all prognosis of bav patients is excellent with regular medical examination and follow - up in the modern era. low incidence of aortic dissection in these series has been re - confirmed by another registry data showing 0.5% at 25 years follow - up13) and raised a challenging clinical issue of adequacy of current preventive or early surgical intervention for patients with bav and aortic dilatation.32)33)34) however, a mere lack of data is responsible for the current controversy surrounding the indication and timing of elective surgical intervention for the aorta in bav patients. it is obvious that aortic size and evidence of medial degeneration are limited risk stratification tools. a collaborative multicenter retrospective and prospective bav patient registry with homogeneous and rigorous entry criteria (i.e., accurate bav diagnosis and phenotyping with exclusion of unclear cases) is absolutely necessary to reconcile the clinical, imaging, pathobiology, genetic, and management pieces of the puzzle.5) bav patients show marked heterogeneity in many different clinical aspects including bav phenotype, severity of valvular dysfunction and bav aortopathy phenotype. despite recent advances in clinical cardiology, risk stratification based on imaging data combined with knowledge of genetics, family history and hemodynamics should be a clinicians ' final goal. a well - characterized bav cohort should be established and followed over time to achieve that goal. the current effort to establish a prospective korean registry of bav should be encouraged to get the fundamental data for evidence - based medical practice. phenotype classification of bav and bav aortopathy based on high quality imaging studies is a key for bav cohort and registry,35)36) which reinforces the leading role of image specialists for this project.	bicuspid aortic valve (bav) is the most common congenital heart disease with marked heterogeneity in many aspects. fusion patterns of the aortic cusp are quite variable with different type and severity of valvular dysfunction. moreover, non - valvular cardiovascular abnormalities are associated with bav. among them, aortic aneurysm / dissection is the most serious clinical condition with variable patterns of segmental aortic dilatation. potential association between bav phenotype and valvulopathy or aortopathy has been suggested, but needs to be tested further. a lack of long - term outcome data at this moment is responsible for unresolved debate regarding appropriate management of patients with bav, specifically to prevent development of aortic dissection. long - term follow - up data of a well - characterized cohort or registry based on standardized classification of bav phenotype and aortopathy are necessary for evidence - based medical practice. advanced imaging techniques such as computed tomography or magnetic resonance imaging offer better opportunities for accurate phenotype classification and imaging specialists should play a central role to establish a collaborative multicenter cohort or registry.
orthokeratology (ok) provides independence of conventional compensation by spectacles or contact lenses during the waking hours. for myopia correction central cornea is flattened to achieve the desired reduction in the power of the anterior corneal surface, while the midperipheral cornea steepens [2, 3 ] as a result of the epithelial thickness redistribution from the center. this treatment deteriorates the quality of vision [58 ] and it has been shown that such changes are dependent on pupil size and strongly affected by the amount of refractive error being corrected. in clinical practice, ok subjects usually complain of subjective perception of dysphotopic phenomena in the form of haloes, ghosting, or glare (here considered as light distortion phenomena or ld). those are more intense at the beginning of treatment and decrease over time [11, 12 ]. we have observed in a cross - sectional study that ok subjects report subjective complaints that are transient during the first days or weeks of treatment. common complaints include perception of haloes and starburst around light sources, either in outdoor (i.e., car lights) or indoor conditions. in a different study, we have quantified the amount of ld over the period of adaptation to ok treatment in a cohort of 29 subjects and observed that ld phenomena increase at the first day but decrease again towards baseline values after 30 days. however, in that study, we were not able to measure ld phenomena beyond the first month. thus, we can not ensure that the adaptation period does not undergo further changes in the long term. other authors found a correlation between corneal irregularity and asymmetry parameters after 1 month of ok treatment, what might suggest that the optical quality of the front corneal surface during ok treatment might affect ld phenomena. considering that our previous study showed that ld improves from 1 day to 1 month of treatment despite the increase in hoa up to 7 days after initiation of treatment, becoming stable thereafter, it has been hypothesized that ld is not related to hoa themselves or that there is an ld adaptation process even when the optical quality of the anterior corneal surface remains significantly affected by spherical - like and coma - like hoa [2, 3 ]. while several studies have addressed the longitudinal changes in hoa and contrast sensitivity (cs) over a period of 1 year, to date no study has addressed the temporal changes in ld phenomena beyond 1 month of treatment. different devices are available for measuring the disturbances surrounding bright spots against dark background ; some of them use software [1620 ] ; others use custom - made or commercially available ones. we use a physical display to present the stimuli as this allows us to have a wider dynamic range of luminance from the main glare source and the detection peripheral stimuli and this instrument has been shown to be consistent with and sensitive to changes in higher - order aberrations (hoa) artificially induced. thus, the purpose of the present study was to evaluate the long - term visual effects of corneal optical quality degradation in ok patients by measuring the ld phenomena and csf. a total of 20 neophyte subjects were recruited and fitted with ok lenses for myopia correction. inclusion criteria required that they were over 18 years of age, had less than 1.00 diopters (d) of refractive astigmatism, were free of ocular disease, had not contraindication for overnight cl wear, and presented a best corrected monocular visual acuity of 0.90 decimals (20/25) or better. subjects were informed of the purpose of the study and signed a consent form after all of their questions had been answered. following the tenets of the declaration of helsinki, the protocol of the study has been reviewed and approved by the irb. subjects underwent a comprehensive optometric examination. in this study, all measures have been obtained monocularly in order to correlate aberration data with clinical data obtained with the different methodologies. subjective baseline refraction and refraction at the time of data collection were determined as the spherocylindrical combination. endpoint criterion was the highest positive (or less negative) refraction that allowed the patient to achieve their maximum visual acuity, in agreement with usual refraction procedures to control for accommodation effects. ld was analyzed with an experimental prototype at a distance of 2.0 m in a darkened room. it consists of an array of 240 1 mm wide leds distributed radially at 15 intervals over 160 mm, with a linear separation of 10 mm around a central 5 mm white led that acts as glare source, as previously described [17, 18, 20 ]. the system is controlled by a custom - made software that interfaces with the patient to detect the peripheral stimuli seen and discriminate them from those hidden by the central glare source. characteristics and examination procedures in the context of assessment of ok patients have been previously described. in brief, in totally darkened room, the instrument presents the central source of glare at maximum intensity while the peripheral leds are randomly turned on and off. the patient provides feedback regarding the stimuli that can be seen by clicking a remote actuator. the system reads and stores the feedback information and within 45 to 75 seconds the instrument provides a drawing of the area where the peripheral stimuli can not be seen by the patient, along with different quantitative metrics. metrics of distortion size included the ldi, calculated as the ratio of the area or points missed by the subject and the total area explored, and is expressed as a percentage (%). best fit circle radius (bfcradius) is defined as the circle that best fits to the distortion area resulting from the linear binding of all points in each meridian of the device. the higher values of ldi and bfcradius are interpreted as a lower ability to discriminate small stimuli surrounding the central source of light. irregularity of the distortion area is derived as the deviation of the actual polygonal shape obtained from the bfc fit and is called the bfc irregularity (bfcirreg). the standard deviation of bfcirreg, called bfcsd, measures how asymmetric is the departure of the actual distortion limits from the perfect circular shape of the bfc. together, bfcirreg and bfcsd can be interpreted as the deviation of the actual distortion from a perfectly rotational symmetric shape. the higher the value of this parameter, the larger the deviation from a circular shape, expressed in mm. the corneal aberrations were derived from topography data using the oculus easygraph (oculus, dutenhofen, germany) for a circular aperture of 6.0 mm. the root mean squares (rms) for 4th- and 6th - order spherical aberration (spherical - like), third- and fifth - order horizontal and vertical coma aberration (coma - like), and fourth- and sixth - order secondary astigmatism (secondary astigmatism) were calculated. decimal high - contrast visual acuity and csf were measured at distance of 5 m under photopic conditions with the lcd screen 22 lcd (topcon cc-100xp, tokyo, japan). frequencies tested were 1.50, 2.12, 3.00, 4.24, 6.00, 8.49, 12.00, 16.97, and 24.00 cycles per degree (cpd). changes in different parameters from baseline to subsequent visits were compared using anova test with bonferroni correction. all subjects included in the analysis showed a monocular visual of 1.0 decimals or better at the 1-month and 1-year visits. figure 1 shows the variations of size - related (ldi and bfcradius) and irregularity - related (bfcirreg) parameters of monocular ld over time. bfcradius worsened after first night recovering normal values after 1 month and 1 year (anova with bonferroni post hoc correction, p < 0.05). bfcirreg followed a similar path with an increase after the first night and a reduction towards baseline values after 1 month (table 2) and remaining stable after 1 year of follow - up. while the size - related parameters of the ld recovered to baseline values, figure 2 shows the variations of optical quality of the anterior corneal surface over time. table 3 summarizes the average differences and statistical significance for those parameters where the rms presented a significant change between two visits as analyzed with anova with bonferroni post hoc correction. spherical - like rms underwent a statistically significant increase after the first night of treatment and worsened further up until the 1-month visit compared to baseline (p < 0.05). coma - like rms, as well as to a less extent secondary astigmatism rms, showed a statistically significant increase up until the first month visit, remaining stable at the 1 year visit. figure 3 shows the variations in csf between baseline and the 3 follow - up visits after 1 day, 1 month, and 1 year of treatment. table 4 presents the average difference and the statistical significance for those spatial frequencies showing statistically significant changes over the study period. bonferroni post hoc correction showed that there was only a significant decrease in cs for frequencies 3.00 and 8.49 cpd from baseline on day 1. furthermore, significant changes from day 1 to 1 month were also observed for 4.24, 8.49, 16.97, and 24.00 cpd spatial frequencies. finally, spatial frequencies of 3.00, 4.24, and 24.00 cpd presented statistically significant changes between 1 day and 1 year. we observed that there was a statistically significant correlation between monocular cs for medium frequencies (3.0, 4.24, and 6.00 cpd) and ld parameters at baseline (r 0.529, p < 0.001). however, after 1 year of treatment, this correlation was only statistically significant for 12 cpd spatial frequency (r 0.565, p < 0.001). in general, however, after one year of treatment, we observed that there was a statistically significant correlation between spherical - like rms for 6 mm pupil size and irregularity of ld (r = 0.420, p < 0.05). regarding correlations between monocular cs and hoa, correlations were statistically significant between the spherical - like rms for 6 mm pupil size and frequencies 4.24 (r = 0.335, p < 0.05) and 12.00 cpd (r = 0.367, p < 0.05) at baseline. after 1 year of treatment, hoa were not significantly correlated with monocular cs values except for the 4.24 cpd spatial frequency against coma - like rms (r = 0.397, p < 0.05) and secondary astigmatism rms (r = 0.419, p < 0.05). with the present study we found that despite a significant and stable deterioration of visual quality, as observed in the aberrometric structure of the anterior corneal surface, visual quality measured through csf returns to baseline values within the first month of