The goal of this project is to evaluate the implementation of an integrated and interdisciplinary program for prevention and management of cardiometabolic risk (PCMR). The intervention is based on the Chronic Care Model. The study will evaluate the implementation of the PCMR in 6 of the 12 health and social services centres (CSSS) in Montréal, and the effects of the PCMR on patients and the practice of their primary care physicians up to 40 months following implementation, as well as the sustainability of the program. Objectives are: 1-to evaluate the effects of the PCMR and their persistence on patients registered in the program and the practice of their primary care physicians, by implementation site and degree of exposure to the program; 2-to assess the degree of implementation of PCMR in each CSSS territory and identify related contextual factors; 3-to establish the relationships between the effects observed, the degree of PCMR implementation and the related contextual factors; 4-to assess the impact of the PCMR on strengthening local services networks.The evaluation will use a mixed design that includes two complementary research strategies. The first strategy is similar to a quasi-experimental "before-after" design, based on a quantitative approach; it will look at the program's effects and their variations among the six territories. The effects analysis will use data from a clinical database and from questionnaires completed by participating patients and physicians. Over 3000 patients will be recruited. The second strategy corresponds to a multiple case study approach, where each of the six CSSS constitutes a case. With this strategy, qualitative methods will set out the context of implementation using data from semi-structured interviews with program managers. The quantitative data will be analyzed using linear or multilevel models complemented with an interpretive approach to qualitative data analysis.Our study will identify contextual factors associated with the effectiveness, successful implementation and sustainability of such a program. The contextual information will enable us to extrapolate our results to other contexts with similar conditions.NCT01326130ClinicalTrials.gov:  Evaluation of effective and sustainable interventions in chronic disease management and of their implementation in various contexts is essential to guide such initiatives in Canada. One initiative that seems promising but presents a challenge to be addressed by decision makers at all levels is the creation of primary care-centred integrated networks that foster and are congruent with the development of local services networks. Our study findings will support efforts to institute local integrated services networks throughout Canada.The Agence de la santé et des services sociaux de Montréal (ASSSM) has commissioned our team to help evaluate the implementation of an integrated and interdisciplinary cardiometabolic risk (diabetes and hypertension) management and prevention program. The intervention is based on the Chronic Care Model (CCM), in a context where integrated services networks are created. Although shorter-term effects can be detected and the implementation process documented over a short time period, longer-term effects on patients registered in the program and on the practices of participating physicians, as well as the sustainability of implemented actions, require a long enough observation period to take place. The project is scheduled to last 40 months.The growing burden of chronic diseases such as diabetes and their impact on the health system are now obvious . PopulatThe most recent clinical practice guidelines for diabetes ,5 and hyFor optimal glycemia and hypertension control, the guidelines target lifestyle modification  and risk factor management (dyslipidemia and obesity), in addition to pharmaceutical treatment. Support for disease self-management is also an integral part of an effective strategy for chronic disease management. Individuals with chronic diseases must cope on a daily basis with the symptoms and consequences of the disease, and are mostly responsible for enacting appropriate strategies  that will enable them to maintain a satisfactory quality of life . Self-maMoreover, the guidelines indicate that although short clinical interventions increase the likelihood that patients adopt and maintain healthy lifestyle habits, interdisciplinary care approaches are more effective ,13. CareA number of care models have been developed to improve chronic disease management. The Chronic Care Model  is the oSeveral studies, particularly of diabetes patients, have shown that interventions based on CCM components improve processes and outcomes, and reduce costs and service utilization among patients with chronic illnesses . HoweverMoreover, the literature indicates that primary care services are best suited to provide chronic disease management, although few studies have analyzed factors facilitating implementation of the CCM in primary care settings ,20. A nuChronic illness management requires better integration of services designed for individuals affected with such diseases. It is in part to meet these needs that, in 2004, 95 health and social services centres (CSSS) were created in Québec, 12 of which being in Montréal. The Centres resulted from the merging of local community health centres, long-term care facilities and, in most cases, a hospital. The goal of this new entity is to bring health services closer to the population, make