Findings generally showed that the participants perceived that they understood themselves better and had stronger resilience after joining the program. Participants also saw themselves as closer to their ideal selves and other positive role figures (but farther away from a loser) after joining the program. This study provides additional support for the effectiveness of the Tier 1 Program of Project P.A.T.H.S. in the Chinese context. This study also shows that the repertory grid test is a useful evaluation method to measure self-identity changes in participants in positive youth development programs.The repertory grid test, based on personal construct psychology, was used to evaluate the effectiveness of Project P.A.T.H.S.  in Hong Kong. One hundred and four program participants ( There are growing adolescent developmental issues, such as mental health problems, abuse of psychotropic substances, adolescent suicide, school violence, and a drop in family solidarity, in Hong Kong that deserve the attention of helping professionals .With refTo promote holistic development among adolescents in Hong Kong, the Hong Kong Jockey Club Charities Trust approved HK$400 million to launch a project entitled “P.A.T.H.S. to Adulthood: A Jockey Club Youth Enhancement Scheme.” P.A.T.H.S. denotes Positive Adolescent Training through Holistic Social Programme. The Trust invited academics of five universities in Hong Kong to form a research team, with The Hong Kong Polytechnic University as the lead institution, to develop a multiyear, universal positive youth development program to promote holistic adolescent development in Hong Kong. Besides developing the program, the research team also provides training for the teachers and social workers who implement the program and carries out longitudinal evaluation of the project . BecauseThere are two tiers of programs (Tier 1 and Tier 2 Programs) in this project. The Tier 1 Program is a universal positive youth development program, where students in secondary 1 to 3 participate in the program, normally with 20 h of training in the school year at each grade. Because research findings suggested that roughly one-fifth of adolescents need help of a deeper nature, the Tier 2 Program is generally provided for at least one-fifth of the students who have greater psychosocial needs at each grade . To promote the positive development of adolescents, a total of 15 adolescent developmental constructs are covered in these two tiers of program, particularly the Tier 1 Program. These include promotion of bonding, cultivation of resilience, promotion of social competence, promotion of emotional competence, promotion of cognitive competence, promotion of behavioral competence, promotion of moral competence, cultivation of self-determination, promotion of spirituality, development of self-efficacy, development of a clear and positive identity, promotion of beliefs in the future, provision of recognition for positive behavior, provision of opportunities for prosocial involvement, and fostering prosocial norms .Although there are many unique attributes of the project, the usefulness of the project remains to be demonstrated. To provide a comprehensive picture of the effectiveness of the project, several evaluation strategies are employed, including objective outcome evaluation, subjective outcome evaluation, qualitative evaluation based on focus groups, student diaries and in-depth interviews, process evaluation, and interim evaluation. To date, research findings based on different evaluation strategies demonstrated that different stakeholders held positive perceptions of the program, and the participants showed positive changes after joining the project. For example, subjective outcome evaluation consistently showed that more than four-fifths of the participants and program implementers perceived the program to be beneficial to the program participants –7. ObjecTo further understand the effectiveness of the project, evaluation based on the approach of personal construct psychology was carried out in this study. According to Kelly , the uniThe repertory grid test was designed by Kelly in order to understand the personal constructs of an individual. In particular, the repertory grid test has been used to understand the self-identity system in numerous studies –15. TherThe repertory grid method as an evaluation strategy, used to examine changes in ex-mental patients who joined a holistic psychiatric rehabilitation program conducted by Luk and Shek , is presT-test analysis showed that the mean distance between E1 and E9 was significantly longer than that between E3 and E9. When the anchoring point was on E10 (an unsuccessful person), the mean distances between E1–E10 and E3–E10 were 0.84 and 1.12, respectively. T-test analysis showed that the mean distance between E1 and E10 was significantly shorter than that between E3 and E10. The findings showed that all related statistical analyses were statistically significant. It should be noted that the findings were still significant after Bonferroni correction.With E5  as the anchoring point, the mean distances between E1–E5 and E3–E5 were 1.12 and 0.81, respectively. Analyses showed that the mean distance between E1 and E5 was significantly longer than that between E3 and E5. With the anchoring point on E9 , the mean distances between E1–E9 and E3–E9 were 1.27 and 0.85, respectively. The repertory grid technique was also used to examine the effectiveness of youth drug prevention programs in Hong Kong. Shek and associates  utilizedAdopting the repertory grid test methodology, the present study attempted to look at how the participants perceived changes in their self-identity system after joining the Tier 1 Program of Project P.A.T.H.S. for 3 years, thus providing an additional perspective on the effectiveness of the program. The basic expectation was that the program should help the participants to identify more with the positive roles but less with the negative roles; hence, enhancing their protective factors to resist negative influences.This study adopted the repertory grid technique originally devised by Kelly  in his RA repertory grid test typically consists of elements and constructs. In the present study, there were 10 constructs elicited from triads. The triads involved selecting groups of three elements from the full list of elements, and the subject was then invited to say in what ways two of the elements were alike and in what way the third element was different from the other two. This procedure was intended to produce two contrasting poles for the construct. The constructs were then linked to elements by a 6-point rating scale. The grid was analyzed by using the INGRID package devised by Slater based on principal components analysis . There aIn the present study, a total of 108 students who had participated in the Tier 1 Program were randomly selected from the participating schools and completed the grids. However, due to missing constructs or elements in four cases, only 104 cases were used for data analysis. The following ten elements were used to elicit the constructs. Element 1: self before joining Project P.A.T.H.S. (past self), Element 2: self after joining Project P.A.T.H.S. for 3 years (present self), Element 3: self at high-school graduation (future self), Element 4: ideal self, Element 5: father, Element 6: mother, Element 7: an important friend, Element 8: a successful person, Element 9: a loser, and Element 10: a mature peer.The constructs were elicited via the triadic method. For every triad , the participant was asked to construct the way that two elements were most alike but were most different from the third one. Each participant was invited to elicit 10 constructs. After the constructs were elicited, the participant was then asked to rate all the elements on the construct  on a 6-point scale, with 1 to 3 representing the contrast pole, and 4 to 6 representing the construct pole.As adolescents are sometimes not very verbal in their responses, two supplied constructs were added. These were “understand oneself” versus “does not understand oneself,” and “high resilience” versus “low resilience.” The raw data for each grid (10 elements by 12 constructs) were analyzed by the INGRID 72 program . In the To examine the perceived changes of the participants after joining Project P.A.T.H.S., differences in the distances between the following pairs of elements were assessed.The mean distance between “self before joining Project P.A.T.H.S.” (Element 1) and “ideal self” (Element 4) versus the mean distance between “self after joining Project P.A.T.H.S.” (Element 2) and “ideal self” (Element 4): it was expected that if the participants had positive changes after joining the program, the distance between E2 and E4 would be shorter than that between E1 and E4 .The mean distance between “self before joining Project P.A.T.H.S.” (Element 1) and “a successful person” (Element 8) versus the mean distance between “self after joining Project P.A.T.H.S.” (Element 2) and “a successful person” (Element 8): it was expected that if the participants had positive changes after joining the program, the distance between E2 and E8 would be shorter than that between E1 and E8 .The mean distance between “self before joining Project P.A.T.H.S.” (Element 1) and “a loser” (Element 9) versus the mean distance between “self after joining Project P.A.T.H.S.” (Element 2) and “a loser” (Element 9): it was expected that if the participants had positive changes after joining the program, the distance between E2 and E9 would be longer than that between E1 and E9 .The mean distance between “self before joining Project P.A.T.H.S.” (Element 1) and “a mature peer” (Element 10) versus the mean distance between “self after joining Project P.A.T.H.S.” (Element 2) and “a mature peer” (Element 10): it was expected that if the participants had positive changes after joining the program, the distance between E2 and E10 would be shorter