treatment. similarly, ld perception measured with an experimental device also returned to baseline over the first month of treatment and remained stable up to 1 year of follow - up. in ok practice, subjects occasionally report visual disturbances, even though their high - contrast visual acuity is excellent. although optical quality of the eye and quality of vision after ok have been investigated [5, 22, 23 ], in the present study, we evaluated hoa, cs, and adaptation to ld simultaneously in subjects undergoing ok for myopia during the first year of treatment. measurement of cs can provide useful information about visual function that may not be revealed by standard visual acuity testing [24, 25 ]. previous studies have reported significant visual quality changes after ok with significant increase of hoa and reduction in cs depending on the amount of myopic correction. in that study, the authors observed that bcva was maintained at baseline over the follow - up period. ocular hoa significantly increased 1 month after the procedure and remained stable thereafter. regarding cs, there was an initial loss during overnight ok, and the loss persisted during the 1-year follow - up. instead, we found that cs recovered to baseline values after 1 month of treatment. similar to the findings previously reported by us in the shorter term monocular csf experienced a significant decrease of 3.00 to 8.49 frequencies from baseline to day 1. in the present study there was a correlation between csf and hoa at baseline and this correlation was lost after treatment onset and during the year of follow - up. on the other hand, ld parameters and hoa were not correlated during the study period, except for spherical - like rms for 6 mm pupil size and bfcirreg after one year of treatment. in a previous work, villa. reported a significant correlation between spherical - like rms and ld. although ld and cs are recovered with respect to baseline point, there was a lack of correlation between them in the present study. this might reflect that ld itself does not in fact impair cs under the conditions of examination. it might be expected that performing cs analysis under glare conditions might result in a significant correlation between ldi and cs, but not for the conditions under which we evaluated cs in the present study. it has been surprising to find an improvement in the higher frequencies for the csf measurement, improving from baseline to 1 year after an initial decrease. this improvement over the best corrected csf before treatment might be related in part to some learning effects after repetitive application of the test. however, this is less likely because the tests were applied for a long period of time apart from each other. several studies have shown that cs significantly correlates with some abilities associated with the patient 's quality of life, such as reading speed or driving performance. this is well recognized in subjects who have undergone corneal refractive surgery and report frequent symptoms of night vision disturbances [26, 29, 30 ] even when high - contrast visual acuity is excellent [3135 ]. in order to evaluate other aspects of the visual quality of the patient, in this longitudinal study, we investigated changes in perception of ld, changes in ocular hoa, and csf as representative parameters of vision quality in eyes undergoing overnight ok during one year. ld analysis showed a transient increase followed by a reduction to baseline levels over the first month of treatment. changes observed in the long - term from 1 month to 1 year are not significant. this is in agreement with the clinical observation of adaptation to the distortion effect. in a recent work we have observed that subjective reports of light distortions increase when treatment starts and decreases over the period of treatment. with the present study we can conclude that adaptation phenomena observed previously in the shorter - term is not expected to change in the longer term. our observations with this experimental device are also in agreement with the time course of night vision disturbances after refractive surgery. though ok and corneal refractive surgery are different procedures, similar time course changes are found with hoa remaining high after the procedure while the complaints and subjective perceptions of the patients seem to improve faster after the procedure. pop and payette showed a reduction in patients reporting night vision disturbances from 25% at 1-month visit to 4.7% at the 12-month visit. our results, however, show that this reduction in night vision complaints might be faster, recovering to baseline after 1 month. this might be explained because the measurement device, such as our ld analyzer, is sensitive to more severe forms of ld phenomena that do not allow to see objects around the bright source of light. even when the patient has recovered to a point where this kind of distortion is no longer present or is attenuated, the patient might still subjectively report it to some degree. evaluated the time course changes in the responses to the quality of vision (qov) questionnaire in patients undergoing myopic and hyperopic lasek corneal surgery. this instrument accounts for frequency, severity, and bothersome of visual symptoms including haloes and starburst and we include these kinds of phenomena below the umbrella of ld phenomena. they also observed a rapid decline in the qov score after prk surgery with the qov scores returning to baseline after 1 month following the treatment. at present there is no objective (and available) method to measure those photic phenomena isolated. the one that approaches such measures in a somewhat specific way is the quality of vision questionnaire. however, this includes other aspects of subjective visual complaints and thus can not be directly compared. indeed, hoa of the anterior corneal surface changed significantly in our study, in agreement with previous work. in a recent paper conducted by our group, where we have evaluated hoa during the first month of ok treatment for one month, and the present results demonstrate the stability in the longer term from 1 month to 1 year of treatment. the time course changes in hoa observed in the present study are in agreement with values reported by other authors [5, 22 ] with spherical - like and coma - like aberrations being the main contributors to the degradation of the image quality. this might be justified by the fact that 3rd- (coma - like) and 4th - order (spherical - like) aberrations are more directly related to the creation and consolidation of the treatment zone, while secondary astigmatism affects more peripheral areas of the cornea presumably more subjected to day - to - day variations as a consequence of slight decentrations or pressure changes at the edge of the transition zone of the lens. we have not measured whole - eye hoa that could correlate better with the visual functions that we aim to evaluate. however, the main changes in aberrations in our study are induced in the front corneal surface, and it is expected that the whole eye aberrations would suffer similar changes. we can not ensure that the ld parameter reflects specifically the haloes, ghost images, starburst, and glare reported subjectively by the patient. however, we have reported in a recent study that the system is sensitive to the photic phenomena induced when we artificially incorporate different amounts and sign of hoa. although we can not quantify each one of the photic phenomena, we consider that such effects are well reflected in the short - term measurements in this study and that adaptation takes place to reduce the size of the ld overtime in the middle and longer term (present study), in agreement with transient subjective complaints expressed by the patient in the clinical setting. on the other side, the lack of correlation between ld and cs values might be related to the fact that we did not use a glare source while measuring the csf. compared to our previous study this does not reflect the actual viewing conditions of the patients, but we aimed to maximize the potential changes to be observed and we know that they would be more likely detected under monocular conditions, as binocular summation will tend to improve the results of ld [14, 37 ] and cs. we also aimed to explore the correlations with hoa specific to each eye, which forced us to do follow a monocular analysis in this part of the study. other factors that might limit the correlation between optical quality (hoa) and visual quality (ld and cs) parameters include the fact that we can not ensure that the pupil area in each condition is exactly the same. furthermore, neural adaptation mechanisms might play a significant role in adaptation to a deteriorated optical quality while preserving a good visual function. this ability to adaptation has been reported previously in the presence of hoa in human eyes [13, 3941 ]. in summary, we have observed that the previously reported adaptation phenomena to increase hoa are maintained in the longer term, at least as far as it concerns the ld and csf. to our knowledge this is the first study addressing the long - term changes in the optical quality of the anterior corneal surface with orthokeratology treatment and the potential impact on visual quality measured through monocular csf and ld measurement.	purpose. to evaluate the corneal higher - order aberrations (hoa), contrast sensitivity function (csf), and light distortion (ld) in patients undergoing orthokeratology (ok). methods. twenty healthy subjects (mean age : 21.40 8 years) with mean spherical equivalent refractive error m = 2.19 0.97 d were evaluated at 1 day, 1 month, and 1 year after starting ok treatment. monocular ld, photopic monocular csf, and corneal hoa for 6 mm pupil size were measured. results. ld showed an increase after the first night (p 0.05). spherical - like, coma - like, and secondary astigmatism hoa rms increased significantly (p 0.022) from baseline to 1-month visit, remaining unchanged over the follow - up. contrast sensitivity for medium frequencies (3.0, 4.24, and 6.00 cpd) was significantly correlated with ld parameters at baseline (r 0.529, p < 0.001). however, after 1 year of treatment, this correlation was only statistically significant for 12 cpd spatial frequency (r 0.565, p < 0.001). spherical - like rms for 6 mm pupil size correlated with irregularity of the ld (r = 0.420, p < 0.05) at the 1-year visit. conclusion. ld experienced by ok patients recovers after one month of treatment and remains stable in the long term while optical aberrations remain significantly higher than baseline.
pregnancy and delivery are among the most stressful events in women s life (1). negative delivery experiences related to unexpected medical interventions, severe pain, or fear from death cause great fear and anxiety for mothers and eventually lead to anxiety disorders (2). fear and anxiety increase muscle tension and direct blood oxygen towards mother s brain and muscles. therefore, mother s tension, fatigue, and sensitivity toward pain are increased and her compatibility with pain is decreased (3). despite the scientific advances regarding the physical problems in pregnancy, psychologic problems are still a major issue in pregnant women s health (4). delivery is an important experience in women s life and the quality of this experience leads to short - term as well as long - term effects. negative delivery experiences cause psychologic problems as well as sexual disorders and affect the emotional relationship between mother and her infant after delivery. on the other hand, fear and anxiety increase the surgical interventions. thus, in order to reduce these interventions, delivery pain associated anxiety must be changed into a desirable experience (5, 6). one other outcome of fear from delivery pain is the increase in the number of elective cesarean sections (7). additionally, studies have shown that abnormal fetal heart rate and reduction of the first- and the fifth- minute apgar scores are more common among the mothers experiencing severe pain and anxiety (8). thus, sedating pain during delivery is highly essential and improves breastfeeding behaviors. consequently, control of delivery pain is among the first and the most important responsibilities of the healthcare staff (9). attachment is defined as a stable, warm, and intimate relationship between mother and child. evidence has shown that improve in attachment results in creation of a stable relationship between mother and child and reduce the mother s anxiety (9). pillitteri believes that attachment can create a desirable relationship between mother and child and improves child s cognitive, emotional, and social growth (10). acupressure is based on the rules of acupuncture and is applied on the same points used in acupuncture (11). it can be easily learned and performed and does not have any negative effect even if it is not performed efficiently. acupressure involves applying stable, gentle pressure on one or some of the 365 energy points on the 12 body meridians and in this way creates balance and releases energy (11). from chinese medicine point of view acupressure as a noninvasive method eliminates imbalance in vital energy, removes pain, reduces muscle tension, improves blood circulation and vital activities, and decreases the anxiety symptoms (12). anxiety leads to physical and psychologic disorders, increase in heart rate, sweating, and worry ; in addition, it has negative effect on individual s function and relationship with others (13). according to a study conducted by kaviani. acupressure on hugo point could decrease the prenatal mean anxiety in comparison with the control group (14). another study showed that attachment and relaxation skills training programs during pregnancy reduced anxiety during pregnancy as well as postpartum depression (15). similarly, a previous study indicated that maternal anxiety was reduced by dry cupping therapy (16). another study demonstrated that higher anxiety levels during labor were accompanied by more pain and less control on maternal behaviors (17). as a practical strategy with no side effects that can control and reduce anxiety, this nonpharmacologic method has motivated researchers to perform studies in this field. moreover, tokumaru. conducted a study to investigate the effect of acupressure on myoelectric activity of the stomach. in that study, acupressure was first applied at p6 point for one minute, rest was taken for a minute, and the process continued for 30 minutes. in addition, changes in the samples heart rate was used to evaluate their autonomic nervous function. overall, the results revealed a significant difference between the intervention and the control groups (19). furthermore, wang. performed a study on the parents of the children undergoing surgery and revealed the effectiveness of acupressure at the third - eye point in reduction of preoperative anxiety (20). since all the previous studies have measured the effects of acupressure on labor pain and have revealed the effect of this method on reduction of pain intensity, the present study aimed to assess the effects of acupressure on maternal - fetal attachment. no study was conducted on this issue up to the time of this report. moreover, the present study compared the effects of acupressure at two acupoints, while previous studies evaluated the effect of acupressure at only one point on labor pain (15, 16, 21, 22). furthermore, evidence has demonstrated that the cause of many elective cesarean deliveries is maternal anxiety and fear of pain. therefore, using alternative medicine not only is effective in reducing anxiety, but also does not impose any costs on families. in addition, pain and anxiety during labor have a negative effect on maternal - fetal attachment. the present study aimed to compare the effects of acupressure at two different acupoints on anxiety level and maternal - fetal attachment in primiparous women. the study aimed to answer the following questions : do different acupressure points have different effects on maternal anxiety ? this randomized controlled clinical trial was conducted on 150 primiparous women with term pregnancy who were referred to the obstetrics wards of shoushtari and hafez hospitals, shiraz, iran, in 2010. the participants with inclusion criteria were consecutively recruited into the study and were allocated into the three groups (a : gb-21 acupoint, b : acupressure at sp-6 acupoint, and c : control group) using a block randomization method. in order to eliminate the psychologic effect of various interventions on the study results, only one type of intervention was performed each day. before the intervention, written informed consents were obtained from all the participants. moreover, the researcher learned the correct way of performing acupressure under the supervision of a specialist in this field. acupressure was applied in the semi - recumbent position on the left arm so that reduction of blood pressure would be prevented. according to the specialist s opinion and since meridian energy flow circulation cycle is approximately 20 minutes, acupressure was performed for 20 minutes (5). the inclusion criteria were being primiparous, being 18 to 35 years old, singleton pregnancy, gestational age of 37 to 41 weeks, cephalic presentation, being before or at the beginning of the active phase of labor (3- to 4-cm dilation), having middle- school or high- school degrees, and signing written informed consents. the exclusion criteria were any psychologic (e.g., psychosis or schizophrenia) or anatomic disorder (e.g., uterine disorders or pelvic stenosis), chronic diseases (e.g., cardiovascular diseases, pulmonary disorders, hypertension, and diabetes), skin disorders (e.g., eczema and superficial skin infections), high - risk pregnancy (e.g., pregnancy hypertension, polyhydramnios, oligohydramnios, membranes rupture for > 12 hours, and history of infertility), using oxytocin for labor induction, abnormal fetal heart rate pattern leading to cesarean delivery, occurrence of any problems during labor necessitating emergency cesarean section, administration of the analgesic methods before the study, and reluctancy to continue participation in the study. the first and the second intervention groups underwent acupressure at gb-21 and sp-6 acupoint (figures 1 and 2), respectively, at 3- to 4-cm cervical dilation. in the control group, the acupoints were only touched, but were not pressed in order to control the psychologic and supportive effects. sp-6 acupoint is located four woman s fingers width above the tip of the medial malleolus (the shinbone inside the ankle). in addition, gb-21 acupoint is located in the middle of an imaginary line between the bony prominence of the neck and the top of the shoulder joint (the acromion process). in acupressure resources, this acupoint has been introduced to be effective in reduction of delivery pain (11). in both intervention groups, the researcher applied pressure by both hands at the beginning of the contractions at 3- to 4-cm dilation. in the first intervention group, gb-21 acupoint on the left shoulder was pressed by the right thumb and the gb-21 point on the right shoulder was pressed by the left thumb. in the second group, on the other hand, sp-6 point on the right leg was pressed by the left thumb and the sp-6 point on the left leg was pressed by the right thumb. after applying pressure for 30 seconds, the researcher took a rest for 30 seconds but the hands were still in touch with the acupoints. this process was continued for 20 minutes. in the control group, however, the acupoints were only touched. in this group, gb-21 acupoint was touched in 25 patients and sp-6 acupoint was touched in the rest. the researcher s hands pressure was measured using a digital scale in order to apply a fixed amount of pressure throughout the intervention. after learning how to apply pressure, right and left thumbs amounts of pressure were calculated at 171 and 1350 mm hg, respectively. in order to make sure about the amount of pressure, it was computed using the following formula : where p is the amount of pressure (mmhg), f stands for force (kg), and a is the area of the thumb (m2). the difference in the amount of applied pressure was reduced to the least possible by repetition and practice. the study data were gathered using demographic information form, and spielberger s anxiety questionnaire (saq). the validity of demographic information form was assessed using content validity. besides, concurrent validity was used to determine the correct location of gb-21 and sp-6 acupoints as well as the method of pressure application. before and one hour after the intervention, anxiety was assessed through saq, which includes two sections, namely, state and trait anxiety. in this study, we only used the state anxiety section, which assesses the transient emotions associated with specific occasions. each questionnaire item included four choices, which were very low, low, high, and very high with respective scores of one to four. saq scale has been widely used in previous studies (23, 24). in a study on 150 patients undergoing surgery, aghamohammadi. reported the reliability coefficient of this instrument to be 0.97 (25). mother s attachment behaviors were evaluated using avant s observation form (aof) during the first breastfeeding and without her awareness. aof consists of a checklist including emotional behaviors (looking, fondling, kissing, talking, smiling, and moving the cradle), proximity behaviors (hugging without touching mother s body, hugging close to mother s body, and hugging by wrapping arms around the baby), and care behaviors (changing diapers, tapping the baby for gastric outlet, and tidying baby s clothes). the researcher observed the behaviors for 30 seconds and recorded them in the next 30 seconds. therefore, one behavior could occur for a maximum of 15 times during 15 minutes. since a total of 11 behaviors were observed for 15 minutes, the maximum score of each study participant would be 165. then mean and standard deviation of the behaviors were calculated. the content validity of this instrument was confirmed in a study by khoramrody. moreover, its reliability coefficient was calculated as 0.85 (27). the collected data were encoded and introduced to the spss (version 13, spss inc., comparisons of the variables before and after the intervention were made using paired - samples t test. independent samples t test was used in order to compare two groups at each stage. this study was approved by the research ethics committee of fasa university of medical sciences. in addition, written informed consent was obtained from each participant before beginning of the study. the researchers tried to observe all the participants rights in accordance with declaration of helsinki. this randomized controlled clinical trial was conducted on 150 primiparous women with term pregnancy who were referred to the obstetrics wards of shoushtari and hafez hospitals, shiraz, iran, in 2010. the participants with inclusion criteria were consecutively recruited into the study and were allocated into the three groups (a : gb-21 acupoint, b : acupressure at sp-6 acupoint, and c : control group) using a block randomization method. in order to eliminate the psychologic effect of various interventions on the study results, only one type of intervention was performed each day. before the intervention, written informed consents were obtained from all the participants. moreover, the researcher learned the correct way of performing acupressure under the supervision of a specialist in this field. acupressure was applied in the semi - recumbent position on the left arm so that reduction of blood pressure would be prevented. according to the specialist s opinion and since meridian energy flow circulation cycle is approximately 20 minutes, acupressure was performed for 20 minutes (5). the inclusion criteria were being primiparous, being 18 to 35 years old, singleton pregnancy, gestational age of 37 to 41 weeks, cephalic presentation, being before or at the beginning of the active phase of labor (3- to 4-cm dilation), having middle- school or high- school degrees, and signing written informed consents. the exclusion criteria were any psychologic (e.g., psychosis or schizophrenia) or anatomic disorder (e.g., uterine disorders or pelvic stenosis), chronic diseases (e.g., cardiovascular diseases, pulmonary disorders, hypertension, and diabetes), skin disorders (e.g., eczema and superficial skin infections), high - risk pregnancy (e.g., pregnancy hypertension, polyhydramnios, oligohydramnios, membranes rupture for > 12 hours, and history of infertility), using oxytocin for labor induction, abnormal fetal heart rate pattern leading to cesarean delivery, occurrence of any problems during labor necessitating emergency cesarean section, administration of the analgesic methods before the study, and reluctancy to continue participation in the study. the first and the second intervention groups underwent acupressure at gb-21 and sp-6 acupoint (figures 1 and 2), respectively, at 3- to 4-cm cervical dilation. in the control group, the acupoints were only touched, but were not pressed in order to control the psychologic and supportive effects. sp-6 acupoint is located four woman s fingers width above the tip of the medial malleolus (the shinbone inside the ankle). in addition, gb-21 acupoint is located in the middle of an imaginary line between the bony prominence of the neck and the top of the shoulder joint (the acromion process). in acupressure resources, this acupoint has been introduced to be effective in reduction of delivery pain (11). in both intervention groups, the researcher applied pressure by both hands at the beginning of the contractions at 3- to 4-cm dilation. in the first intervention group, gb-21 acupoint on the left shoulder was pressed by the right thumb and the gb-21 point on the right shoulder was pressed by the left thumb. in the second group, on the other hand, sp-6 point on the right leg was pressed by the left thumb and the sp-6 point on the left leg was pressed by the right thumb. after applying pressure for 30 seconds, the researcher took a rest for 30 seconds but the hands were still in touch with the acupoints., gb-21 acupoint was touched in 25 patients and sp-6 acupoint was touched in the rest. the researcher s hands pressure was measured using a digital scale in order to apply a fixed amount of pressure throughout the intervention. after learning how to apply pressure, right and left thumbs amounts of pressure were calculated at 171 and 1350 mm hg, respectively. in order to make sure about the amount of pressure, it was computed using the following formula : where p is the amount of pressure (mmhg), f stands for force (kg), and a is the area of the thumb (m2). the difference in the amount of applied pressure was reduced to the least possible by repetition and practice. the study data were gathered using demographic information form, and spielberger s anxiety questionnaire (saq). the validity of demographic information form was assessed using content validity. besides, concurrent validity was used to determine the correct location of gb-21 and sp-6 acupoints as well as the method of pressure application. before and one hour after the intervention, anxiety was assessed through saq, which includes two