them more accessible and improve their coordination and continuity in each territory. CSSS have also been given the mandate to set up local services networks by encouraging collaboration among organizations and partners in their territories, more particularly primary care services .Several strategies can be adopted to create local services networks, one of which is to set up integrated services networks based on disease or population target groups. Studies have shown that implementation of health networks is facilitated by the development of initiatives that foster integration of services for targeted groups of patients with chronic illnesses or with distinct demographic characteristics -29. In aThe Agence de la santé et des services sociaux de Montréal (ASSSM) has undertaken activities designed to improve management of chronic illnesses and inspired by the Chronic Care Model. In 2006, following a strategic initiative launched by the ASSSM, the CSSS Sud-Ouest-Verdun set up a diabetes referral centre. Since then, the program has become a model for the other CSSS in Montréal, who have shown an interest in developing similar programs in their territories. Due to the frequent association between diabetes and hypertension, the ASSSM has recommended adding the latter to the program. The ASSSM proposes to continue to implement the integrated and interdisciplinary program for prevention and management of cardiometabolic risks (PCMR) first in six CSSS territories in Montréal (including the CSSS Sud-Ouest-Verdun), and then eventually in all CSSS in Montréal. Each CSSS will implement the program with the perspective of having an integrated care network in its territory, taking into account its specific organizational context. The program aims to improve diabetes and hypertension control among the target clientele ; enhance the quality of life of people with dysglycemia and/or hypertension; consolidate primary care case management and optimize use of specialized services; and strengthen links with various partners to offer integrated services in the CSSS territory. Targeted clienteles are adults with marginal fasting glycemia, or glucose intolerance, or diabetes treated with diet only, or diabetes treated with monotherapy, or diabetes treated with more than one medication if HbA1c ≤ 8.0%; as well as adults with hypertension with BP in the doctor's office ≥ 140/90 mm Hg .clinical process in each CSSS that aims, over a two-year period, to change lifestyle habits, control biological indicators, prevent complications and support patient self-management. Clinical interventions include group educational sessions given by the nutritionist, nurse, kinesiologist, pharmacist and psychosocial worker; individual follow-up by the nutritionist and the nurse; participation in a physical activity program; monitoring of biological parameters; and referral to specialists, as needed. In each CSSS, follow-up and educational activities are provided by a cardiometabolic risk education centre's interdisciplinary team, complementarily with medical follow-up provided by the primary care physician. Details of the program are available from the authors upon request.The PCMR anticipates implementing a practice support to primary care physicians for management of targeted patients by consolidating continuing medical education programs and developing clinical tools and documentation to ensure that practices are standardized in accordance with the guidelines. Lastly, the PCMR plans, in a perspective of integrated services networks, to establish and consolidate links among various partners  and especially to consolidate service pathways that facilitate primary care access to specialized health services. Moreover, mechanisms will be put in place to ensure the clinical information needed for effective patient management and monitoring circulates fluidly among professionals. The ASSSM plans to implement a computerized regional registry for chronic diseases that includes anthropometric and biochemical data on the clientele being followed. For professionals of the cardiometabolic education centres, the registry will serve as a clinical tool, while it will be used as a communication tool by attending physicians and for project evaluation.In addition to implementing the clinical process, the PCMR provides Participating CSSS are responsible for implementation of the PCMR, and for setting up local coordination committees to oversee project planning and implementation, and to follow up on the project evaluation. In each CSSS, the local committee will set up a clinical committee responsible for the clinical tools and contents needed to implement the project. The ASSSM will provide regional leadership and organizational support required to implement the program in CSSS. Mechanisms will be put in place to encourage primary care physicians to participate . Furthermore, financial compensation is available from the RAMQ  for general practitioners who help organize clinical projects in a CSSS territory.The starting point for our conceptual framework Figure  is the iTo evaluate the effects of the PCMR and their persistence on patients registered in the program and the practices of their primary care physicians, by implementation site and degree of exposure to the program.For patients, the program should result in improved lifestyle habits and better control of their diabetes and/or their hypertension. Chronic illness management and follow-up should be noticeably improved, as should patient