than that between E1 and E10 .For the supplied constructs, it was expected that the self after joining Project P.A.T.H.S. (E2) would have higher mean ratings in the constructs of “understand oneself” versus “does not understand oneself,” and “high resilience” versus “low resilience.” Repertory grid test data can be analyzed by both quantitative and qualitative methods –24. For Group analyses of the grid data of the 104 informants showed that the expectations outlined in the abovementioned hypotheses were supported.t(103) = 15.801, P < 0.0001; effect size = 1.60. In other words, the informants psychologically identified themselves more with the “ideal self” after joining the program.The mean distance between Element 1 (self before joining Project P.A.T.H.S.) and Element 4  was significantly longer than the mean distance between Element 2 (self after joining Project P.A.T.H.S.) and Element 4 : mean distance = 1.32 versus 0.88; t(103) = 10.325, P < 0.0001; effect size = 1.01. In other words, the informants psychologically identified themselves more with “a successful person” after joining the program.The mean distance between Element 1 (self before joining Project P.A.T.H.S.) and Element 8 (a successful person) was longer than the mean distance between Element 2 (self after joining Project P.A.T.H.S.) and Element 8 (a successful person): mean distance = 1.20 versus 0.90; t(103) = −9.027, P < 0.0001; effect size = 1.10. In other words, the informants psychologically identified themselves less with “a loser” after joining the program.The mean distance between Element 1 (self before joining Project P.A.T.H.S.) and Element 9 (a loser) was significantly shorter than the mean distance between Element 2 (self after joining Project P.A.T.H.S.) and Element 9 (a loser): mean  distance = 0.85 versus 1.10; t(103) = 9.027, P < 0.0001; effect size = 1.01. In other words, the informants psychologically identified themselves more with “a mature peer” after joining the program.The mean distance between Element 1 (self before joining Project P.A.T.H.S.) and Element 10 (a mature peer) was significantly longer than the mean distance between Element 2 (self after joining Project P.A.T.H.S.) and Element 10 (a mature peer): mean distance = 1.10 versus 0.85; t(103) = −15.333, P < 0.0001; effect size = 1.61). The participants also saw themselves as having a higher sense of resilience after joining the program  = −14.844, P < 0.0001; effect size = 1.57).Consistent with the hypotheses, findings showed that selves after joining Project P.A.T.H.S. were seen to be significantly different from selves before joining program. The participants saw themselves as understanding themselves after joining the program : like talking, indifferent, extrovert, care for friends, stick to established practice, hold on to one's own view, has no sense of humor, willing to share innermost feelings with others, mean, calculating, does not understand oneself, and low resilience see . HoweverThe informant perceived himself to have the following qualities before joining the Project P.A.T.H.S. (Element 1): absent minded, pessimistic, bad temper, complexity in thinking, impetuous, insincere, nonrestrained, flexible in thinking, direct in talking, selfish, does not understand oneself, and low resilience see . HoweverThe informant perceived himself to have the following qualities before joining Project P.A.T.H.S. (Element 1): relaxed, does not like thinking, not good at teaching, self-centered, easy going, irritable, hold on to one's own view, a follower, introverted, does not understand oneself, and high resilience see . HoweverThere are several unique features of this study. First, a large sample was employed to look at the self-representation of the informants. Second, participants who joined the Tier 1 Program were randomly selected from the participating schools, thus reinforcing the generalizability of the findings. Third, both quantitative and qualitative data were collected. Fourth, this is the first known evaluation study in different Chinese communities utilizing the repertory grid technique based on a large sample size. Finally, this is also the first known scientific study utilizing the repertory grid technique in the field of positive youth development to evaluate program effectiveness using this methodology.The evaluation findings based on the repertory grid tests clearly revealed the beneficial effects of P.A.T.H.S. Project. Using data based on 104 informants, group analyses showed that the informants identified more with the “ideal self” and “a successful person,” but less with “a loser” after joining the program. Individual analyses using three exemplar cases also illustrated the perceived positive changes in the informants after joining the program. The use of the repertory grid data provides an additional perspective to understand the program effects. Because the repertory grid technique is a “disguised” form of assessment in which the informants would find it difficult to figure out the purposes of the assessment, the chance of having demand characteristics in the assessment process was not high. As such, the positive findings point to the beneficial effects of the program. In particular, the increased distance between the “present self” and the “loser” suggests that the informants were less motivated to identify with the loser. Because the repertory grid techniques have been rarely used in the evaluation of positive youth development programs, the present attempt is a pioneering effort in the literature. If resources permit, it is recommended that this method should be used to assess the perceived self-identity systems of all program participants.One unique characteristic of Project P.A.T.H.S. is the systematic evaluation of the program. A wide range of evaluation strategies have been used to evaluate the Tier 1 Program as follows;Objective outcome evaluation: a randomized group trial with 24 experimental schools and 24 control schools initially has been carried out.Subjective outcome evaluation (Tier 1 Program): both students and program implementers are invited to complete subjective outcome evaluation forms  after completion of the program.Process evaluation: systematic observations are carried out in randomly selected schools to understand the program implementation details.Interim evaluation: to understand the process of implementation, interim evaluation is conducted by randomly selecting roughly half of the participating schools in the experimental and full implementation phases.Qualitative evaluation (focus groups based on students): focus groups involving students based on schools randomly selected from the participating schools are carried out.Qualitative evaluation (focus groups based on program implementers): focus groups involving instructors based on schools randomly selected from the participating schools are carried out.Qualitative evaluation (in-depth interviews with program implementers): Prolonged in-depth interviews with teachers are carried out.Qualitative evaluation (case study based on focus groups): case studies documenting the implementation experience of schools that have incorporated the Tier 1 Program into school formal curriculum are carried out.Qualitative evaluation (student logs): students are invited to reflect on their experiences after attending P.A.T.H.S. lessons and to apply things learned to their real lives.Qualitative evaluation (student products): students' weekly diaries are collected after completion of the program. Students' drawings are also collected to reflect the experiences of the program participants.Management information collected from the cowalker scheme: Because the cowalkers conducted classroom observations and completed observation forms, such information can give an overall picture about the implementation details in different schools.Evaluation based on the repertory grid tests: students are randomly selected to complete repertory grid tests that assess their self-identity systems before and after joining the program and perceived changes across years.Borrowing concepts from navigation and military disciplines, Denzin  used theThe present study underscores the utility of using the repertory grid test to assess changes in program participants. According to some researchers, the repertory grid technique can provide data from the informants' own views and thus represents a better method than questionnaire-type assessments, which may elicit only socially desirable responses. With some exceptions, the use of the repertory grid technique is not common in social work or related professions in Hong Kong. Because of the wide applicability of the repertory grid technique in clinical settings , helpingOf course, it is noteworthy that there are several limitations of the study. First, although a representative sample used in this study may permit generalizability of the findings, replication of the findings is necessary. In particular, it would be exciting to look at the prolonged changes in a person over time. Second, although statistically significant findings were obtained, it should be noted that because of the retrospective nature of the design , the clinical significance of the findings cannot be easily assessed. As such, data collected in real-life contexts would be helpful. Third, because there was no comparison group in the study, there is the possibility that the perceived changes in the participants can be attributed to factors other than holistic intervention . Finally, because no manipulation of the independent variable was carried out, the design does not permit us to draw the conclusion that the positive youth development program caused positive changes in the informants. Of course, the counterargument to this view is that looking at the self-representation from the perspective of the informants may give a clearer and more comprehensive picture about the beneficial effects of the Tier 1 Program of Project P.A.T.H.S. in Hong Kong.