sections, namely, state and trait anxiety. in this study, we only used the state anxiety section, which assesses the transient emotions associated with specific occasions. each questionnaire item included four choices, which were very low, low, high, and very high with respective scores of one to four. saq scale has been widely used in previous studies (23, 24). in a study on 150 patients undergoing surgery, aghamohammadi. mother s attachment behaviors were evaluated using avant s observation form (aof) during the first breastfeeding and without her awareness. aof consists of a checklist including emotional behaviors (looking, fondling, kissing, talking, smiling, and moving the cradle), proximity behaviors (hugging without touching mother s body, hugging close to mother s body, and hugging by wrapping arms around the baby), and care behaviors (changing diapers, tapping the baby for gastric outlet, and tidying baby s clothes). the researcher observed the behaviors for 30 seconds and recorded them in the next 30 seconds. therefore, one behavior could occur for a maximum of 15 times during 15 minutes. since a total of 11 behaviors were observed for 15 minutes, the maximum score of each study participant would be 165. then mean and standard deviation of the behaviors were calculated. the collected data were encoded and introduced to the spss (version 13, spss inc., comparisons of the variables before and after the intervention were made using paired - samples t test. independent samples t test was used in order to compare two groups at each stage. this study was approved by the research ethics committee of fasa university of medical sciences. in addition, written informed consent was obtained from each participant before beginning of the study. the researchers tried to observe all the participants rights in accordance with declaration of helsinki. the study results revealed no significant difference among the three groups regarding gestational age (range, 37 - 42 weeks) and level of education (p > 0.05). the mean age of the participants was 25.88 3.47, 24.86 3.90, and 25.02 4.65 years in the gb-21, sp-6, and in the control groups, respectively, and the difference was not statistically significant. in addition, the mean gestational age was 38.26 0.82, 38.68 0.74, and 38.52 0.88 weeks in the acupressure at gb-21, sp-6, and the control group, respectively (p 0.05). maternal - fetal attachment score was higher in the intervention groups in comparison to the control group (p < 0.001). the mean score of the attachment behaviors was 85.1 9.9, 84.2 9.2, and 65.8 11.5 in the gb-21, sp-6, and in the control group, respectively, and the difference was statistically significant (p < 0.001). nevertheless, no significant difference was observed between the two intervention groups (p = 0.65). the means scores of the three types of behaviors were also separately calculated for the study groups. the highest and lowest means in the three groups were related to proximity and care behaviors, respectively (table 3). the results of this study showed that the anxiety level decreased in the two intervention groups, but increased in the control group. showed that acupressure while transferring the patients to hospital was effective in reducing anxiety level (28). moreover, fassoulaki. indicated that acupressure was effective in reducing anxiety and elevating mood (29). in addition, wang. investigated the effect of acupressure on anxiety and reported that acupressure had reduced anxiety level of the intervention group (20). in agreement with aforementioned studies, barker. demonstrated that acupressure reduced the patients anxiety level (30) the mechanism of acupressure has not been identified up to now ; however, the type of used touch and pressure seems to have a considerable effect beyond a simple touch. acupressure causes physiologic, systemic, and local changes in the body and creates calmness and balance throughout body and mind. although the main mechanism of acupressure has not been known yet, several studies have shown that this method leads to release of neurotransmitters such as serotonin, which can affect individuals tranquility (31, 32). some other studies have indicated that acupressure results in release of specific peptides with analgesic effects and reduces the activity of the sympathoadrenal system, which is activated in stressful conditions (33). furthermore, lewith. believed that the interventions used in the studies on complementary medicine could change the rate of fear, anxiety, bronchoconstriction, heartbeat, blood pressure, and skin temperature. the reduction of anxiety might be due to the effectiveness of the intervention, self - hypnosis, or even the feeling of security resulting from the presence of the researcher as one of the healthcare staff (34). to date, acupuncture and acupressure have attracted considerable attention as nonpharmacologic methods and world health organization has confirmed application of these two methods in 100 conditions. when these methods are performed accurately, they can even be performed by the individuals themselves, do not have high costs, and do not require any special facilities (35). up to this date, nurses and midwives have investigated various interventions on the effect of acupressure on the different problems and have employed various interventions to prevent anxiety disorders (36). the findings of the present study showed that acupressure could enhance maternal - fetal attachment after delivery. in addition, the results revealed a significant difference between the intervention groups and the control group regarding the attachment behaviors. this difference might result from reduction of anxiety and mother s feeling of tranquility and health after delivery. thus, maternal - fetal attachment might have increased due to the effect of acupressure on anxiety. in fact, acupressure leads to release of endogenous opioids that eventually result in reduction of anxiety (33). in general, performance of various interventions during pregnancy and delivery can be effective in reduction of anxiety and increase of attachment after delivery. complementary medicine interventions were shown to decrease anxiety and enhance maternal - fetal attachment after delivery in the studies by kim and cho, griffith., and bellieni. the findings of the current study demonstrated that acupressure as a simple and noninvasive method was effective in reducing the pain and anxiety and could be easily used in labor rooms. reduction of pain, in turn, increases maternal - fetal attachment. regarding no significant difference between the two acupoints in reducing pain intensity one of the limitations of this study was the frequent vaginal examinations in the labor room by residents and medical students. in addition, the study might have low external validity and generalizability due to its large number of inclusion and exclusion criteria. nevertheless, the strong points of the study were the large number of samples and its double - blind design.	background : delivery is one of the most stressful events in women s life. excessive anxiety, in turn, increases delivery and pregnancy complications. mother s positive experience of delivery leads to more effective maternal - fetal attachment in the first few hours of birth.objectives:the present study aimed to compare the effects of acupressure at two different acupoints on anxiety level and maternal - fetal attachment in primiparous women.materials and methods : in this study, 150 primiparous women were allocated to acupressure at gb-21 acupoint, acupressure at sp-6 acupoint, and control group. the women in their active phase of delivery were enrolled in the study and pressure was applied to the acupoints for 20 minutes. mother s anxiety level was assessed using spielberger s questionnaire before and one hour after the intervention. in addition, maternal - fetal attachment behaviors were evaluated using avant s questionnaire during the first breastfeeding. then the data were introduced to the spss (v. 13) and were analyzed using t test and one way anova.results:the results revealed no significant difference among the three groups regarding the anxiety level before the intervention (p > 0.05). one hour after the intervention, this measure was significantly lower in the intervention groups in comparison to the control group (p 0.05). moreover, maternal - fetal attachment was higher in the intervention groups in comparison with the control group (p < 0.001).conclusions : acupressure at both acupoints reduced anxiety level and increased maternal - fetal attachment. this method can be easily used in the delivery room.
all patients provided written informed consent according to german law as confirmed by the local committees (1442008 and 837.211.12 - 8312-f). glioblastoma tissue not required for neuropathological diagnostic procedures was obtained after surgical resection at the department of neurosurgery (leipzig or mainz). an overview of the samples used in this study is given in table 1. the tissue was transported to the laboratory in minimal essential medium (mem ; invitrogen). slice cultures were prepared using a vibratome (leica vt 1000) or a tissue chopper (mcilwain tc752) at a thickness of 350 m under sterile conditions. before preparations, a standard razor blade was wiped with ethanol to remove any oil and then sterilized by autoclaving. additionally, a normal glass pipette and a pipette with the fine tip broken off were autoclaved. if needed, biopsy specimens were cut into appropriate - size pieces first to obtain evenly shaped slices of 5 5 mm. when the tissue chopper was used, the tissue was put on a stack of sterile filter membranes, cut, and then transferred carefully into ice - cold mem by forceps. in most of the preparations, the slices stuck together after cutting, so they were separated under a stereomicroscope with 2 scalpels without cutting into the tissue. slices were then transferred by the glass pipette with the wide opening onto membrane culture inserts (millipore) in 6-well plates at a maximum of 34 slices per insert depending on size. the cultivation medium consisted of mem (gibco), 25% hank 's balanced salt solution (with ca and mg ; gibco), 25% n - hydroxysuccinimide (gibco), 1% l - glutamine (braun), 1% glucose (stock solution 45%, final concentration 0.45% ; braun), and 1% penicillin / streptomycin (sigma). slices were cultivated on a liquid / air interface in a humidified incubator at 37c and 5% co2. after time points ranging from 1 h to 4 weeks, slices were fixed in 4% paraformaldehyde and processed for paraffin embedding. paraffin sections (8 m) were cut and stained with hematoxylin and eosin (h&e) for histology. histology of these sections was compared with the diagnostic histopathology of the same tumor to detect tissue culture induced changes. for immunocytochemistry, sections were dewaxed in xylene, rehydrated in a decreasing alcohol series, and (if needed) pretreated for antibody staining with citrate buffer (ph 6) in a microwave. then, sections were washed in phosphate buffered saline (pbs), permeabilized with 1.5% triton / pbs for 10 min, blocked with 10% normal goat serum in 1.5% triton / pbs for 1 h, and incubated overnight at 4c with primary antibodies against ki67 (rabbit, 1:100 ; dcs), cleaved caspase 3 (rabbit, 1:400 ; cell signaling), glial fibrillary acidic protein (gfap ; dako, rabbit, 1:600 ; dako), nestin (rabbit, 1:600 ; chemicon), vimentin (mouse, 1:100 ; dako), neurofilament (mouse, 1:100 ; dako), or h2ax (mouse, 1:100 ; millipore). visualization was achieved by incubation either with appropriate fluorescent - labeled secondary antibodies (goat anti - mouse or anti - rabbit alexa 488) or with biotinylated anti - rabbit immunoglobulin g followed by streptavidin - conjugated horseradish peroxidase and developing by adding diaminobenzidine for the color reaction. for fluorescent staining, photographs were taken using an olympus bx51 fluorescent microscope or a zeiss lsm 510 confocal microscope. in addition to the green fluorescent channel, the red channel was included because some of the samples exhibited a strong autofluorescence, which is normal in the nonjuvenile human brain. only cells devoid of red fluorescence were used for further analysis. for h&e and diaminobenzidine staining, a zeiss axioplan 2 microscope was used. table 1.glioblastoma samples used for slice culture experiments in this studysampleexperimentdata shown in03082010long - term culturefig. 112082010long - term culturefig. 125072012long - term culture and x - irradiationfigs. 1 and 312122011tmz treatmentfigs 505042011carbon ion irradiation and tmz treatmentfigs. 