self-management and quality of life. Closer and better coordinated follow-up should also improve certain elements of the patients' experience of care with their primary care physicians . Finally, controlling their condition is likely to lead patients to use health services more appropriately . In all likelihood, these effects on patients will vary depending on the setting (CSSS) and degree of exposure to the PCMR. As regards medical practice, PCMR implementation should be reflected in participation in the program , interprofessional collaboration and patient management interventions. Persistence of effects on both patients and medical practice remains to be demonstrated.To assess the degree to which the PCMR has been implemented in each participating CSSS territory  and identify the context-related factors  that could explain the level of implementation.The aim is to answer the following research questions: To what degree has each setting implemented the program? What contextual factors facilitate or hinder program implementation? To what extent is program sustainability assured: does the PCMR fit into CSSS program activities?To establish the relationships between the observed effects (Objective 1), degree of implementation of the program and contextual factors (Objective 2).This objective implies the following research questions: How do variations in PCMR implementation influence the effects observed? To what extent do the contextual factors that facilitate or hinder program implementation influence the observed effects?To assess the impact of implementing the PCMR on strengthening local services networks.This objective concerns the following research question: To what degree does implementing this program help consolidate local networks by establishing closer and long-lasting links among service providers in the CSSS territory?PCMR evaluation includes analysis of its implementation and of its effects. We posit that the program concept and implementation will adjust to the different CSSS settings. The proposed evaluation will use a mixed design that involves two complementary research strategies . The firThe second strategy is consistent with a multiple case study approach, where each of the six settings constitutes a case. This strategy is based on qualitative methods and will characterize the variable represented by the implementation context. A monograph for each setting will be produced following analysis and will include data on the various indicators described below. The monographs will explain the processes that have led to the effects observed and the particular contextual conditions that can be linked to differences in implementation and effects. Adding this component to the evaluation will therefore increase both internal and external validity.The two strategies will generate data that we will triangulate to provide a deeper explanation of the link between degree of program implementation, context and observed effects. Triangulation will be based on an interpretative analysis of the data collected and on a validation process with individuals responsible for implementing the PCMR through discussion groups that will also serve as mediums for knowledge translation and exchange.To meet this objective, the indicators measuring the effects of the PCMR  and lifestyle habits . These indicators include measures of change during the intervention as well as measures of attainment of clinical targets at the end of the evaluation (e.g. proportion of patients with HbA1c ≤ 7%). Data needed to calculate indicators of biological parameters and lifestyle habits for each patient will be taken from the regional computerized registry for chronic illnesses. These data will be compiled in the registry at program entry and then at the 3rd, 6th, 12th, 18th and 24th month of follow-up.Several of the Patient Assessment of Chronic Illness Care questionnaire [th section is on patient self-management and quality of life related to their diabetes and/or hypertension. Self-management is measured using questions adapted from the Summary of Diabetes Self-Care Activities, and assesses the frequency of self-management behaviours during a typical week  [Audit of Diabetes Dependent Quality of Life [Indicators for health services utilization by patients in the PCMR, their experience of care, follow-up of diabetes and/or hypertension, self-management and quality of life will be measured using a 20-minute self-administered questionnaire, in French or English, given to patients by the nurse or nutritionist at the cardiometabolic education centre upon program entry, and at 12 and 24 months of follow-up  . Questio of Life -40. DataUse of health services by patients in the PCMR will also be documented with medical administrative data , using the patient's health insurance number to match medical administrative data to data from the regional computerized registry on chronic illnesses and to his or her survey data.Regression models will be built to assess the program's effects on patients, in terms of biological parameters, lifestyle habits, health services utilization, chronic illness follow-up, self-management, quality of life and care experience, based on the CSSS, degree of exposure to the program, patient characteristics, and organizational characteristics of primary care clinics where patients are seen. Particular attention will be paid to patients' sociodemographic characteristics  in terms of equity of access to services. Linear models will be constructed for