The ‘Doctors to the Barrios’ (DTTB) Program was launched in 1993 in response to the shortage of doctors in remote communities in the Philippines. While the Program has attracted physicians to work in such areas for the prescribed 2-year period, ongoing monitoring shows that very few chose to remain there for longer and be absorbed by their Local Government Unit (LGU). This assessment was carried out to explore the reasons for the low retention rates and to propose possible strategies to reverse the trend.A mixed methods approach was used comprising a self-administered questionnaire for members of the current cohort of DTTBs, and oral interviews with former DTTBs.Among former DTTBs, the wish to serve rural populations was the most widely cited motivation. By comparison, among the current cohort of DTTBs, more than half joined the Program due to return of service obligations; a quarter to help rural populations, and some out of an interest in public health. Those who joined the Program to return service experienced significantly less satisfaction, whilst those who joined out of an interest in public health were significantly more satisfied with their rural work. Those who graduated from medical schools in the National Capital Region were significantly more critical about their compensation and perceived there to be fewer options for leisure in rural areas. With regard to the factors impeding retention, lack of support from the LGU was most frequently mentioned, followed by concerns about changes in compensation upon absorption by the LGU, family issues and career advancement.Through improved collaboration with the Department of Health, LGUs need to strengthen the support provided to DTTBs. Priority could be given to those acting out of a desire to help rural populations or having an interest in public health, and those who have trained outside of the National Capital Region. Whether physicians should be able to use the Program to fulfil return service obligations should be critically assessed. The problem of attracting, recruiting and retaining skilled health workers in rural areas has risen high on the agenda of policy-makers. It is a global problem, the effects of which are most pronounced in countries where staffing deficits are severe and where rural areas are particularly inaccessible and difficult places to work.The literature on the mobility of health workers suggests that an interplay of “push” and “pull” factors influence an individual’s decision to leave or stay in a rural workplace. Pull factors can attract health workers to urban workplaces or even abroad. These include career advancement, such as positions in centres of medical and educational excellence, higher financial rewards and improved living conditions. Concurrently, push factors, such as professional isolation, poorly resourced facilities and limited recreational possibilities, can provoke health workers to leave.In general, retention is known to be influenced by personal origin, family and community factors, financial considerations, career development, working and living conditions, as well as mandatory service requirements,3. RuralThe Philippines suffer from a maldistribution of health workforce, with only 10% of doctors, dentists, and pharmacists found in rural areas where more than half of the population resides. In 1993The DTTB Program regularly assigns a cohort of physicians to underserved and difficult-to-access municipalities for a period of 2 years. Initially, the deployment was twice or three times a year. During this assignment, the physicians receive good salaries and full benefits as employees of the national DOH. Later on, they have priority access to a Master’s degree or to clinical residency programs. These physicians have the role of Municipal Health Officer (MHO) which includes technical and managerial functions that cover national and local policy and program implementation, financial effectiveness, human resource for health management and development, provision of health services, information management, and infrastructure development and preservation. After completion of the two years, the DTTBs have the choice to remain in their position and to be “absorbed” as MHO of the LGU. However, LGU employees in low-income (5th and 6th class) municipalities are only entitled to part (65-70%) of what they would normally receive from a national agency under the same salary grade. Consequently, DTTBs experience a drop in their total compensation upon absorption.The DOH eventually expanded the deployment criteria to take into account population growth such that more than one doctor is required to meet the needs of the municipality, and to temporarily replace MHOs on study leave. A scheme of biannual Continuing Medical Education (CME) as a further incentive for participants was also introduced. By 2005 the Philippines were experiencing alarming levels of out-migration of doctors. The media contrasted the image of doctors in high paying jobs abroad with that of altruistic doctors choosing to serve in rural villages across the Philippines. This heroic portrayal of DTTBs increased attention for the Program leading to higher application rates and greater political backing. In the light of these developments, public medical schools began to make the receipt of scholarships dependent upon a period of mandatory service after qualification as a physician. The current cohort of DTTBs is the second batch that can make their CME sessions count towards a Master’s degree in Public Management with a major in Health Systems and Development offered through a partnership between the DOH and the Development Academy of the Philippines.Ongoing monitoring by the DOH showed that of the 452 DTTBs who took part in the Program between 1993 and 2011, only 81 (18%) chose to remain in their rural posts and to be absorbed by their respective LGUs. According to the DTTB Alumni Database, the numbers of those choosing to be absorbed have in fact declined since 2006  on the occasion of a forthcoming national CME session, and to a DTTB alumni database of the 452 former Program participants – although, as explained below, the contact information was later found to be outdated which severely compromised its usefulness for random sampling and tracing purposes. Given this constellation, the changes in the design of the Program that had taken place during the years of its existence and constraints of time and other resources, a mixed methods approach was selected: a self-administered questionnaire for all current DTTB, and oral interviews by telephone with available former DTTBs. The findings from both methods were analysed separately and later consolidated for the final interpretation.The development of the questionnaire for current DTTBs was based on a review of formally published as well as grey literature on staff retention in rural areas. A review of policy documents from the Philippines and the DTTB Program was also conducted. These included the DOH Revised Operational Guidelines for the Implementation of the DTTB Program and the Memorandum of Agreement between the DTTB, the LGU, and the DOH. Moreover, explorative interviews were conducted with key informants including the Director of the DOH Health Human Resource Development Bureau, and the past and present DTTB Program coordinators in order to guide the development of the questionnaire.The questionnaire used in this study was inspired by The ‘Stayers Questionnaire’ used in a health workforce assessment manual in Uganda. PermissThe variables retained and explored from the demographics section of the original Stayers Questionnaire were sex, marital status, number of dependents, and city/municipality of residence. After perusing the responses from the key informant interviews it was decided to expand these variables to include whether respondents graduated from a medical school within or outside the National Capital Region (NCR), whether their main reason to join the Program was to return service for a scholarship or not, whether their main reason for joining was an interest in Public Health/Community Medicine or other reasons, and whether they planned to remain in the area of assignment for more than one year or indefinitely after the Program, or alternatively to leave.To reach the current DTTBs in an efficient manner, advantage was taken of a CME session to request their participation in the assessment. All of the current 71 DTTBs were in attendance and all agreed to take part. Time was allocated at the session to introduce the study and the use of the Likert scale. Completing the questionnaire took approximately 30 minutes. In all but one case, the participants responded to all items of the questionnaire.t-tests were run for the statements and questions, and the differences between the mean scores were tested for statistical significance .Epi Info was used to analyse data obtained by the questionnaires from current DTTBs. The descriptive aspect of the analysis measured the frequencies by which the respondents gave a rating of “agree strongly” or “agree” to a specific question and the corresponding mean score. For the inferential component of the analysis, independent two-sample The topic guideline for interviews with former DTTBs was developed to broadly align with the sections of the self-administered questionnaire and made use of open questions so as to obtain further, clarifying and explanatory information. Both tools were tested by a small panel including two former DTTBs, a former DTTB Program Coordinator and the researchers conducting the assessment. The topic guideline provided the key themes for the analysis of the interviews with former DTTBs. Sub-themes emerging from the responses were then identified.Tracing the former DTTBs proved to be extremely difficult, as the contact information in the Alumni Database had not been maintained. Snowball sampling was, therefore, used over a one-month period to generate a productive sample, that inFormer DTTBs (n=26) were interviewed either face-to-face or, in the majority of cases for practical reasons, by phone. All the interviews were conducted by the same interviewer (JAL). None of those approached refused to be interviewed. The interviews lasted an average of 20 minutes. The responses were translated into English and directly transcribed by the interviewer.This assessment was suggested and facilitated by the Human Resources Development Bureau which obtained ethical clearance from the Department of Health in the Philippines. Informed consent was gained from all those who agreed to take part in either the questionnaire or the interviews. Respondents were allowed to withdraw at any time or skip questions without having to give a reason. However, no one made use of this possibility. All data were de-identified during the analysis so that responses could not be traced back to a particular informant.Of the 71 current DTTBs who filled out the self-administered questionnaire, 46 (65%) were female and 25 (35%) male. The mean age of the respondents was 29 years. Twenty-nine (41%) of the respondents had dependents, with an average of 2 dependents each. Eleven (15%) of them were married. Fifty-eight (82.9%) were from urban areas and 45 (63.4%) graduated from medical schools in the NCR.Of the 452 DTTBs who had graduated from the Program, a total of 26 DTTBs took part in the interviews; 14 of whom were male and 12 were female. The mean age of the interviewees was 38 years, with a range of 28 to 64 years. Fifteen of them came from a rural background. Nine of the interviewees were employed as local government health officers, 7 worked under the DOH Central Office, 5 were hospital clinicians, 3 were private practitioners, and 2 worked in other public health agencies. Twelve of the interviewees had chosen to work as municipal health officers for an average of 6 years after completing the DTTB Program, while 6 had re-entered the Program for a further rural deployment but to a different municipality. The remaining 8 had left the rural assignment after finishing the Program.The perception of current DTTBs with respect to their work situation is detailed in Table Former DTTBs derived personal satisfaction from getting to know a new part of the country with different cultures, meeting new people and being accepted in a rural community.