3, 8, 4, and 611102011carbon ion irradiation and tmz treatmentfigs. glioblastoma samples used for slice culture experiments in this study all patients had a diagnosis of world health organization grade iv gbm. paraffin sections (8 m) of slices were dewaxed as previously described here, and proliferating cells were stained with the ki67 antibody., at least 12 images were acquired of 46 different sections per group and manually analyzed using imagej and the plugin cellcounter. the percentage of ki67-positive cells in relation to the total cell number was referred to as the proliferation index. glioblastoma tissue slices maintained on cell culture inserts were incubated with tmz (dissolved in dimethyl sulfoxide) at a final concentration of 50 or 200 m of the compound. control slices were incubated with the corresponding amount of dimethyl sulfoxide (0.2% v / v). after 72 h, tissue slices were removed from the membranes and incubated in 500 l of medium and 10 g / ml of hoechst 33342 (sigma) at 37c and 5% co2 for 1 h to allow for nuclear staining. the tissues were then transferred into 1 ml pbs containing 2 g / ml propidium iodide (pi ; invitrogen) and gently fixated between 2 glass coverslips. for confocal imaging and quantification of viable and dead cells within the tissues, an lsm-510 meta inverted laser scanning microscope and a 20/0.8 plan - apochromat objective (carl zeiss microimaging) were used. viable cells with hoechst - positive and pi - negative nuclei, and dead cells with hoechst - positive and pi - positive nuclei, were counted in 2 or 3 images of each tissue slice per group. one - way anova was used, and p <.05 was considered to be statistically significant. photon irradiation of slices was performed at the department for radiation therapy and radio - oncology, university of leipzig, with a 150-kv x - ray unit (darpac 150-mc) with an energy of 13.2 ma and a dose rate of 0.86 gy / min. cell culture plates were placed under a specially constructed plate device and irradiated until the desired dose was reached. alternatively, photon irradiation was performed using the gsi x - ray device (ge isovolt titan 320, 250 kv, 16 ma) at a dose rate of 1.4 gy / min. hi irradiation with a carbon beam was performed at gsi (gesellschaft fr schwerionenforschung), darmstadt, at the former patient irradiation site. the ion beam was generated at the sis18 synchrotron facility and delivered in a spread - out bragg peak (sobp) as used in carbon ion therapy. the dose applied to the slices was 2 or 4 gy in a 50-mm - width sobp corresponding to a linear energy transfer range of 5070 kev/m. with this method, then, the ion beam is directed at the 3-dimensional tumor volume, using active energy variation and the raster scanning technique. for experiments with slice cultures, the volume was defined as the area and the height of 1 well. before and after irradiation, slice cultures were kept in an incubator as previously described here and were removed for only about 15 min for transport and to place them on the irradiation belt. after irradiation, slices were fixed in 4% paraformaldehyde after one of several time points, washed in pbs, and further processed for paraffin embedding or cryosectioning. paraffin sections were prepared at 8 m, dried, and stored at room temperature. for staining of dna dsbs, cryosections were dried for 20 min at room temperature and then washed twice in pbs and incubated with 1.5% triton / pbs for 10 min. then sections were blocked with 10% normal goat serum in 1.5% triton / pbs for at least 1 h, followed by incubation overnight at 4c with h2ax primary antibody (mouse monoclonal, 1:100 ; millipore). then, sections were washed 3 times with pbs and incubated with the secondary antibody (goat anti - mouse 1:1000 ; alexa 488, invitrogen) for 1 h, washed again, counterstained with hoechst 33342, and mounted with dako fluorescent mounting medium. z - stacks were taken using a zeiss lsm 510 confocal microscope at 400 magnification at intervals of 2 m. all patients provided written informed consent according to german law as confirmed by the local committees (1442008 and 837.211.12 - 8312-f). glioblastoma tissue not required for neuropathological diagnostic procedures was obtained after surgical resection at the department of neurosurgery (leipzig or mainz). an overview of the samples used in this study is given in table 1. the tissue was transported to the laboratory in minimal essential medium (mem ; invitrogen). slice cultures were prepared using a vibratome (leica vt 1000) or a tissue chopper (mcilwain tc752) at a thickness of 350 m under sterile conditions. before preparations, a standard razor blade was wiped with ethanol to remove any oil and then sterilized by autoclaving. additionally, a normal glass pipette and a pipette with the fine tip broken off were autoclaved. if needed, biopsy specimens were cut into appropriate - size pieces first to obtain evenly shaped slices of 5 5 mm. when the tissue chopper was used, the tissue was put on a stack of sterile filter membranes, cut, and then transferred carefully into ice - cold mem by forceps. in most of the preparations, the slices stuck together after cutting, so they were separated under a stereomicroscope with 2 scalpels without cutting into the tissue. slices were then transferred by the glass pipette with the wide opening onto membrane culture inserts (millipore) in 6-well plates at a maximum of 34 slices per insert depending on size. the cultivation medium consisted of mem (gibco), 25% hank 's balanced salt solution (with ca and mg ; gibco), 25% n - hydroxysuccinimide (gibco), 1% l - glutamine (braun), 1% glucose (stock solution 45%, final concentration 0.45% ; braun), and 1% penicillin / streptomycin (sigma). slices were cultivated on a liquid / air interface in a humidified incubator at 37c and 5% co2. after time points ranging from 1 h to 4 weeks, slices were fixed in 4% paraformaldehyde and processed for paraffin embedding. paraffin sections (8 m) were cut and stained with hematoxylin and eosin (h&e) for histology. histology of these sections was compared with the diagnostic histopathology of the same tumor to detect tissue culture induced changes. for immunocytochemistry, sections were dewaxed in xylene, rehydrated in a decreasing alcohol series, and (if needed) pretreated for antibody staining with citrate buffer (ph 6) in a microwave. then, sections were washed in phosphate buffered saline (pbs), permeabilized with 1.5% triton / pbs for 10 min, blocked with 10% normal goat serum in 1.5% triton / pbs for 1 h, and incubated overnight at 4c with primary antibodies against ki67 (rabbit, 1:100 ; dcs), cleaved caspase 3 (rabbit, 1:400 ; cell signaling), glial fibrillary acidic protein (gfap ; dako, rabbit, 1:600 ; dako), nestin (rabbit, 1:600 ; chemicon), vimentin (mouse, 1:100 ; dako), neurofilament (mouse, 1:100 ; dako), or h2ax (mouse, 1:100 ; millipore). visualization was achieved by incubation either with appropriate fluorescent - labeled secondary antibodies (goat anti - mouse or anti - rabbit alexa 488) or with biotinylated anti - rabbit immunoglobulin g followed by streptavidin - conjugated horseradish peroxidase and developing by adding diaminobenzidine for the color reaction. for fluorescent staining, photographs were taken using an olympus bx51 fluorescent microscope or a zeiss lsm 510 confocal microscope. in addition to the green fluorescent channel, the red channel was included because some of the samples exhibited a strong autofluorescence, which is normal in the nonjuvenile human brain. only cells devoid of red fluorescence were used for further analysis. for h&e and diaminobenzidine staining, a zeiss axioplan 2 microscope was used. table 1.glioblastoma samples used for slice culture experiments in this studysampleexperimentdata shown in03082010long - term culturefig. 112082010long - term culturefig. 125072012long - term culture and x - irradiationfigs. 1 and 312122011tmz treatmentfigs 505042011carbon ion irradiation and tmz treatmentfigs. 3, 8, 4, and 611102011carbon ion irradiation and tmz treatmentfigs. glioblastoma samples used for slice culture experiments in this study all patients had a diagnosis of world health organization grade iv gbm. paraffin sections (8 m) of slices were dewaxed as previously described here, and proliferating cells were stained with the ki67 antibody., at least 12 images were acquired of 46 different sections per group and manually analyzed using imagej and the plugin cellcounter. the percentage of ki67-positive cells in relation to the total cell number was referred to as the proliferation index. glioblastoma tissue slices maintained on cell culture inserts were incubated with tmz (dissolved in dimethyl sulfoxide) at a final concentration of 50 or 200 m of the compound. control slices were incubated with the corresponding amount of dimethyl sulfoxide (0.2% v / v). after 72 h, tissue slices were removed from the membranes and incubated in 500 l of medium and 10 g / ml of hoechst 33342 (sigma) at 37c and 5% co2 for 1 h to allow for nuclear staining. the tissues were then transferred into 1 ml pbs containing 2 g / ml propidium iodide (pi ; invitrogen) and gently fixated between 2 glass coverslips. for confocal imaging and quantification of viable and dead cells within the tissues, an lsm-510 meta inverted laser scanning microscope and a 20/0.8 plan - apochromat objective (carl zeiss microimaging) were used. pi was excited with the 543-nm laser line. viable cells with hoechst - positive and pi - negative nuclei, and dead cells with hoechst - positive and pi - positive nuclei, were counted in 2 or 3 images of each tissue slice per group. one - way anova was used, and p <.05 was considered to be statistically significant. photon irradiation of slices was performed at the department for radiation therapy and radio - oncology, university of leipzig, with a 150-kv x - ray unit (darpac 150-mc) with an energy of 13.2 ma and a dose rate of 0.86 gy / min. cell culture plates were placed under a specially constructed plate device and irradiated until the desired dose was reached. alternatively, photon irradiation was performed using the gsi x - ray device (ge isovolt titan 320, 250 kv, 16 ma) at a dose rate of 1.4 gy / min. hi irradiation with a carbon beam was performed at gsi (gesellschaft fr schwerionenforschung), darmstadt, at the former patient irradiation site. the ion beam was generated at the sis18 synchrotron facility and delivered in a spread - out bragg peak (sobp) as used in carbon ion therapy. the dose applied to the slices was 2 or 4 gy in a 50-mm - width sobp corresponding to a linear energy transfer range of 5070 kev/m. with this method, then, the ion beam is directed at the 3-dimensional tumor volume, using active energy variation and the raster scanning technique. for experiments with slice cultures, the volume was defined as the area and the height of 1 well. before and after irradiation, slice cultures were kept in an incubator as previously described here and were removed for only about 15 min for transport and to place them on the irradiation belt. after irradiation, slices were fixed in 4% paraformaldehyde after one of several time points, washed in pbs, and further processed for paraffin embedding or cryosectioning. paraffin sections were prepared at 8 m, dried, and stored at room temperature. for staining of dna dsbs, cryosections were dried for 20 min at room temperature and then washed twice in pbs and incubated with 1.5% triton / pbs for 10 min. then sections were blocked with 10% normal goat serum in 1.5% triton / pbs for at least 1 h, followed by incubation overnight at 4c with h2ax primary antibody (mouse monoclonal, 1:100 ; millipore). then, sections were washed 3 times with pbs and incubated with the secondary antibody (goat anti - mouse 1:1000 ; alexa 488, invitrogen) for 1 h, washed again, counterstained with hoechst 33342, and mounted with dako fluorescent mounting medium. z - stacks were taken using a zeiss lsm 510 confocal microscope at 400 magnification at intervals of 2 m. slices were at first cut with a vibratome and survived well, with histological preservation of the main features of the original tumor for at least 16 days (fig. 1). at later stages, cell density appeared to decline in some tumor slices, whereas cells in other slices survived longer (fig., however, were difficult or even impossible to cut due to their viscous texture, which may have resulted from altered collagen expression. using a tissue chopper resolved this problem with equally good histological preservation and maximal survival time. histological examination of cultured gbm slices and comparison with the original neuropathology used for diagnosis confirmed striking maintenance of the general and individual hallmarks of the tumor (pleomorphic nuclei, palisading, invading vessels / neovascularization, and necrotic areas). as expected, gbm stained positive for gfap, nestin (intermediate filament protein, gbm stem cell marker), and vimentin (mesenchymal intermediate filament protein) and differed strongly in their cell density and content of necrotic areas (fig. 2). slices were cultured on membrane inserts in six - well plates with no signs of degeneration in acute (a) slices at 1 day or at 3 days (b), 6 days (c), or 12 days original h&e neuropathology (e and i) and h&e - stained paraffin - embedded sections (8 m ; f h and j l) prepared from slices after various culture periods. two different tumors (e h and i note that typical features of individual tumors were maintained at least from 1 to 16 days (f g) and 1 to 13 days (j k) in vitro ; massive cell loss was observed after 20 days in vitro (h and l). slices were fixed after 7 days in culture and processed for paraffin sectioning (8 m). characteristic marker proteins were visualized by antibody staining for gfap (a), nestin (b), vimentin (c), and neurofilament (d). slices were cultured on membrane inserts in six - well plates with no signs of degeneration in acute (a) slices at 1 day or at 3 days (b), 6 days (c), or 12 days (d) in vitro. original h&e neuropathology (e and i) and h&e - stained paraffin - embedded sections (8 m ; f h and j l) prepared from slices after various culture periods. two different tumors (e h and i note that typical features of individual tumors were maintained at least from 1 to 16 days (f g) and 1 to 13 days (j k) in vitro ; massive cell loss was observed after 20 days in vitro (h and l). slices were fixed after 7 days in culture and processed for paraffin sectioning (8 m). characteristic marker proteins were visualized by antibody staining for gfap (a), nestin (b), vimentin (c), and neurofilament (d). if dna repair can not be conducted properly, cell proliferation is arrested at a cell cycle checkpoint and either is inactivated (eg, in postmitotic cells) or proceeds into programmed cell death. here, we tested the dose- and time - dependent decrease of the proliferation index after sobp carbon or x - irradiation. gbm slices were fixed 6 or 24 h after irradiation and processed for paraffin sectioning. 