continuous dependent variables (e.g. before-after intervention differences in HbA1c or BP). Logistic models will be used for dichotomous dependent variables . Longitudinal analyses will enable us to model the evolution of dependent variables throughout the follow-up period. These analyses will be performed using multilevel models   in whichWe plan on recruiting about 250 patients a year in each participating CSSS, for a total of 3500 patients over 28 months. Assuming that recruitment rate is constant, we estimate that the 2000 patients recruited in the first 16 months could be the object, during the study, of at least three observations for data collected using questionnaires, and at least six observations for clinical data  completeness of the components with regard to the program initially planned by the ASSSM; to assess the degree of implementation, we will measure the differences between the program planned by the ASSSM and the one actually implemented in CSSS; 2) intensity of the effort made to conduct program activities ; 3) integration of the PCMR into CSSS program activities; and 4) program penetration rate among the targeted populations. This will be estimated by calculating the proportion of patients with diabetes or hypertension followed in the PCMR among patients with diabetes or hypertension in each participating CSSS territory .The second element includes an analysis of factors influencing program implementation . The goaData will be collected in three phases, as per the program implementation stages. Semi-structured interviews with individuals from the ASSS and CSSS responsible for program implementation are scheduled for the beginning of the project (start-up phase). They will provide an initial portrait of regional services organization related to the activities of the program being implemented and will document the program components . They will also be used to define the dynamics in each implementation site. Interviews with respondents from each territory will focus on local historical contexts of interventions with people at risk of or who have diabetes or hypertension; organizational contexts of the CSSS; PCMR activities already in place and their components; local resources allocated; number of medical clinics and physicians involved; and number of patients targeted by the program. A second and third round of semi-structured interviews with local and regional respondents will be conducted 20 and 40 months after the beginning of the program. They will document PCMR activities actually implemented and degree of program implementation, activities set up to integrate the PCMR into the CSSS's regular activities, and obstacles and facilitators encountered. The questionnaires for physicians will also document barriers and facilitators. These contextual determinants will be grouped by dimension and conveyed with descriptive variables. The interview grids used at the beginning of the implementation of the project are included in Additional files Based on the information collected throughout the project, a descriptive analysis of each case (implementation setting) will be performed. Six monographs, one for each setting, will describe the phases of PCMR implementation and its explanatory factors.Two approaches will be used to attain this objective. The first is designed to complement the analysis proposed in Objective 2. Using the material compiled in the monographs, quantitative variables and categorical taxonomic variables will be created and integrated into regression analyses explaining the effects of the program. Regression models will be built to assess the influence of the variables pertaining to PCMR set up and implementation in the various CSSS on the effects observed among patients, controlling for degree of exposure to PCMR, patient characteristics and the characteristics of primary care clinics where they are seen. Given the low number of upper level units (6 CSSS), variables linked to CSSS will be considered as fixed effects in regression models. Linear regressions will be constructed for continuous dependent variables, while logistic models will be used for dichotomous dependent variables and multilevel longitudinal models for dependent variables that are the object of repeated measures. A similar analysis strategy will be used to assess the influence on medical practice of variables that reflect PCMR set up and implementation in various CSSS.The second approach will be interpretive: from the material used to produce the monographs, participants' statements on the relationship between program effects and components will be noted and analyzed. Conclusions drawn from these two approaches will be validated in discussion groups.This objective concerns the PCMR's influence on interorganizational collaboration and networking among local services networks. We are interested in mechanisms for collaboration that exist between CSSS and primary care organizations in the territories and health care and services providers, as well as their evolution over time. We will use the framework developed for our previous project . InterorThe project has received approval from the ASSSM's research ethics committee.Our project has both strengths and weaknesses. Strictly speaking, for a comparative analysis, the number of CSSS in our study is relatively small. However, for a multiple case analysis strategy, this number is sufficient to show contrasts and can rather be seen as a strength of our study. The absence of a control group is a