“[What I find to be most satisfying is] experiencing new cultures and learning to live with them, while at the same time making friends from the DTTB Program.” ""Most of the current DTTBs found their work meaningful and stimulating as shown in Table Former DTTBs derived job satisfaction from applying their training in practice, gaining experience, successfully lobbying for staff benefits and development of their respective health centres, as well as from improvement in local health indicators. Some former DTTBs perceived a decline in DOH support once they were absorbed by their LGU. More than half of them enjoyed opportunities for further education while being on the Program. However, others mentioned that training on legal issues was lacking. Others were dissatisfied with their administrative roles as MHO, had concerns that their clinical skills could become out-dated, or had left to undergo further training in clinical areas.“I want to have more career options so that after [clinical] residency I can choose between private practice or public health.” ""Current DTTBs considered their workloads to be manageable and that they had the flexibility to attain a reasonable work-life balance as shown in Table . Staff mThe former DTTBs described the difficulties of their role brought about by weaknesses in local governance, perceived inadequacy of LGU support, and local politics.“It is hard to work with many bosses – you have to satisfy many politicians who sometimes have conflicting political interests.” .""Relations with the municipality mayor, as the MHO’s direct superior, emerged as extremely important in this regard. Former DTTBs who re-entered the Program rather than choosing to be absorbed felt that, as part of the LGU, it would be harder to critique the system and create positive change. Former DTTBs who stayed after the 2 years all stated that support from their respective LGUs was crucial to their decision to stay.Current DTTBs reported having comfortable accommodation with a clean toilet, regular electricity and, to a lesser extent, running water as shown in Table . Most coMany of the former DTTBs agreed that problems with living conditions influenced their decision to leave their rural post and those who stayed on reported having good accommodation. Negative issues raised in the interviews included difficulties with reaching and living in storm-prone regions and political violence during election times.“I did not feel very safe in my area of assignment. Elections were approaching and there were reports of politically-motivated killings.” ""Physical separation from one’s family and relatives was frequently mentioned as a push factor. However, some indicated that with the improved means of communication offered by internet and mobile phones the situation today may be less difficult.Most current DTTBs considered their salary and benefits to be fair. Less agreed regarding the representation and travel allowances as shown in Table .Former DTTBs considered conditions to have been good but felt better compensation was justified, as they were practically on-call for 24 hours and carried a great deal of responsibility. Some of the former DTTB who were absorbed, described having received inadequate allowances and incentives from the LGU and how they engaged in private enterprise activities as a response. As such possibilities were limited in rural areas, the constrained rural economy ultimately became a reason to leave.”). Almost a quarter entered the Program to help those living in rural areas, while several respondents cited their interest in public health and community medicine.Table By contrast, former DTTBs attached most importance to having the opportunity to help rural populations. Many described how their decision to join the Program was influenced by community exposure and encounters with rural physician role-models during their medical studies and rotations in government hospitals. Several mentioned joining the Program for adventure and travel, although only one with this motivation remained in service after the two years. Former DTTBs make no mention of return service. This was to be expected, as this reflects a more recent trend of conditionality that has been subsequently introduced by public medical schools and scholarship foundations.Associations between variables were next analysed from the data collected from the current DTTBs. Only those that were found to be statistically significant are considered in the following:Differences between sexes Table  revealedDTTBs who joined the Program because of return service enjoyed significantly lower personal satisfaction than those who joined the Program for other reasons Table . Other lBy contrast, DTTBs who joined the Program because of their interest in public health or community medicine were significantly more likely to find fulfilment in their work with rural communities Table .t-tests.Two thirds of the DTTBs who graduated from a medical school in the NCR joined the Program to return service. To avoid confounding, the participants whose main reason for joining the Program was return service were excluded from these Graduates of medical schools from the NCR were less satisfied with DOH support compared to those who graduated from schools in the provinces Table . The graOnly 7 of the 71 current DTTBs declared plans to remain in their rural post for more than one year or indefinitely after completing the Program. The mean age of these physicians was 28 years, 4 of them were male, 3 were married, 4 had dependents, 5 originated from urban areas, 4 trained in the NCR and 3 had entered the Program to return service.DTTBs who planned to remain in the Program gave significantly higher ratings to the support they received from the DOH Regional Office and the municipal government Table . They weLGU support was the most frequently cited factor followed by compensation, factors related to family concerns and career advancement opportunities as shown in Table . Former However, most of the former DTTB who initially remained in their rural post after the Program eventually left. The most frequently cited reasons for this were related to family and career development. Specific family reasons included the need to spend more time with their children, to raise their children in the city or at least in their hometowns, and to be within reach of their aging parents or relatives as the doctor in the family.“My father died and I was not there [for him]. I don’t want this to happen to my mother.” ""Those who did not continue in their rural post after completing the Program also stated career advancement as a major reason for leaving. Several preferred to undertake further clinical training to have more flexibility in their choice of work in the future. Some left because they felt they had done their part for the underserved, or from the frustration of witnessing the poverty in the rural areas.Better compensation was the most frequent suggestion made by both former and current DTTBs to encourage remaining in their rural posts. The under-provision of prescribed benefits and the previously described reduction in total compensation upon absorption by the LGU were shown to be key issues. Increased remote-assignment allowances and benefits, including support for dependents, were suggested as ways to mitigate this situation.Former DTTBs who chose to remain in their posts highlighted how the DTTB Program itself was instrumental in their decision by having enabled them to face the challenges of their role or facilitated a smooth transfer to the LGU. In this regard, the DOH, as the implementer of the DTTB Program, was considered to play a crucial role in lobbying for LGU support. In some cases, the experience derived during the 2 years in the Program drove the desire to serve and was also instrumental in the decision to stay. Former DTTBs who decided not to remain in their posts, nonetheless attributed to the Program their interest in pursuing a career in public health.Lack of LGU and DOH support were frequently mentioned as impediments to retention. However, no concrete measures to improve LGU support were proposed by the DTTBs that took part in this study. As for the DOH, it was proposed that they improve the technical, operational, career development, legal, and even moral support  provided to DTTBs. Further suggestions included the targeted recruitment of physicians with rural backgrounds, particularly those who are from the areas included in the Program, tailored career-development coaching for those who choose to stay in the rural areas, improved pre-deployment orientation to better prepare the DTTBs for their roles and the introduction of a rural physician network.Job satisfaction among health workers has been found to positively correlate with greater work commitment and retention,13. WhilOn the other hand, those who joined out of an interest in public health were more satisfied with their rural work. It is arguable that the current DTTBs have a stronger focus upon themselves and their individual needs and wishes. This might be the effect of the changing profile of Program applicants over time, or actually reflect a shift in societal values.The LGU was shown to play a pivotal role in retention and has the most important influence on DTTBs’ decision-making. It becomes apparent that, if LGU support is in place, then the MHO can rely on the availability of utilities, infrastructure, logistics, services, funding, and human resources – thus considerably facilitating the management of the local health system. Conversely, where LGU support is lacking, personal security, the availability of drugs, medical supplies, health program funds and even the provision of DTTB benefits become problematic. Moreover, once DTTBs are absorbed by the LGUs, they become more susceptible to the pressures of local politics. This would explain why some former DTTBs preferred to re-enter the Program and be assigned to another area and to retain their more neutral DOH employee status than to become an LGU employee.DOH support is also important for retention, particularly when DTTBs consider or embark upon absorption. Some of the former DTTBs expressed regret that DOH support waned once they became part of the LGU despite efforts from the DOH to continually contact and invite former DTTBs to CME sessions. This implies that former DTTBs may have either been poorly informed of the support available to them or they may have expected support in other areas, particularly in dealing with local governance issues. DTTBs graduating from medical schools in NCR were significantly less positive about DOH support. This could be because these physicians are overall less likely to remain in their rural post and perceive the linkage with the DOH to be crucial for their immediate and future careers.Career advancement is one of the most common reasons why former DTTBs gave up being a rural physician. Many of those who joined the Program were doctors at the start of their careers and still quite mobile career-wise. Meanwhile, DTTBs who choose to be absorbed face less opportunities of going up the career ladder, especially because the position of MHO is typically the highest non-elected position for a doctor in the municipal government. The lack of career mobility is an established problem in decentralized settings. Aside from the difficulties involved in moving up to the national level and between devolved administrative units, information regarding career opportunities is hardly shared.The majority of the current cohort are females, yet female DTTBs are significantly less likely to feel respected and have good friends at work than their male counterparts. This may indicate that women find it more difficult to deal with local politics in rural areas. It is also likely that men dominate the key LGU positions in such areas.Most current