3a d), which marks cells in every state of the cell cycle except the g0 resting phase. the percentage of ki67-positive cells in relation to the total number of nuclei was calculated to express the proliferation index. carbon ions did not significantly diminish proliferation at 6 h, but after 24 h a reduction of 40% was found (p =.018 ; fig. this is the first demonstration of specific effects of hi on human gbm tissue ex vivo. photons also showed the anticipated time - dependent reduction of proliferation (here, 50% after 24 h ; fig. thus, gbm - derived slice cultures can be irradiated and the biological effects of photons and hi on tumor cells and mechanisms of resistance can be studied in this model. 3.proliferation index of human gbm slice cultures irradiated with x - rays or carbon ions. slices were treated with either 4 gy of sobp carbon ions at gsi or 4 gy of x - rays and fixed 6 or 24 h later. proliferating cells were then visualized in paraffin - embedded sections (8 m) using a ki67 antibody (green) combined with nuclear counterstaining (hoechst 33342 ; blue) for quantitative analysis. (a), non - irradiated ; (b), irradiated with 4 gy carbon ions ; (c), non - irradiated ; (d), irradiated with 4 gy x - rays ; scale bar = 50 m). the proliferative fraction in relation to total cell number was determined in at least 12 pictures per group by using imagej software. after 6 h, the effect on proliferation was not yet significant after both irradiation types, but after 24 h both treatments resulted in a significant decrease of ki67-positive cells (e and f). proliferation index of human gbm slice cultures irradiated with x - rays or carbon ions. slices were treated with either 4 gy of sobp carbon ions at gsi or 4 gy of x - rays and fixed 6 or 24 h later. proliferating cells were then visualized in paraffin - embedded sections (8 m) using a ki67 antibody (green) combined with nuclear counterstaining (hoechst 33342 ; blue) for quantitative analysis. (a), non - irradiated ; (b), irradiated with 4 gy carbon ions ; (c), non - irradiated ; (d), irradiated with 4 gy x - rays ; scale bar = 50 m). the proliferative fraction in relation to total cell number was determined in at least 12 pictures per group by using imagej software. after 6 h, the effect on proliferation was not yet significant after both irradiation types, but after 24 h both treatments resulted in a significant decrease of ki67-positive cells (e and f). after induction of dna dsbs by ionizing radiation, repair proteins are rapidly recruited to the sites of damage. in mammalian cells, this involves a cascade of proteins that ultimately allows repair of the breaks in the form of rejoining the loose dna ends. when we established the irradiation setup of gbm slice cultures, we wanted to test whether the slices were evenly hit by the beam, and therefore we used h2ax as an early dsb marker for the visualization of dna damage. h2ax describes a phosphorylation of histone 2ax at serine 139 around the region of the dsb, which allows binding of further repair proteins, such as mdc1 and 53bp1. once the repair process is completed, the repair proteins dissociate and h2ax is dephosphorylated by a distinct phosphatase complex. slices were irradiated with 4 gy carbon ions in an sobp to match therapeutic conditions, fixed after 1 h, and embedded in paraffin, and sections were stained with an antibody to h2ax. h2ax was mostly absent in controls but appeared in a punctuated nuclear pattern in all sections, confirming induction of dna damage throughout the irradiated tissues (fig. 4). slices were irradiated with 4 gy of carbon ions in an sobp and fixed 1 h later. paraffin sections (8 m) were assembled and dna dsbs were visualized by immunocytochemistry with a h2ax antibody (green) and nuclei with hoechst (blue). although h2ax was rarely detected in non - irradiated controls (a), exposure to therapeutic heavy ions caused massive induction of phosphorylation of h2ax (b). slices were irradiated with 4 gy of carbon ions in an sobp and fixed 1 h later. paraffin sections (8 m) were assembled and dna dsbs were visualized by immunocytochemistry with a h2ax antibody (green) and nuclei with hoechst (blue). although h2ax was rarely detected in non - irradiated controls (a), exposure to therapeutic heavy ions caused massive induction of phosphorylation of h2ax (b). it is an alkylating agent that, in an aqueous solution at physiological ph, dissolves into its bioactive form mtic (5-(3-methyl-1-triazeno)imidazole-4-carboxamide), which is capable of penetrating the blood whether gbm tissue in culture responds to tmz treatment, slices were incubated with vehicle control or tmz (50 or 200 m). after 72 or 96 h, hoechst 33342 and pi were added to the cultures to visualize intact and dying nuclei using confocal live imaging. microimages were taken in which dying cells (hoechst / pi double labeled) were counted and their number related to the total number of nuclei (hoechst single labeled), allowing calculation of a cell death rate. tmz - induced cell death was highly significant at 72 h, with little or no further increase until 96 h. this effect was more pronounced in slices treated with 200 m compared with 50 m of tmz (fig. thus, chemotherapeutic effects can be mimicked in gbm slices and observed over time in situ. slices were treated with either 50 or 200 m tmz and then incubated for 72 or 96 h (a). after determination that an incubation of 72 h was sufficient, the experiment was repeated 3 times with 50 or 200 m tmz (b). dying cells were labeled with pi (red), and all nuclei were counterstained with hoechst 33342 (blue) for quantitative analysis (c). grid distance= student 's t - test was performed, and p <.05 was considered statistically significant. both concentrations showed a significant increase in dying cells compared with controls, with the effect slightly more pronounced at 200 m. slices were treated with either 50 or 200 m tmz and then incubated for 72 or 96 h (a). after determination that an incubation of 72 h was sufficient, the experiment was repeated 3 times with 50 or 200 m tmz (b). dying cells were labeled with pi (red), and all nuclei were counterstained with hoechst 33342 (blue) for quantitative analysis (c). grid distance= student 's t - test was performed, and p <.05 was considered statistically significant. both concentrations showed a significant increase in dying cells compared with controls, with the effect slightly more pronounced at 200 m. slices were exposed to either tmz or carbon ions in an sobp alone or in combination and fixed 48 h after irradiation. tmz treatment was started 24 h before irradiation, and a second dose was applied with the regular change of medium 48 h later. at the end of the treatment phase, induction of programmed cell death was determined using cleaved caspase 3 and h&e staining. all treatment regimens caused a decrease in cell density and nuclear alterations (fig. therefore, instead of relating damaged cells to a total number of cell nuclei, the area coverage of caspase 3positive (green) and hoechst - positive (blue) pixels (fig. 6e, caspase 3positive cells ; 6h, nuclear fragments) was calculated as a measure of treatment - induced damage. after all treatments, the area of caspase 3positive pixels was significantly increased, whereas hoechst - positive pixels were decreased (fig. a combination of both treatments did not show a synergistic or additive effect in the cases studied. irradiation with x - rays, however, also activated caspase 3 but did not cause significant cell loss (fig. thus, effects of established treatment options on cell death can be studied in gbm slices. 6.combined treatment of tmz and irradiation with carbon ions and x - rays on human gbm slice cultures. gbm slices were treated with tmz (200 m), x - ray irradiation (4 gy), sobp carbon ions (2 gy), or irradiation + tmz. tmz treatment started 24 h before irradiation and was maintained throughout the entire incubation time. slices were fixed 2 days after irradiation, and h&e staining was performed for neuropathological assessment (a d). in addition, activated caspase 3 was labeled (green in e), and fragmented nuclei were visualized using hoechst 33342 (blue in e and h). pictures of immunofluorescent stainings (in rgb format) were split into the three single channels of red, green, and blue, resulting in 3 gray - value pictures. the area coverage is represented by white pixels in the respective channels and can be fine - tuned by threshold adjusting. in the green channel, the positive area represents the caspase 3positive cell population, and in the blue channel, the area covered by nuclei is displayed (e j). carbon significantly reduced cell numbers (g), whereas x - rays did not (j). both treatments result in a significant increase in cell death and morphological alterations compared with the vehicle - treated control. combined treatment of tmz and irradiation with carbon ions and x - rays on human gbm slice cultures. gbm slices were treated with tmz (200 m), x - ray irradiation (4 gy), sobp carbon ions (2 gy), or irradiation + tmz. tmz treatment started 24 h before irradiation and was maintained throughout the entire incubation time. slices were fixed 2 days after irradiation, and h&e staining was performed for neuropathological assessment (a d). in addition, activated caspase 3 was labeled (green in e), and fragmented nuclei were visualized using hoechst 33342 (blue in e and h). pictures of immunofluorescent stainings (in rgb format) were split into the three single channels of red, green, and blue, resulting in 3 gray - value pictures. the area coverage is represented by white pixels in the respective channels and can be fine - tuned by threshold adjusting. in the green channel, the positive area represents the caspase 3positive cell population, and in the blue channel, the area covered by nuclei is displayed (e j). carbon significantly reduced cell numbers (g), whereas x - rays did not (j). both treatments result in a significant increase in cell death and morphological alterations compared with the vehicle - treated control. original magnification : 400 in a f. lack of promoter methylation of o - methylguanine - dna methyltransferase (mgmt) has been reported to significantly decrease patients ' susceptibility to tmz and thus survival, but there are also patients with nonmethylated promoter who benefit from tmz treatment. in fact, we identified one tumor in which tmz did not significantly induce cell death and we therefore requested the promoter methylation status, which is assessed by quantitative pcr and sequencing techniques of tumor material obtained from surgery. however, in line with the clinical observations that some patients with nonmethylated mgmt respond to tmz, we subsequently identified other tumors in which tmz significantly enhanced activation of caspase 3 to levels comparable to those of the methylated tumor (fig. gbm slice cultures with different mgmt promoter methylation states were tested for their response to tmz treatment. in a gbm specimen with methylated promoter sequence (a), tmz treatment resulted in significant induction of caspase 3 cleavage (gbm 1011), whereas caspase 3 activation was not significantly induced (b) in gbm with an unmethylated promoter sequence (gbm 0611). immunocytochemistry is shown for the methylated (c, right) and unmethylated (c, left) tumor for cleaved caspase 3 (green) and nuclei (hoechst, blue). after tmz treatment, caspase 3 activation was detected in some specimens, whereas others seemed to be resistant. this was independent of mgmt promoter methylation. from left to right : gbm 1011 with methylated mgmt promoter (meth +) and significant caspase 3 activation ; gbm 0411 with nonmethylated mgmt promoter (meth -) and significant caspase 3 activation ; gbm 1211 with nonmethylated mgmt promoter (meth -) and significant caspase 3 activation ; gbm 0812 with nonmethylated mgmt promoter (meth -) and significant caspase 3 activation ; gbm 0611 with nonmethylated mgmt promoter (meth -) and nonsignificant (ns) caspase 3 activation ; gbm 0512 with nonmethylated mgmt promoter (meth -) and nonsignificant (ns) caspase 3 activation. gbm slice cultures with different mgmt promoter methylation states were tested for their response to tmz treatment. in a gbm specimen with methylated promoter sequence (a), tmz treatment resulted in significant induction of caspase 3 cleavage (gbm 1011), whereas caspase 3 activation was not significantly induced (b) in gbm with an unmethylated promoter sequence (gbm 0611). immunocytochemistry is shown for the methylated (c, right) and unmethylated (c, left) tumor for cleaved caspase 3 (green) and nuclei (hoechst, blue). after tmz treatment, caspase 3 activation was detected in some specimens, whereas others seemed to be resistant. this was independent of mgmt promoter methylation. from left to right : gbm 1011 with methylated mgmt promoter (meth +) and significant caspase 3 activation ; gbm 0411 with nonmethylated mgmt promoter (meth -) and significant caspase 3 activation ; gbm 1211 with nonmethylated mgmt promoter (meth -) and significant caspase 3 activation ; gbm 0812 with nonmethylated mgmt promoter (meth -) and significant caspase 3 activation ; gbm 0611 with nonmethylated mgmt promoter (meth -) and nonsignificant (ns) caspase 3 activation ; gbm 0512 with nonmethylated mgmt promoter (meth -) and nonsignificant (ns) caspase 3 activation. slices were at first cut with a vibratome and survived