limitation but in this type of study, it is impossible to use an experimental design; we replaced it with a quasi-experimental design  and a hySome of the strong points of our study include the fact that it will enable us to fill certain gaps in knowledge and, more particularly, to evaluate the application of the CCM to a primary care "case management" approach rather than to "disease management". It will also allow us to evaluate CCM implementation from a systemic perspective based on the will of the ASSSM to completely implement the proposed program in all Montréal CSSS. Mostly, our study will identify the contextual factors that can modulate the effects of the CCM and that can be associated with successful implementation and with the effectiveness of such a program. These contextual elements will enable us to extrapolate our results to other contexts with similar conditions as well as to other chronic illnesses. The study will also allow us to assess the impact of implementing a program inspired from the CCM on consolidation of local services networks. Finally, in terms of the methodology, we should add that other strengths of the project are sample size, repeated number of observations and use of numerous tools (questionnaires) that have either been validated or used in our previous studies.Since this project involves decision makers, clinicians and researchers, the knowledge translation and exchange plan is built into the study and consists in having mutual exchanges throughout the duration of the study. This close proximity fosters knowledge exchange and appropriation. The evaluation project is formative and many knowledge transfer activities are planned throughout the study.To actualize some of these knowledge transfer activities, two regional steering committees  will be set up to support the research team during various stages of the project. Moreover, local coordination and clinical committees will be potential sources of information to document the degree of implementation and of venues to engage in dialogue to validate and interpret the results obtained. Halfway through the project, we will organize a meeting of local and regional decision-makers, managers and clinicians, who will be asked to react to results regarding the degree of program implementation in various CSSS and identification of factors that facilitate or hamper implementation.We will also set up an advisory committee composed of representatives from among the project researchers and decision makers as well as other nationally and internationally known researchers and decision makers (some from other provinces) with expertise in research on primary care services organization and chronic illnesses. We plan to have two meetings with this committee to help us focus data collection and result analyses to promote the project's specific contribution to current knowledge and increase the potential to adapt our results to other contexts.At the end of the project and with the help of the Institut national de santé publique du Québec (INSPQ), we will set up an exchange forum that will allow us to add more decision makers and researchers to the advisory committee; they will enhance adaptation of research results in the contexts of Québec and of other Canadian provinces, as well as internationally. The forum will present an opportunity to set up a more permanent structure for collaboration among researchers and decision makers, and thus enhance the outcome and value of our research by developing prolonged and sustainable partnerships. Our team has proved its capacity to implement such a structure. The exchange forum will bring together decision makers from the areas of primary care services organization and chronic diseases  as well as researchers from various backgrounds with whom the research team has collaborated for several years.Moreover, we will benefit from the network the INSPQ developed through the Initiative sur le partage des connaissances et le développement des compétences, a consortium of provincial, regional and local partners (including CSSS) whose mandate is to implement population-based responsibility. Results will also be presented at conferences in Québec  as well as at national and international meetings. A research report will be written and scientific articles submitted for publication.The authors declare that they have no competing interests.An evaluation of the implementation of integrated services networks requires a range of research experience and skills. The research team includes knowledgeable researchers who have extensive research experience pertaining to health services organization and performance, and experience in developing innovative population-based interventions.re ligne (2005-2010) dans deux régions du Québec: Montréal et Montérégie . RP, coprincipal investigator is also Fellow in the Department of Social and Preventive Medicine at Université de Montréal. He will be mentor to the research team. SP is co-principal investigator. She has expertise in clinical preventive services, chronic illness management and surveys. PT is the nominated principal investigator and a researcher in epidemiology at McGill University. He possesses special expertise in the analysis of large medical administrative databases. The three principal investigators, affiliated with the Research Centre of the Centre Hospitalier de l'Université de Montréal (CHUM), have been lead researchers or co-researchers or have collaborated in the three projects listed above, and