DTTBs consider their workloads to be manageable and agree that utilities such as water and electricity are generally available at their respective health facilities. However there is strong disagreement with the statements regarding the availability of medical supplies and equipment. DTTB who intend to remain in their rural posts are significantly more likely to be satisfied with the availability of essential drugs. It is worrying that the majority of current DTTB are found to be dissatisfied with the quality of care that their health centres provide. Whilst not specifically articulated, it is possible that this reflects a frustration with the possibilities of rural practice that may erode DTTB readiness to remain in their post.Most current DTTB have adequate accommodation and feel safe in their communities. The interviews with former DTTBs revealed that insecurity, due to natural hazards or political tensions, can rapidly undermine a physician′s readiness to remain, as documented in other studies in the Philippines.Family issues also considerably influence retention. The DTTBs mentioned the need to be closer to aged parents as a greater constraint to retention than the needs of spouses and children, which are more commonly cited in the literature,17. It iThe findings suggest that the DTTB Program could gain from exploring physicians’ backgrounds and reasons for joining. There could be potential to more actively recruit physicians from rural areas, or to consider strategies, including advocacy and scholarships, to encourage rural students to consider medicine as a future area of study. Reasons for joining could be examined during interviews or through letters of motivation. Priority should be given to qualified physicians with an interest in public health and community medicine and who trained outside of NCR. In particular, a decision should be taken whether to continue to automatically accept return-service physicians. Given that these physicians have already enjoyed the benefit of a scholarship based on an agreement to undertake rural service, it can be argued that they can be directly assigned to rural municipalities as part of the terms of their contract, thus foregoing the issues related to the transition of employment from the DOH to the LGU. For physicians with no obligation to return service, the DOH could consider involving LGUs in the choice of DTTBs from the outset to maximise the chances of a harmonious relationship between the two parties. The experience of the University of the Philippines Manila School of Health Sciences which promotes close early contact, and the elaboration of a social contract, between health workers and partner LGUs could be drawn upon here.On the other hand, it should be noted that 3 of the 7 physicians who planned to extend beyond the initial 2 years had originally joined the Program to return service. The number of those planning to continue is low and their profiles are so disparate that generalisations cannot be made. Yet, every physician counts in such a context as observed in the rural Zamboanga province of the Philippines, where the presence of just a small number of physicians already had a positive effect on community health outcomes. A netwo. For DTTBs who intend to work as clinical specialists in the future, the DOH needs to offer more opportunities for clinical updates and rotations in centres of medical excellence. For those that see their future in more managerial roles, the currently offered Masters course in Public Management appears to be a good choice.The role of MHO involves both administrative and clinical skillsThis assessment suggests that the DTTB Program needs to work on enhancing the factors that drive retention, whilst mitigating those that impede it. Through improved collaboration with the DOH, LGUs need to strengthen the support provided to DTTBs. The findings suggest 5 areas for future attention:The motivation to join the DTTB Program was, in some cases, awakened through meetings with inspiring rural physicians and rotations undertaken during training. This underlines the importance of continued advocacy at medical schools, with a particular focus on those located outside of the NCR, and of ensuring that all medical students are routinely afforded exposure to rural practice.DTTB Program coordinators would be well advised to invest in exploring why individuals seek to join. Priority should be given to physicians motivated by a wish to help rural populations or by an interest in public health, to those who have trained outside of NCR and those originating from rural or disadvantaged areas. Whether physicians should be able to use the Program to fulfil return service obligations should be critically assessed.The DTTB preparation provided by the DOH needs to be intensified with regards to political, legal and administrative aspects and made more gender-sensitive. Modern communication technology should be made available in MHO offices.Greater efforts are needed to ensure that absorbed DTTBs continue to receive DOH support so they can keep abreast with new clinical developments, and access new positions readily once the agreed retention period has been completed.Finally, most DTTBs will not remain in a rural area for an open-ended period of time. The retention period that is aspired to needs to be more clearly defined and aligned with LGU timetables, career development options and succession planning. The assessment highlights the importance for the DTTB Program to invest in an effective monitoring and tracking system.CME: Continuing Medical Education; DOH: Department of Health; DTTB: Doctor to the Barrios; LGU: Local Government Unit; MHO: Municipal Health Officer; NCR: National Capital Region.The authors all declare that they have no competing interests.JAL conceived the study, undertook the literature review, adapted the questionnaire, developed the interview guideline, collected the data, conducted the analysis, and wrote the first draft of the manuscript and took the lead for all later versions. HP assisted with the literature review, with data analysis, contributed at all stages of manuscript preparation including submission. KR contributed to the study design, provided clearance for the study, supervised the thesis, coordinated the data collection, and contributed to the background and discussion. RN contributed to the study design, tested the tools, coordinated the data collection, and contributed to the background and discussion. AR contributed to the study design, oversaw development of the tools, supervised the thesis, revised the later stages and final version of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/12/411/prepub


They could not recognize the scrambled H31–19, which contained the same amino acid composition but a different sequence as H31–19. In comparison, healthy subjects in general did not produce IgG against H31–19. However, about 70% of the healthy subjects produced IgM Abs against mono-methylated K9 of H31–19 (H31–19K9me). Our further studies revealed that ε-amine mono-methylated lysine could completely inhibit the IgM binding to H31–19K9me, but lysine had no inhibitory effect. In addition, the IgM Abs could bind peptides containing a mono-methylated lysine residue but with totally different sequences. Thus, mono-methylated lysine was the sole epitope for the IgM. Interestingly, SLE patients had much lower levels of this type of IgM. There was no obvious correlation between the IgM levels and disease activity and the decreased IgM was unlikely caused by medical treatments.We also found that the IgM Abs were not polyreactive to dsDNA, ssDNA, lipopolysaccharide (LPS) or insulin and they did not exist in umbilical cord serum, implying that they were not natural Abs. The IgM Abs against mono-methylated lysine are present in healthy subjects but are significantly lower in SLE patients, suggesting a distinct origin of production and special physiological functions.Post-translational modifications on proteins are important in biological processes but may create neo-epitopes that induce autoimmune responses. In this study, we measured the serum IgG and IgM response to a set of non-modified or acetyl- and methyl-modified peptides corresponding to residues 1–19 of the histone 3 N-terminal tail in systemic lupus erythematosus (SLE) patients and healthy subjects. Our results indicated that the SLE patients and healthy subjects produced antibodies (Abs) to the peptides, but the two groups had different Ab isotype and epitope preferences. Abs to the non-modified form, H3 Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of various autoantibodies (autoAbs). Many of these antibodies (Abs) are reactive to components in the cell nuclei, such as dsDNA, nucleosomes, Sm, La, Ro, etc. Histones can be grouped into linker histones (H1), core histones  and other variants. The core histones are small basic proteins consisting of a globular domain and a flexible N-terminal tail, which comprises about 25–30% of the mass of the individual histones. Two copies of each core histone are assembled into an octamer that has 146 base pairs of DNA wrapped around to form the nucleosome core. The histone N-terminal tails protrude from the nucleosome and are exposed on the surface et al. reported that ubiquitinated H2A and H2B were targets for SLE autoAbs et al. reported that apoptosis-associated histone modifications generated the epitopes H2BK12ac, H3K27me3, H4K8ac3, H4K12ac3 and H4K16ac3 that were recognized by autoAbs in SLE patients and lupus mice Histones can be extensively modified by acetylation, methylation, phosphorylation, ubiquitination, sumoylation, ADP-ribosylation, deimination and proline isomerization There are many sites on the 1–19 residues of the H3 N-terminal tail that can be modified to generate neo-epitopes. Although autoAbs to the non-modified H3 N-terminal tail have been reported In this study, we tested the reactivity of IgG and IgM from SLE patients and healthy subjects against a set of synthetic peptides corresponding to the 1–19 residues of the H3 N-terminal tail with or without methylation and acetylation. We found that both SLE patients and healthy controls could produce Abs to the peptides, but the two groups had different isotype and epitope preferences. IgG Abs were mainly produced by SLE patients with specificities to the non-modified peptide, whereas the healthy controls contained mostly IgM Abs recognizing epitopes containing mono-methylated lysine. Interestingly, SLE patients had significantly lower levels of this type of IgM than the healthy subjects.This study was performed in accordance with the Declaration of Helsinki and approved by the ethic committees of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences . All the personal privacy was well protected throughout the work. All the serum samples used in this work were leftover samples after clinical examinations of patients or routine health check of healthy people. As these samples were treated as abandoned samples and used in anonymous codes, the informed consent was exempted. Patient’s clinical information was obtained through a doctor who had the master key of coding and he would strictly abide by the confidentiality agreement. The umbilical cord blood samples were treated as medical waste and could be disposed by the hospitals. They were also used anonymously and coded without identifiable information.62 pediatric-onset SLE (pSLE) patients  were recruited between 2001 and 2012 from the Department of Pediatric Rheumatology, Capital Institute of Pediatrics, Beijing, China. 