well, with histological preservation of the main features of the original tumor for at least 16 days (fig. 1). at later stages, cell density appeared to decline in some tumor slices, whereas cells in other slices survived longer (fig., however, were difficult or even impossible to cut due to their viscous texture, which may have resulted from altered collagen expression. using a tissue chopper resolved this problem with equally good histological preservation and maximal survival time. histological examination of cultured gbm slices and comparison with the original neuropathology used for diagnosis confirmed striking maintenance of the general and individual hallmarks of the tumor (pleomorphic nuclei, palisading, invading vessels / neovascularization, and necrotic areas). as expected, gbm stained positive for gfap, nestin (intermediate filament protein, gbm stem cell marker), and vimentin (mesenchymal intermediate filament protein) and differed strongly in their cell density and content of necrotic areas (fig. 2). slices were cultured on membrane inserts in six - well plates with no signs of degeneration in acute (a) slices at 1 day or at 3 days (b), 6 days (c), or 12 days original h&e neuropathology (e and i) and h&e - stained paraffin - embedded sections (8 m ; f h and j l) prepared from slices after various culture periods. two different tumors (e h and i note that typical features of individual tumors were maintained at least from 1 to 16 days (f g) and 1 to 13 days (j k) in vitro ; massive cell loss was observed after 20 days in vitro (h and l). slices were fixed after 7 days in culture and processed for paraffin sectioning (8 m). characteristic marker proteins were visualized by antibody staining for gfap (a), nestin (b), vimentin (c), and neurofilament (d). slices were cultured on membrane inserts in six - well plates with no signs of degeneration in acute (a) slices at 1 day or at 3 days (b), 6 days (c), or 12 days (d) in vitro. original h&e neuropathology (e and i) and h&e - stained paraffin - embedded sections (8 m ; f h and j l) prepared from slices after various culture periods. two different tumors (e h and i note that typical features of individual tumors were maintained at least from 1 to 16 days (f g) and 1 to 13 days (j k) in vitro ; massive cell loss was observed after 20 days in vitro (h and l). slices were fixed after 7 days in culture and processed for paraffin sectioning (8 m). characteristic marker proteins were visualized by antibody staining for gfap (a), nestin (b), vimentin (c), and neurofilament (d). if dna repair can not be conducted properly, cell proliferation is arrested at a cell cycle checkpoint and either is inactivated (eg, in postmitotic cells) or proceeds into programmed cell death. here, we tested the dose- and time - dependent decrease of the proliferation index after sobp carbon or x - irradiation. gbm slices were fixed 6 or 24 h after irradiation and processed for paraffin sectioning. 3a d), which marks cells in every state of the cell cycle except the g0 resting phase. the percentage of ki67-positive cells in relation to the total number of nuclei was calculated to express the proliferation index. carbon ions did not significantly diminish proliferation at 6 h, but after 24 h a reduction of 40% was found (p =.018 ; fig. this is the first demonstration of specific effects of hi on human gbm tissue ex vivo. photons also showed the anticipated time - dependent reduction of proliferation (here, 50% after 24 h ; fig. thus, gbm - derived slice cultures can be irradiated and the biological effects of photons and hi on tumor cells and mechanisms of resistance can be studied in this model. 3.proliferation index of human gbm slice cultures irradiated with x - rays or carbon ions. slices were treated with either 4 gy of sobp carbon ions at gsi or 4 gy of x - rays and fixed 6 or 24 h later. proliferating cells were then visualized in paraffin - embedded sections (8 m) using a ki67 antibody (green) combined with nuclear counterstaining (hoechst 33342 ; blue) for quantitative analysis. (a), non - irradiated ; (b), irradiated with 4 gy carbon ions ; (c), non - irradiated ; (d), irradiated with 4 gy x - rays ; scale bar = 50 m). the proliferative fraction in relation to total cell number was determined in at least 12 pictures per group by using imagej software. after 6 h, the effect on proliferation was not yet significant after both irradiation types, but after 24 h both treatments resulted in a significant decrease of ki67-positive cells (e and f). proliferation index of human gbm slice cultures irradiated with x - rays or carbon ions. slices were treated with either 4 gy of sobp carbon ions at gsi or 4 gy of x - rays and fixed 6 or 24 h later. proliferating cells were then visualized in paraffin - embedded sections (8 m) using a ki67 antibody (green) combined with nuclear counterstaining (hoechst 33342 ; blue) for quantitative analysis. (a), non - irradiated ; (b), irradiated with 4 gy carbon ions ; (c), non - irradiated ; (d), irradiated with 4 gy x - rays ; scale bar = 50 m). the proliferative fraction in relation to total cell number was determined in at least 12 pictures per group by using imagej software. after 6 h, the effect on proliferation was not yet significant after both irradiation types, but after 24 h both treatments resulted in a significant decrease of ki67-positive cells (e and f). after induction of dna dsbs by ionizing radiation, repair proteins are rapidly recruited to the sites of damage. in mammalian cells, this involves a cascade of proteins that ultimately allows repair of the breaks in the form of rejoining the loose dna ends. when we established the irradiation setup of gbm slice cultures, we wanted to test whether the slices were evenly hit by the beam, and therefore we used h2ax as an early dsb marker for the visualization of dna damage. h2ax describes a phosphorylation of histone 2ax at serine 139 around the region of the dsb, which allows binding of further repair proteins, such as mdc1 and 53bp1. once the repair process is completed, the repair proteins dissociate and h2ax is dephosphorylated by a distinct phosphatase complex. slices were irradiated with 4 gy carbon ions in an sobp to match therapeutic conditions, fixed after 1 h, and embedded in paraffin, and sections were stained with an antibody to h2ax. h2ax was mostly absent in controls but appeared in a punctuated nuclear pattern in all sections, confirming induction of dna damage throughout the irradiated tissues (fig. 4). slices were irradiated with 4 gy of carbon ions in an sobp and fixed 1 h later. paraffin sections (8 m) were assembled and dna dsbs were visualized by immunocytochemistry with a h2ax antibody (green) and nuclei with hoechst (blue). although h2ax was rarely detected in non - irradiated controls (a), exposure to therapeutic heavy ions caused massive induction of phosphorylation of h2ax (b). slices were irradiated with 4 gy of carbon ions in an sobp and fixed 1 h later. paraffin sections (8 m) were assembled and dna dsbs were visualized by immunocytochemistry with a h2ax antibody (green) and nuclei with hoechst (blue). although h2ax was rarely detected in non - irradiated controls (a), exposure to therapeutic heavy ions caused massive induction of phosphorylation of h2ax (b). it is an alkylating agent that, in an aqueous solution at physiological ph, dissolves into its bioactive form mtic (5-(3-methyl-1-triazeno)imidazole-4-carboxamide), which is capable of penetrating the blood brain barrier. to test whether gbm tissue in culture responds to tmz treatment, slices were incubated with vehicle control or tmz (50 or 200 m). after 72 or 96 h, hoechst 33342 and pi were added to the cultures to visualize intact and dying nuclei using confocal live imaging. microimages were taken in which dying cells (hoechst / pi double labeled) were counted and their number related to the total number of nuclei (hoechst single labeled), allowing calculation of a cell death rate. tmz - induced cell death was highly significant at 72 h, with little or no further increase until 96 h. this effect was more pronounced in slices treated with 200 m compared with 50 m of tmz (fig. thus, chemotherapeutic effects can be mimicked in gbm slices and observed over time in situ. slices were treated with either 50 or 200 m tmz and then incubated for 72 or 96 h (a). after determination that an incubation of 72 h was sufficient, the experiment was repeated 3 times with 50 or 200 m tmz (b). dying cells were labeled with pi (red), and all nuclei were counterstained with hoechst 33342 (blue) for quantitative analysis (c). grid distance= 50 m. student 's t - test was performed, and p <.05 was considered statistically significant. both concentrations showed a significant increase in dying cells compared with controls, with the effect slightly more pronounced at 200 m. slices were treated with either 50 or 200 m tmz and then incubated for 72 or 96 h (a). after determination that an incubation of 72 h was sufficient, the experiment was repeated 3 times with 50 or 200 m tmz (b). dying cells were labeled with pi (red), and all nuclei were counterstained with hoechst 33342 (blue) for quantitative analysis (c). grid distance= 50 m. student 's t - test was performed, and p <.05 was considered statistically significant. both concentrations showed a significant increase in dying cells compared with controls, with the effect slightly more pronounced at 200 m. slices were exposed to either tmz or carbon ions in an sobp alone or in combination and fixed 48 h after irradiation. tmz treatment was started 24 h before irradiation, and a second dose was applied with the regular change of medium 48 h later. at the end of the treatment phase, induction of programmed cell death was determined using cleaved caspase 3 and h&e staining. all treatment regimens caused a decrease in cell density and nuclear alterations (fig. therefore, instead of relating damaged cells to a total number of cell nuclei, the area coverage of caspase 3positive (green) and hoechst - positive (blue) pixels (fig. 6e, caspase 3positive cells ; 6h, nuclear fragments) was calculated as a measure of treatment - induced damage. after all treatments, the area of caspase 3positive pixels was significantly increased, whereas hoechst - positive pixels were decreased (fig. both irradiation and tmz alone induced cell death, but a combination of both treatments did not show a synergistic or additive effect in the cases studied. irradiation with x - rays, however, also activated caspase 3 but did not cause significant cell loss (fig. thus, effects of established treatment options on cell death can be studied in gbm slices. 6.combined treatment of tmz and irradiation with carbon ions and x - rays on human gbm slice cultures. gbm slices were treated with tmz (200 m), x - ray irradiation (4 gy), sobp carbon ions (2 gy), or irradiation + tmz. tmz treatment started 24 h before irradiation and was maintained throughout the entire incubation time. slices were fixed 2 days after irradiation, and h&e staining was performed for neuropathological assessment (a d). in addition, activated caspase 3 was labeled (green in e), and fragmented nuclei were visualized using hoechst 33342 (blue in e and h). pictures of immunofluorescent stainings (in rgb format) were split into the three single channels of red, green, and blue, resulting in 3 gray - value pictures. the area coverage is represented by white pixels in the respective channels and can be fine - tuned by threshold adjusting. in the green channel, the positive area represents the caspase 3positive cell population, and in the blue channel, the area covered by nuclei is displayed (e j). carbon significantly reduced cell numbers (g), whereas x - rays did not (j). both treatments result in a significant increase in cell death and morphological alterations compared with the vehicle - treated control. combined treatment of tmz and irradiation with carbon ions and x - rays on human gbm slice cultures. gbm slices were treated with tmz (200 m), x - ray irradiation (4 gy), sobp carbon ions (2 gy), or irradiation + tmz. tmz treatment started 24 h before irradiation and was maintained throughout the entire incubation time. slices were fixed 2 days after irradiation, and h&e staining was performed for neuropathological assessment (a d). in addition, activated caspase 3 was labeled (green in e), and fragmented nuclei were visualized using hoechst 33342 (blue in e and h). pictures of immunofluorescent stainings (in rgb format) were split into the three single channels of red, green, and blue, resulting in 3 gray - value pictures. the area coverage is represented by white pixels in the respective channels and can be fine - tuned by threshold adjusting. in the green channel, the positive area represents the caspase 3positive cell population, and in the blue channel, the area covered by nuclei is displayed (e j). carbon significantly reduced cell numbers (g), whereas x - rays did not (j). both treatments result in a significant increase in cell death and morphological alterations compared with the vehicle - treated control. lack of promoter methylation of o - methylguanine - dna methyltransferase (mgmt) has been reported to significantly decrease patients ' susceptibility to tmz and thus survival, but there are also patients with nonmethylated promoter who benefit from tmz treatment. in fact, we identified one tumor in which tmz did not significantly induce cell death and we therefore requested the promoter methylation status, which is assessed by quantitative pcr and sequencing techniques of tumor material obtained from surgery. however, in line with the clinical observations that some patients with nonmethylated mgmt