will be involved in all stages of the project.SP, RP, PT are members of a research team  linked to the Direction de santé publique (DSP) de Montréal and to the Institut national de santé publique du Québec (INSPQ). They have carried out many studies on primary care, its organization and its contribution to population health, including the following three: 1) Accessibility and Continuity of Care: A study of primary care services in Québec ; 2) Association of primary health care service models with perceived health status, utilisation of health services, ability for self-care and perceived quality of services in patients with chronic disease ; and 3) L'évolution de l'organisation et de la performance des services de 1The research team also includes co-researchers associated with the ESPSS team. RBDS, health economist and postdoctoral trainee with the ESPSS team, is co-investigator on project 3. Her primary interests are general practitioners' practice methods and analyses performed with medical administrative databases. MH has been coordinator for project 1 and co-investigator on projects 1 and 3, to which she has brought an expertise in primary care organization, integration of services and multidimensional analyses. She will be involved in the program implementation analysis.All the authors have been involved in drafting the manuscript; they also read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2296/12/126/prepubList of indicators and measures. This file lists the indicators and measures related to the effects of the program on patients and medical practice along with their sources of data.Click here for fileQuestionnaire for patients at their entry into the program. Questions relate mainly to utilization of health services, experience of care with the primary care physician, management and follow-up of chronic illnesses, self-management and quality of life related to diabetes and/or hypertension, sociodemographic characteristics and health status.Click here for fileQuestionnaire for patients at 12, 24 and 36 months after entry into the program. This questionnaire includes questions on utilization of health services, experience of care with the primary care physician, management and follow-up of chronic illnesses, self-management and quality of life related to diabetes and/or hypertension.Click here for fileQuestionnaire for patients who dropped out of the program during follow-up. Includes questions aimed at documenting the reasons for dropping out.Click here for filest patient to the cardiometabolic risk programQuestionnaire for primary care physicians at registration of a 1. This questionnaire contains questions pertaining to physicians' sociodemographic and professional characteristics, to the organizational characteristics of their clinics and to the physicians' practices regarding management of patients with diabetes or hypertension.Click here for filest patient to the cardiometabolic risk programQuestionnaire for primary care physicians 12-24-36 months after registration of a 1. This questionnaire includes questions on their assessment of the program: participation in the program, impacts on their patients and on their practice, relationships with specialized services, strengths and weaknesses of the program, as well as barriers and facilitating factors.Click here for fileInterview guide with individuals in charge of program implementation. These files contain discussion themes for the semi-structured interviews conducted at the beginning of the project, with individuals from the ASSS and CSSS who are responsible for program implementation. Discussion themes include an initial portrait of the services organization related to the activities of the program being implemented, the program components , the dynamics in each implementation site.Click here for fileCSSS Questionnaire. This file contains the questionnaire used to document interorganizational collaboration within local services networks in a previous project [ project .Click here for fileCSSS Questionnaire - End of project evaluation. This questionnaire, administered to key CSSS informants 40 months after project onset, will document the evolution of interorganizational collaboration within local services networks in relation to the program implementation.Click here for file


The following information was missing from the Funding section: B.L.M. was supported by the Childhood Infections Research Program (ChIRP) training program (1T32AI095202-01).


Lymphatic vessel invasion (LVI) exerts an important process in the progression and local spread of cancer cells. However, LVI as a prognostic factor for survival in non-small cell lung cancer (NSCLC) remains controversial.A meta-analysis of published studies from PubMed and EMBASE electronic databases was performed to quantity the effects of LVI on both relapse-free survival and overall survival for patients with NSCLC. Hazard ratios (HRs) with 95% confidence intervals (95% CIs) were used to assess the strength of these effects. This meta-analysis included 18,442 NSCLC patients from 53 eligible studies. LVI appeared in 32.1%  of tumor samples. In all, patients with LVI were 2.48 times more likely to relapse by univariate analysis (95% CI: 1.92–3.22) and 1.73 times by multivariate analysis (95% CI: 1.24–2.41) compared with those without LVI. For the analyses of LVI and overall survival, the pooled HR estimate was 1.97 (95% CI: 1.75–2.21) by univariate analysis and 1.59 (95% CI: 1.41–1.79) by multivariate analysis. Multivariate