75 adult SLE (aSLE) patients  were recruited in 2012 from the Department of Nephrology, Peking Union Medical College Hospital, Beijing, China. Information for the patients and healthy controls is listed in the Sera from SLE patients and healthy controls were obtained from whole blood and stored at −80°C until use.1–19 and H31–19K9me peptides onto Sepharose CL-4B beads was performed by Absea. The beads were finally suspended in PBS/0.1% NaN3 as a 50% slurry. The histones were obtained by acid extraction from Hela cells as described The peptides  were syn2CO3, 35 mM NaHCO3, pH 9.6. After washing with PBS containing 0.05% Tween-20 (PBST), the plates were blocked with 200 µl of PBST containing 2% BSA for 2 h at room temperature. Then, 100 µl/well of the serum samples (1∶100 diluted in PBST containing 2% BSA) were added and incubated at room temperature for 1 h. After washing, the mouse anti-human IgG monoclonal Ab (mAb) KT47 or mouse anti-human IgM mAb KT16 (Absea) diluted at 1∶1000 was added. After incubating at room temperature for 1 h, the plates were washed with PBST and HRP-conjugated goat anti-mouse IgG  diluted at 1∶5000 was added. After incubating at room temperature for 1 h, the wells were washed and the substrate 2, 2′-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid  was added. Color was developed for 20 min (for IgM) or 40 min (for IgG), and the OD values were read at 405nm.96-well microtiter plates  were coated with H3 peptides cross-linked to BSA  at 4°C overnight in coating buffer containing 15 mM Na96-well microtiter plates (Kelongda) were coated with 100 µl/well of KT16 as a capture Ab (2.5 µg/ml) at 4°C overnight. The plates were then washed with PBST and blocked with 200 µl/well of PBST containing 2% BSA for 2 h at room temperature. The samples diluted in PBST containing 2% BSA  were then added. After incubating at room temperature for 1 h, the plates were washed and HRP-conjugated mouse anti-human IgM mAb KT38 (Absea) diluted at 1∶1000 was added as the detection Ab. After incubating for 1 h at room temperature, the plates were washed and the color was developed for 30 min using ABTS as the substrate.1–19 Abs, 1.5 ml of the filtered serum samples were absorbed with 10 µl of the H31–19 beads at 4°C for 10 h with rotation. The beads were separated by centrifugation using microcentrifuge spin columns . Then the sera were absorbed for the second time with 5 µl of the H31–19 beads to ensure the complete absorption of the anti-H31–19 Abs. The beads were then washed twice with PBS and 5 times with 1 M NaCl. Finally, the beads were treated twice with 50 µl of 0.1 M glycine (pH 2.5) for 10 min. The eluates were collected and immediately neutralized with 2 M Tris·Cl (pH 8.5). The sera that did not bind to the H31–19 beads were used for the purification of anti-H31–19K9me Abs, and the procedures were essentially the same as above.4 or 5 serum samples from each subgroup as indicated in 1–19 or H31–19K9me beads were run on gradient 5–18% SDS-PAGE gels under reducing conditions. The proteins were then transferred onto nitrocellulose membranes . After blocking with 5% nonfat milk for 2 h at room temperature, the membranes were cut along the 58 kDa position into two pieces. The piece with molecular weights smaller than 58 kDa was incubated with KT47, and the piece with molecular weights larger than 58 kDa was incubated with KT16 at 4°C overnight. After washing 5 times with PBST, the membranes were incubated with HRP-conjugated goat anti-mouse IgG for 1 h at room temperature. The membranes were washed and the bands were visualized using enhanced chemiluminescence (Product No. 34079. Pierce).10 µl of the eluates from the H31–19K9me beads were diluted at 1∶100 in PBST containing 2% BSA and different concentrations of lysine or ε-amine mono-methylated lysine. After incubating for 1 h at room temperature with rotation, 100 µl of the Abs was added to each well and incubated at room temperature for 1 h. KT16 and HRP-conjugated goat anti-mouse IgG were used for the detection of bound IgM as described above.96-well microtiter plates (Kelongda) were coated with peptides cross-linked to BSA in coating buffer  at 4°C overnight. The plates were washed with PBST and blocked with PBST containing 2% BSA for 2 h at room temperature. The Abs purified from the H3et al.The tests were performed as described by Tiller 1–19K9me Abs (1∶20 dilution) that were incubated in the presence or absence of lysine or mono-methylated lysine (5 mg/ml) were added and incubated at room temperature for 1 h. KT16 was used as the primary Ab and HRP-conjugated goat anti-mouse IgG was used as the secondary Ab.96-well microtiter plates (Kelongda) were coated with histones  at 4°C overnight in coating buffer. After washing with PBST, the plates were blocked in 200 µl of PBST containing 2% BSA for 1 h at room temperature. 100 µl/well of the purified IgM anti-H3t test was performed to analyze the differences between two groups and a paired t test was used to compare differences within a group. The Mann-Whitney test was used to compare data with a non-normal distribution. Percentages were compared using the Chi-square test or Fisher’s exact test. Pearson test was used for calculating correlation between data with normal distribution. P<0.05 was considered significant.All of the data were analyzed using the GraphPad Prism software (Version 5.01). For data with a normal distribution, an unpaired 1–19), acetylation on lysine 9 (H31–19K9ac) and mono-, di-, or trimethylation on lysine 4 or lysine 9 ), we performed an ELISA using sera from 62 pSLE and 75 aSLE patients. The age- and sex-matched healthy controls were tested in parallel.To determine if SLE patients produced IgG or IgM against a set of synthetic peptides corresponding to the 1–19 N-terminal residues of H3 , although these levels were still the highest among all of the peptides tested. IgM reactivity in the pSLE patients against H31–19K4me, H31–19K4me3 and H31–19K9me3 was also lower than the levels in the healthy controls (P≤0.029). Similar results occurred for the adult samples, except that the IgM Abs against H31–19K4me2, H31–19K9me2 and H31–19K9ac from the aHC were also higher than the IgM levels in the aSLE patients  IgM(1–19high91high), IgG(1–19low91low)IgM(1–19high91low), IgG(1–19low91low)IgM(1–19low91high), IgG(1–19low91low)IgM(1–19low91low) and IgG(1–19high91x)IgM(1–19x91x) IgM(1–19x91x) had high levels of IgG reactivity against H31–19 but varied IgG reactivity against H31–19K9me and varied IgM reactivity against both peptides. These five subgroups included samples from100% of the healthy adults and 95% of the aSLE patients.These results suggest that IgM and IgG might have different epitope preferences for the peptides. However, this is unclear because the raw serum samples would inevitably result in non- or low-specific signals. Therefore, we used affinity purified Abs to test the specificities. We were only able to purify adult Abs against H3–19x91x) . 1–19 re1–19 beads. The unbound Abs in the sera were then absorbed with H31–19K9me beads. The beads were washed with 1 M NaCl to remove the non- or low- specific Abs bound on the beads. The Abs were then separately eluted and analyzed by Western blot and ELISA.For Ab purification, 4 or 5 serum samples within a subgroup were pooled together. Equal volumes (1.5 ml) of the pooled sera were absorbed twice with H31–19 beads, IgG was barely detectable in the eluates from the healthy controls. However, the IgG Abs were clearly detectable in the IgG(1–19high91x)IgM(1–19x91x) subgroup of aSLE patients. IgG Abs were also detectable in the IgG(1–19low91low)IgM(1–19high91low) subgroup, which was categorized as low IgG reactivity against H31–19. IgM was undetectable in both the SLE and healthy controls, including the IgG(1–19low91low)IgM(1–19high91high), IgG(1–19low91low)IgM(1–19high91low) and IgG(1–19high91x)IgM(1–19x91x) subgroups, indicating that the high IgM reactivity in Western blot was performed to detect the Ab isotypes and amounts of IgG and IgM in the eluates from each subgroup. The results are shown in 1–19K9me beads, a heavy IgM band, but not IgG band, appeared in the IgG(1–19low91low)IgM(1–19high91high) subgroup of the healthy controls (low91low)IgM(1–19high91low) and IgG(1–19low91low)IgM(1–19low91low), which were categorized as low IgM reactivity against H31–19K9me. In comparison, anti-H31–19K9me IgM was detectable in all the subgroups of the SLE patients except for the IgG(1–19low91low)IgM(1–19low91low) subgroup, but in lower quantities. No IgM could be detected in the IgG(1–19low91low)IgM(1–19low91low) subgroup, which represented 60% of the patient samples. IgG could also be detected in the H31–19K9me eluates from the IgG(1–19high91x)IgM(1–19x91x), IgG(1–19low91low)IgM(1–19high91high) and IgG(1–19low91low)IgM(1–19high91low) subgroups, although in lower quantities.For the Abs purified using the H3controls , which r1–19 eluates nor the H31–19K9me eluates (1–19K9me eluates were clearly detectable in the IgG(1–19low91low)IgM(1–19high91high), IgG(1–19low91low)IgM(1–19low91high) and IgG(1–19high91x)IgM(1–19x91x) subgroups (1–19K9me eluates were even detectable in the IgG(1–19low91low)IgM(1–19high91low) and IgG(1–19low91low)IgM(1–19low91low) subgroups IgM(1–19x91x) subgroup, but not the other subgroups, were detectable and able to cross-react with all of the peptides IgM(1–19high91high) and IgG(1–19low91low)IgM(1–19low91high) subgroups, which represented 13.3% of aSLE patients, although the signals were weaker TGSAGKQKPR, was tested. This peptide contained the same amino acid composition but a different sequence as H31–19K9me. Our results indicated that IgM from healthy adults and aSLE patients purified using the H31–19K9me beads reacted to the scrambled peptide . No significant correlations were found except for IgG in the pediatric patients, which was weakly correlated with SLEDAI  are produced without exogenous antigen stimulation, as they are present in the cord blood from newborn humans and mice as well as in mice housed in germ-free conditions and fed an antigen-free diet + memory B cells 1–19 IgG and anti-Kme IgM, although both of these Abs recognize the H3 N-terminal peptides. We did observe that SLE patients produced little but detectable anti-H31–19K9me IgG  and the pathogenic autoAbs produced in the patients (mostly the IgG isotype) is still unclear. It is speculated that natural poly−/auto-reactive Abs may occasionally promote autoimmunity by serving as a template for the high affinity pathogenic autoAbs. These poly−/auto-reactive Abs may also suppress autoimmunity by regulating excessive autoimmune and inflammatory responses K9me IgG . HoweverIn conclusion, we have identified mono-methylated lysine as a novel epitope for IgM autoAbs in a majority of healthy subjects, and the levels of these IgM Abs in SLE patients are significantly lower than that in the healthy controls. These IgM autoAbs are acquired after birth and can recognize peptides or proteins containing mono-methylated lysine residues but not di- or tri-methylated lysine. Although the serum IgM levels against mono-methylated lysine do not associate with disease activity, they may still be useful in discriminating SLE patients from healthy persons.Figure S1SDS-PAGE of H3 peptides cross-linked to BSA. Synthetic peptide was cross-linked onto BSA. 2 µg (according to the BSA concentration) of each peptide/BSA run on 10% SDS-PAGE under reducing conditions.(TIF)Click here for additional data file.Figure S21–19K9me and GGKme.Serum reactivity to H3 Microtiter plates were coated with H31–19K9me and GGKme conjugated to BSA. Serum samples (n = 25) from the healthy adults or aSLE patients were diluted 1∶100 and tested. KT16 anti-human IgM was used as the primary Ab and HRP-conjugated goat anti-mouse IgG was used as the secondary Ab. Data are expressed as mean±SEM. (A) H31–19K9me coated. (B) GGKme coated. The results are shown as representative of two separate experiments.(TIF)Click here for additional data file.Table S1Information about the SLE patients and healthy controls. a, M, male; F, female. Ratios were compared by Chi-square test. b, Ages were compared by Mann-Whitney test. P<0.05 was considered significant.(DOCX)Click here for additional data file.Table S2Treatments received by SLE patients at blood collection. GC, Glucocorticoids; IS, Immunosuppressants; AM, Antimalarials; mAbs, monoclonal antibodies; IVIG, Intravenous immunoglobin. a, The number of patients treated with/without that medicine.(DOCX)Click here for additional data file.Table S3Peptides and their modifications.(DOCX)Click here for additional data file.