respond to tmz, we subsequently identified other tumors in which tmz significantly enhanced activation of caspase 3 to levels comparable to those of the methylated tumor (fig. gbm slice cultures with different mgmt promoter methylation states were tested for their response to tmz treatment. in a gbm specimen with methylated promoter sequence (a), tmz treatment resulted in significant induction of caspase 3 cleavage (gbm 1011), whereas caspase 3 activation was not significantly induced (b) in gbm with an unmethylated promoter sequence (gbm 0611). immunocytochemistry is shown for the methylated (c, right) and unmethylated (c, left) tumor for cleaved caspase 3 (green) and nuclei (hoechst, blue). after tmz treatment, caspase 3 activation was detected in some specimens, whereas others seemed to be resistant. this was independent of mgmt promoter methylation. from left to right : gbm 1011 with methylated mgmt promoter (meth +) and significant caspase 3 activation ; gbm 0411 with nonmethylated mgmt promoter (meth -) and significant caspase 3 activation ; gbm 1211 with nonmethylated mgmt promoter (meth -) and significant caspase 3 activation ; gbm 0812 with nonmethylated mgmt promoter (meth -) and significant caspase 3 activation ; gbm 0611 with nonmethylated mgmt promoter (meth -) and nonsignificant (ns) caspase 3 activation ; gbm 0512 with nonmethylated mgmt promoter (meth -) and nonsignificant (ns) caspase 3 activation. gbm slice cultures with different mgmt promoter methylation states were tested for their response to tmz treatment. in a gbm specimen with methylated promoter sequence (a), tmz treatment resulted in significant induction of caspase 3 cleavage (gbm 1011), whereas caspase 3 activation was not significantly induced (b) in gbm with an unmethylated promoter sequence (gbm 0611). immunocytochemistry is shown for the methylated (c, right) and unmethylated (c, left) tumor for cleaved caspase 3 (green) and nuclei (hoechst, blue). after tmz treatment, caspase 3 activation was detected in some specimens, whereas others seemed to be resistant. this was independent of mgmt promoter methylation. from left to right : gbm 1011 with methylated mgmt promoter (meth +) and significant caspase 3 activation ; gbm 0411 with nonmethylated mgmt promoter (meth -) and significant caspase 3 activation ; gbm 1211 with nonmethylated mgmt promoter (meth -) and significant caspase 3 activation ; gbm 0812 with nonmethylated mgmt promoter (meth -) and significant caspase 3 activation ; gbm 0611 with nonmethylated mgmt promoter (meth -) and nonsignificant (ns) caspase 3 activation ; gbm 0512 with nonmethylated mgmt promoter (meth -) and nonsignificant (ns) caspase 3 activation. organotypic slice cultures derived from early postnatal rodent brain are widely used in neuroscience due to their easy access for pharmacological intervention, electrophysiological studies, and live imaging. we have employed entorhinohippocampal preparations, which, due to the orientation of the trisynaptic pathway (perpendicular to the longitudinal axis of the hippocampus), allow for maintenance of the major connectivity. in this study, we adjusted cutting and culturing methods to prepare slices from human gbm and demonstrated evidence for their suitability as a test system for novel therapies including irradiation with hi. we irradiated gbm slices with photons and carbon ions and in both instances found that radiation induced dna damage and strongly affected proliferation. carbon ion radiation also induced activation of caspase 3, a potent inductor of programmed cell death (figs. in contrast to photon radiation, which delivers energy all the way through the body (eg, in an anterior - posterior direction), the depth of energy deposition of hi can be adjusted and limited to distinct areas of a few millimeters. in fact, much hope derives from successful intensity - modulated carbon therapy of chondrosarcomas of the skull base, a treatment first established at gsi. an accelerator specialized for medical applications has been constructed in heidelberg and opened for patients in 2009. currently, clinical trials and work with cell lines are aimed at testing the effects of carbon ion radiation in tumors other than chondrosarcomas. our data support observations in gbm - derived cell lines and in a small group of patients, and our approach may lead to a more detailed understanding of the biological effects of hi and additional novel therapies. moreover, surviving tumor cells can be studied to understand their mode of resistance. we also applied tmz to gbm slices and analyzed the effect on cell survival using pi staining and live imaging, as well as labeling of activated caspase 3 in paraffin - embedded sections. tmz is an alkylating agent widely used to treat gbm that, in combination with radiation therapy, helps prolong patients ' survival time. methylation was significantly more frequent in patients who survived longer than 36 months after surgery (p <.05). statistically, methylation status apparently affects susceptibility to tmz, but some patients with nonmethylated status also seem to benefit from tmz. of note, determining methylation status is not trivial, as only a few of the 109 potential sites have been tested. a recent survey among 1053 members of the neuro - oncology community in the united states found that only a small percentage (10.9%) of clinicians regard mgmt status as always or almost always thus, the next challenge will be to relate tmz - induced cell death rates obtained in slices to (progression - free) survival times ; we will address this issue with tumor - derived samples during the next 2 years to test the predictive value of this assay. it may be trivial to state that only in vitro systems allow different therapeutic options to be tested for an individual patient. our data demonstrate that tumor - derived gbm - slice cultures in principle are suitable for that task as a step on the way to more personalized therapies while also helping unravel basic mechanisms of tumor resistance. this work was supported by the messer foundation, the kassel foundation, and the dr.	backgroundglioblastoma multiforme is the most common lethal brain tumor in human adults, with no major therapeutic breakthroughs in recent decades. research is based mostly on human tumor cell lines deprived of their organotypic environment or inserted into immune - deficient animals required for graft survival. here, we describe how glioblastoma specimens obtained from surgical biopsy material can be sectioned and transferred into cultures within minutes.methodsslices were kept in 6-well plates, allowing direct observation, application of temozolomide, and irradiation. at the end of experiments, slice cultures were processed for histological analysis including hematoxylin - eosin staining, detection of proliferation (ki67), apoptosis / cell death (cleaved caspase 3, propidium iodide), dna double - strand breaks (h2ax), and neural subpopulations. first clinical trials employed irradiation with the heavy ion carbon for the treatment of glioblastoma patients, but the biological effects and most effective dose regimens remain to be established. therefore, we developed an approach to expose glioblastoma slice cultures to 12c and x-rays.resultswe found preservation of the individual histopathology over at least 16 days. treatments resulted in activation of caspase 3, inhibition of proliferation, and cell loss. irradiation induced h2ax. in line with clinical observations, individual tumors differed significantly in their susceptibility to temozolomide (0.4%2.5% apoptosis and 1%15% cell loss).conclusionglioblastoma multiforme slice cultures provide a unique tool to explore susceptibility of individual tumors for specific therapies including heavy ions, thus potentially allowing more personalized treatments plus exploration of mechanisms of (and strategies to overcome) tumor resistance.
osseous bridging of adjacent lumbar transverse processes has been reported as a rare complication following trauma to the back in adults2,6). after trauma, heterotopic ossification develops in surrounding soft tissues, and can lead to bridging or pseudarthrosis of transverse processes4,5). in the literature, congenital bridging has been reported much less frequently than traumatic bridging and usually these reports concern adults. here, we present the simple radiographic and computed tomographic findings of a youngest girl with congenital transverse process bridging. the clinical features of this rare anomaly are discussed with a review of the literature. a 13-year - old girl was referred to our hospital due to low back pain (lbp) and stiffness in the left lower back region. the pain had gradually increased in severity over 1 year, and at presentation she was unable to sit comfortably. she reported that the pain was aggravated by extension of the lower back and prolonged by hours of standing. her detailed medical history was unremarkable, and there was no history of back injury. a deep, firm fullness was palpable in the paravertebral muscles in the left lumbar area, and was tender to percussion. lumbar motion was limited in all directions, but the findings of a neurological examination, including the straight leg - raising test (slrt) were within normal limits. an anteroposterior simple radiographs of the lumbar spine demonstrated the presence of osseous bridging between the left transverse processes of the third and fourth lumbar vertebrae with minimal scoliosis at the bridging site and simultaneous disc space narrowing at the l3-l4 level (fig. 1). computed tomography revealed articulation between left transverse processes of the third and fourth lumbar vertebrae (fig. the left lateral contour of the vertebral body and the bridging transverse processes resembled the shape of the letter ' o ', and the components of bone bridge had smooth, regular contours. because of her young age and parents ' wishes, the patient was treated conservatively, that is, by medical and physical therapy. after 6 months of follow up, although her symptoms were still present, they had ameliorated in some degree. osseous bridging between two transverse processes of the lumbar spine mostly has a traumatic origin in middle aged patients3). yoslow.7), in their review of lumbar osseous bridging in the literature, found that an association exists with back injury and that pseudarthrosis may form in heterotopic bone. in 1991 billet.1) also found that only 15% of their cases had a congenital etiology. in this study, the youngest patient treated was 23 year old, but the origin of the osseous bridge was not obvious. in addition, criteria were suggested for the differentiation of congenital and traumatic osseous bridges. the localizations of hematoma and myositis ossificans play important roles during bony bridge modeling, and the configurations and shapes of traumatic osseous transverse process bridges show acute angles and irregular, asymmetric outlines. on the other hand, osseous bridges of a congenital - developmental etiology are ' o ' shaped with symmetrical, contour with convexity of the lumbar spine on the contralateral side and the absence of degenerative changes in the corresponding intervertebral space. other congenital skeletal anomalies, such as, spina bifida occulta, sacralization of the fifth lumbar vertebra may accompany congenital bone bridges but these findings are not obligatory6). in the present case, the patient had experienced no evident traumatic episode before admission and had a symmetrical smooth regular contour, which was considered indicative of a congenital - developmental etiology. bone bridging is usually asymptomatic, but low back pain is the most frequent symptom when it becomes symptomatic. although, the exact mechanism of pain in bony bridge is still not clear, we believe congenital bone union itself, radiculopathy or spinal deformity caused by imbalanced mechanical stress, can contribute to manifestation of symptoms. our patient showed motion restriction and back pain, but surgical management was not attempted due to improvement of symptoms after medical and physical therapy. restricted motions of adjacent joints are known to be ameliorated by freeing muscles from the heterotopic mass and by subsequent physical therapy. however, when symptoms are not relieved by conservative treatment, surgical removal of pseudarthrosis should be considered2). the elimination of pain following pseudarthrosis excision is related to local denervation, increased lumbar motion, and the re - establishment of pseudarthrosis in a plane that does not restrict lumbar motion. it would seem appropriate to remove bony bridge when magnetic resonance image or discography fails to show any significant abnormality of the intervertebral disc. we present the imaging findings of this rare anomaly in a young girl with lbp. this is the youngest case reported to date of congenital bridging of transverse processes without back injury. although rare, this rare anomaly should be borne in mind when evaluating the lumbar spine for the etiology of lbp even in children or adolescents.	osseous bridging between lumbar transverse processes is an uncommon condition that may cause low back pain. in most cases, its etiology is alleged to be trauma to the back and only rarely has a congenital origin been indicated. furthermore, most reported cases involved adults, the majority of whom were middle - aged. here, the authors describe the case of the youngest girl reported to date with congenital transverse process bridging. as far as the authors ' knowledge, there has been no report of congenital bridging of transverse processes in children or adolescents in korea.

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