The endothelial specific cell-cell adhesion molecule, VE-cadherin, modulates barrier function and vascular homeostasis. In this context, we have previously characterized that VEGF  leads to VE-cadherin phosphorylation, β-arrestin2 recruitment and VE-cadherin internalization in mouse endothelial cells. However, exactly how this VE-cadherin/β-arrestin complex contributes to VEGF-mediated permeability in human endothelial cells remains unclear. In this study, we investigated in-depth the VE-cadherin/β-arrestin interactions in human endothelial cells exposed to VEGF.First, we demonstrated that VEGF induces VE-cadherin internalization in a clathrin-dependent manner in human umbilical vein endothelial cells (HUVEC). In addition to the classical components of endocytic vesicles, β-arrestin1 was recruited and bound to phosphorylated VE-cadherin. Molecular mapping of this interaction uncovered that the C-terminus tail of β-arrestin1, that comprises amino acids 375 to 418, was sufficient to directly interact with the phosphorylated form of VE-cadherin. Interestingly, the expression of the C-terminus tail of β-arrestin1 induced loss of surface exposed-VE-cadherin, promoted monolayer disorganization and enhanced permeability. Finally, this effect relied on decreased VE-cadherin expression at the transcriptional level, through inhibition of its promoter activity.Altogether, our results demonstrate that β-arrestin1 might play multiple functions collectively contributing to endothelial barrier properties. Indeed, in addition to a direct implication in VE-cadherin endocytosis, β-arrestin1 could also control VE-cadherin transcription and expression. Ultimately, understanding the molecular mechanisms involved in VE-cadherin function might provide therapeutic tools for many human diseases where the vascular barrier is compromised. Macromolecules and cells larger than 3 nm use the paracellular pathway, chiefly orchestrated through cell-cell junctions . Endothen in vivo,14-19. HVEGF stimulation leads to VE-cadherin phosphorylation, β-arrestin2 recruitment and VE-cadherin internalization in murine cells . To deteBecause β-arrestin1 and β-arrestin2 share redundant functions, we next asked whether β-arrestin1 was similarly involved in VE-cadherin internalization ,19. In rin vitro with a 40 kDa fluorescent tracer , along with empty plasmid (mock), full-length (FL) and deleted C-tail (ΔC-tail). The stable expression of CFP-myc-tagged β-arrestin1 constructs was validated by flow cytometry and confocal analysis  HUVEC were cultivated on collagen-coated slides, either sparse or confluent, serum deprived (Ctrl), subjected to VE-cadherin internalization assay and stimulated with VEGF . Cells were fixed and stained for VE-cadherin  and analyzed by confocal microscopy. Scale bar: 10 μm. (B) Graph represents the mean ± s.e.m. of the percentage of cells with iVEC staining; n > 200; T-test: ** p < 0.01. (C) Confocal analysis of iVEC (green), together with caveolin, cholera toxin (CTX), or rab11 (red). Scale bar: 10 μm. (D) Pearson’s coefficients were calculated for each indicated staining with iVEC; n > 10. Panels are representive of 3 independent experiments.Characterization of internalized VE-cadherin. (Click here for fileA-B) HUVEC were transfected with the indicated CFP-fused β-arrestin1 constructs, and analyzed by flow cytometry (A) and confocal microscopy (B). Scale bar: 10 μm. Panels are representive of 3 independent experiments. (C) Chromatin immunoprecipitations (ChIP) were performed with anti-GFP and preimmune (lg pAb) antibodies on shear cross-linked chromatin preparation from C-tail HUVEC. Three sets of primers flanking regions -541/-390, -132/+18 and -785/-670 on cadherin-5  promoter were used. Additional controls included anti-PoIII IP and premmune (lg mAb) and GADPH primers. Panels are representative of 3 independent experiments.β-arrestin1 expression and localization. (Click here for fileA) HEK-293 T transfected with luciferase reporter for the indicated promoter activity (NF-KB and AP1) and Renilla, together with mock, full lenght (FL) and C-tail β-arrestin1 (C-tail). Alternatively, cells were treated with the TNFα . Graphs shows the mean + s.e.m. of 3 independent experiments, normalized to Renilla. Two-way ANOVA test: *** p < 0.001. (B) HeLa transfected with C-tail β-arrestin1 (green) were stained (red) for p65 and cFos and analyzed by confocal. Alternatively, cells were treated with TNFα for 1 h. (C) RT-PCR for ICAM1 and VCAM1 was performed in starved HUVEC (-), treated with TNFα  or expressing C-tail and FL β-arrestin1. GAPDH serves as an internal control. (D) HEK-293 T (-) transfected with FL and C-tail β-arrestin1 were analyzed by western-blot for phosphorylated (p) p38. Anisomycin-treated cells  were used as a positive control. Total ERK2 serves as a loading control. All panels are representive of 3 independent experiments.Status of inflammatory signaling pathways in β-arrestin1 C-tail expressing cells. (Click here for fileA-C) HeLa were transfected with luciferase reporter for VE-cadherin (VEC) promoter activity and Renilla, together with either, a control CFP-myc plasmid (mock) and CFP-myc-tagged β-arrestin1 (βarr1) comprising amino acids 317-410 (C-tail). (B-C) Alternatively, HeLa (B) and HUVEC (C) received non-silencing duplexes (nsi) and β-arrestin1 (βarr1), β-arrestin2 (βarr2), and β-arrestin1/2 (βarr1/2)-targeting siRNA. VEC promoter activity was measured in a luciferase-based assay (B), and siRNA efficiency was evaluated by western-blot (C), using anti-βarr1 and anti-βarr2 antibodies. Tubulin serves as a loading control. Graph shows the mean ± s.e.m. of 3 independent experiments. Two-way and one-way ANOVA tests: ***p < 0.001; ** p < 0.01;* p < 0.05. Panels are representative of 3 independent experiments.Impact of β-arrestin1 and β-arrestin2 on VE-cadherin promoter activity. (Click here for file


Interim evaluation studies were carried out in order to examine the implementation details of the Tier 1 Program of the Project P.A.T.H.S.  in Hong Kong. Quantitative results of the interim evaluation findings based on eight datasets collected from 2006 to 2009 are reported in this paper. Three hundred and seventy-eight schools were randomly selected to provide information on the implementation details of the program via face-to-face interviews, telephone interviews, and self-completed questionnaires. Results showed that a majority of the workers perceived that the students had positive responses to the program and the program was helpful to the students. In conjunction with other process evaluation findings, the present study suggests that the implementation quality of the Tier 1 Program of the Project P.A.T.H.S. is high. The present study also provides support for the effectiveness of the Tier 1 Program of the Project P.A.T.H.S. in Hong Kong. In the positive youth development literature, many researchers argue that building developmental assets in adolescents is critical to the promotion of adolescent health , 2. In aTo promote holistic adolescent development, The Hong Kong Jockey Club Charities Trust earmarked HK$400 million for a positive youth development program entitled “P.A.T.H.S. to Adulthood: A Jockey Club Youth Enhancement Scheme” for junior secondary school students (secondary 1 to 3 students) in Hong Kong. The word “P.A.T.H.S.” denotes Positive Adolescent Training through Holistic Social Programmes. The project consists of two tiers of programs. While the Tier 1 Program is a universal program aimed at all secondary school students in Hong Kong, the Tier 2 Program targets around one-fifth of the students with greater psychosocial needs. The focus of the present study was to report evaluative findings on the Tier 1 Program.There are two implementation phases in this project—the experimental implementation phase and the full implementation phase. For the experimental implementation phase (January 2005 to August 2008), 52 secondary schools were invited to participate in the project with the objectives of accumulating experience in program implementation and familiarizing the front-line workers with the program design and philosophy. The full implementation phase started in January 2006. In the 2006/2007 school year, the programs were implemented on a full scale at the secondary 1 level. In the 2007/2008 school year, the programs were implemented at the secondary 1 and 2 levels. In the 2008/09 school year, the programs were implemented at the Secondary 1, 2, and 3 levels [In the Tier 1 Program, students in secondary 1 to 3 participate, normally with 20 h of training in the school year at each grade. The research team has developed a set of curriculum manuals, which includes curriculum materials based on 15 positive youth development constructs identified from the existing successful positive youth development programs: bonding, resilience, social competence, emotional competence, cognitive competence, behavioral competence, moral competence, self-determination, spirituality, self-efficacy, clear and positive identity, beliefs in the future, recognition for positive behavior, prosocial involvement, and prosocial norms . In eachBecause adolescent development is influenced by the interactions between young people and their surrounding environment, the ecological perspective was adopted in the Project P.A.T.H.S. when designing the teaching units that are intended to cultivate students' development in five different domains—individual, family, peer, school, and society. For example, some units in the secondary 1 curriculum were designed to promote students' relationships with their teachers and classmates, which helps the secondary 1 students to adapt to their new school life. Moreover, because there are worrying trends and phenomena related to the development of adolescents in Hong Kong, such as mental health problems, abuse of psychotropic substances, adolescent suicide, school violence, and a drop in family solidarity , some teTo evaluate the effectiveness of the Project P.A.T.H.S., several mechanisms involving different stakeholders have been used with multiple types of data collected. These include objective outcome evaluation, subjective outcome evaluation, qualitative evaluation, management information collected from the cowalker scheme, process evaluation, interim evaluation, and evaluation based on personal construct psychology. Among these approaches, process evaluation and interim evaluation constitute an indispensable part of program evaluation that helps researchers to monitor program adherence, create an infrastructure that supports the project, evaluate how effectively that process functions, and assess changes in skills, attitudes, and knowledge of the participants and program implementers , 10. ThrNevertheless, in the context of evaluation of positive youth development programs, a survey of the literature shows that findings on program implementation quality are rarely reported , 11, 12.In the same vein, there are several arguments for conducting process evaluation . First, Weinbach  providedThere are many forms of process evaluation studies. One possible form is to send observers to monitor the actual implementation of the program, where the implementation details, such as fidelity, student involvement, and implementer's skills, are rated. Another possibility is to collect feedback about the implementation process from program workers and participants so that some interim assessments can be carried out to obtain more information about the implementation process. Interim evaluation serves at least four purposes. First, it helps the program developers to identify whether there are any problems in implementation so that corrective measures or adjustments to the program can be stepped up where appropriate. Second, interim evaluation as a potential asset to program management provides valuable information on the progress of the implementation. Third, interim evaluation serves as a gesture of concern and encouragement, and it serves as a bridge between the program developers and the program implementers. Finally, the instant feedback of the interim evaluation findings to the program implementers can help to boost the morale and ownership of the program implementers.In evaluating the Project P.A.T.H.S. in Hong Kong, while process evaluation has been conducted through systematic observation on the program implementation details, interim evaluation in several cohorts has also been carried out during the program implementation process. Based on the program implementers' comments regarding the whole process of program implementation, more understandings of the reactions of the participants and workers to the program are gained. Via both face-to-face interviews and telephone interviews, interim evaluation information in the following areas is collected: (1) program workers' perceptions of the responses of the participants to the program, (2) experiences of the program workers delivering the program, (3) program implementers' perceived helpfulness of the program, (4) program implementers' perceived positive aspects of the program, (5) aspects of the program that require improvement, (6) difficulties encountered during program implementation, and (7) overall evaluation of the program. Several interim evaluation studies on the Project P.A.T.H.S.. have been published in peer-reviewed, international journals. Results show that both the program implementers and participants had positive comments on the program, although the workers also encountered problems and difficulties in the implementation process –19. In tFrom 2005 to 2009, the total number of schools that participated in the Project P.A.T.H.S. was 244. Among them, 46.27% of the respondent schools adopted the full program , whereas 53.73% of the respondent schools adopted the core program .Among all the participating schools, 236 schools that joined the 20-hour full program and 167 schools that joined the 10-hour core program were randomly selected to participate in the interim evaluation study. A total of 265 teachers and 178 social workers were invited to participate in face-to-face interviews on a voluntary basis during school visits. If the respondents were not available for the face-to-face interviews during the school visits, they were invited to participate in telephone interviews. Otherwise, they were asked to complete a self-administered questionnaire and return it to the research team via e-mail or fax. The random sampling method increased the validity of the findings. Descriptions of the datasets collected over the 4 years can be seen in In the 2005/2006 school year, a self-constructed, semistructured interview guide with six open-ended questions was used to collect information on the program implementation process. In the 2006/2007 to 2008/2009 school years, a modified, self-constructed, semistructured interview guide was developed and used with five closed-ended questions as follows.Question 1: what do you think about students' involvement in the program? .Question 2: do you think the students like the program? .Question 3: to what degree do you think the Tier 1 Program is helpful to students? .Question 4: do you like this program? .Question 5: your overall satisfaction to the program is? . Seven open-ended questions were also used to collect information on the program implementation process. The open-ended questions were as follows.Question 1: what are the responses of the students to this program?Question 2: do you think this program is beneficial to the students? If yes, what are the benefits?Question 3: what are the good aspects of the program?Question 4: which areas of the program require improvement? Question 5: have you encountered any difficulties during the program implementation process? If yes, what problems have you encountered?Question 6: what are your perceptions of the “Cowalker scheme”?Question 7: do you have other opinions?The qualitative data were analyzed by two trained research assistants. For the quantitative data (closed-ended questions), frequencies and percentages of responses were calculated.Since the instrument used for data collection in the 2005/2006 school year was different from others, no data collected in the 2005/2006 school year was presented. In the present paper, only the quantitative results of the interim evaluation in the 2006–2009 school years are reported. First, 92.86% of 378 respondent schools reported that students were involved in the program, which included 94.17, 94.48, and 89.38% of the workers implementing the secondary 1, 2, and 3 programs, respectively . Second,Based on several datasets collected in the experimental implementation phase and the full Implementation phase, the present paper integrates, analyzes, and interprets interim evaluation findings of the Tier 1 Program of the Project P.A.T.H.S. over time. There are several unique features of this study. First, a large sample involving a large number of teachers and social workers was used in this study. Second, data collected over different cohorts were utilized. Third, in view of the paucity of interim evaluation findings in both western and Chinese contexts, the present study is a pioneering study in the literature. Actually, this is the first known scientific interim evaluation study based on a series of evaluation studies in the Chinese evaluation literature.Several phenomena can be highlighted from the present study. First, the program implementers perceived that the students were involved in the program. This finding is consistent with the previous findings on process evaluation where students were observed to be highly involved in the Tier 1 Program. Because the activities in the Project P.A.T.H.S. strongly encourage student participation and the implementers are expected to teach in an interactive manner, it is not surprising to find that the students were highly involved in the program. This observation strongly suggests that when designing positive youth development programs, how to promote student involvement is an important consideration.The findings also show that both students and the program implementers indicated that they liked the program. This observation is generally consistent with the previous subjective outcome evaluation findings where both program participants and program implementers indicated that they liked the Tier 1 Program. As indicated, the interactive and participative nature of the program is quite unlike the regular subjects in the formal curriculum. The findings are also consistent with the qualitative evaluation findings that the program was perceived in a positive manner by the program participants and implementers. Taken as a whole, the present findings are consistent with the subjective outcome and qualitative evaluation findings based on different stakeholders.n = 3, 170 students) and 24 control schools  participated in a randomized group trial. Utilizing the 6-wave longitudinal data, analyses of covariance and linear mixed models controlling for differences between the two groups in terms of wave 1 pretest scores revealed that participants in the experimental schools showed significantly better developmental outcomes than did participants in the control schools at posttest (wave 6) based on different indicators of positive youth development derived from the Chinese Positive Youth Development Scale and other measures. Students in the experimental schools also displayed a lower level of intention to engage in problem behavior and better school adjustment than did students in the control schools. Similarly, differences between experimental participants who perceived the program to be beneficial and control participants were found. Similar analyses based on linear mixed models via the Statistical Package for Social Sciences showed that participants in the experimental schools displayed better positive youth development than did participants in the control schools in terms of different positive youth development indicators, including positive self-identity, prosocial behavior, and general positive youth development attributes [Finally, both the program participants and implementers perceived the Tier 1 Program to be beneficial to the program participants. This observation also echoes the subjective outcome and qualitative findings reported previously. In addition, this observation is in line with the objective outcome evaluation findings. For example, Shek and Sun  reportedtributes , and thatributes .When a psychosocial intervention program is designed, one basic question is whether the developed program is effective. In the evaluation literature, many strategies have been proposed to evaluate the effectiveness of a psychosocial intervention program, such as objective outcome evaluation and subjective outcome evaluation . While tDespite the positive evaluation findings, there are two limitations of the study that should be taken into account when the findings are interpreted. First, because the findings are based on the subjective perceptions of the program implementers only, subjective biases involved must be considered. Second, from the qualitative findings of interim evaluation previously reported, some problems encountered in the implementation process and recommendations for improvement were noted. Notwithstanding these limitations, together with other evaluation findings , the intAccording to Meyer et al. , the dev