analysis showed a risk was 91% higher for recurrence  and 70% higher for mortality  in LVI-positive I stage patients compared with LVI-negative I stage patients. Subgroup analyses showed similar significant adjusted risks for recurrence and death in adenocarcinomas, and a significant adjusted risk for death in studies that utilized elastic staining with or without immunohistochemistry in defining LVI.The present study indicates that LVI appears to be an independent poor prognosticator in surgically managed NSCLC. NSCLC patients with LVI would require a more aggressive treatment strategy after surgery. However, large, well-designed prospective studies with clinically relevant modeling and standard methodology to assess LVI are required to address some of these important issues. Non-small cell lung cancer (NSCLC) accounts for approximately 80% of lung cancers and is the most common cause of cancer-related death worldwide Prognostic factors may be useful for identifying subgroup of patients with a worse outcome and selecting a more aggressive treatment strategy such as adjuvant chemotherapy RASSF1A could serve as an independent prognostic marker for NSCLC Pathological and biological factors involving in cancer development and progression, and genetic alterations have been identified to predict survival and improve treatment strategies of patients with NSCLC during the past decades Lymphatic vessels are regarded as the important route by which neoplastic cells reach local lymph nodes We searched the electronic databases PubMed  and EMBASE  between 1978 and 2012. Key words included non-small cell lung cancer, NSCLC, lymphatic vessel invasion, lymphatic involvement, lymphatic permeation, relapse, recurrence, prognostic, prognosis and outcome. The last search was updated in April 2012. The search was limited to English-language papers. This meta-analysis was limited to studies that dealt with the prognostic implications of LVI. The following criteria for eligibility among studies were set before selecting articles: (i) LVI was determined at least by hematoxylin and eosin (H&E) stain in surgically resected primary human lung tumors that had not received irradiation or chemotherapy prior to surgery, (ii) the relationship between LVI and survival was evaluated, and the results were published as a full paper, and (iii) available hazard ratio (HR) and 95% confidence interval (CI), or sufficient data are useful for examining HR and 95% CI. If a study detected lymphovascular invasion (including LVI or/and BVI) but did not analyze the association of LVI or BVI with survival separately, it will not be included in final meta-analysis.et alThe search and identification was independently conducted by three authors  according to a standardized approach, and the selection of a study was reached by discussion. Abstracts, reviews, other diseases and case reports were not included in this meta-analysis because of insufficient. When more than one of the same or overlapping publications was reported in several studies, only the most recently reported data or complete data were used for further combined analysis. We also performed a manual search from the references of relevant publications, including original articles and reviews, to identify additional records. For every study, last name of first author, year of publication, country of origin, patient resources, study size, methods for LVI evaluation, histology, and disease stage were collected. Three investigators  also independently performed methodological assessment. Disagreements were resolved by a third investigator (B. Wang). Quality scoring for each study was made according to the European Lung Cancer Working Party scale reported by Steels et alP value, or data from Kaplan-Meier survival curves. An observed HR >1 indicated a poor survival for the population with LVI. The χ2-based Q test was used to assess the heterogeneity of included studies P-value <0.05 was considered to indicate significant heterogeneity. When the test of heterogeneity was significant, the random-effect model based on Mantel–Haenszel method would be used. A funnel plot and Egger’s linear regression test were used to investigate any possible publication bias P value of <0.05 was considered statistically significant. We read the Kaplan–Meier curves using Engauge Digitizer version 2.11 (free software downloaded from http://sourceforge.net). All analyses were performed using STATA version 11.0 software .We performed separate meta-analyses using an adjusted or unadjusted hazard ratio (HR) for RFS and OS. In some studies, LVI was determined to be an independent prognostic indicator using multivariate analysis; HRs and 95% CIs were generally reported. Some studies reported the HR but did provide sufficient information on survival by LVI status; we thus calculated the HR and CIs according to the methods described by Parmar We perform an electronic data search in PubMed and EMBASE databases and yielded 154 citations. Additional 31 records were further identified via manually reviewing references. Five studies were excluded because an identical patient cohort occurred within another selected cohort The individual characteristics of the 53 eligible studies are summarized in et al., the significant risk for multivariate RFS was reported, but HR and 95% CI was not presented A total of 9.4% (5/53) of eligible publications In univariate analysis for OS, 26 studies identified LVI as a significant prognostic factor P = 0.67) irrespective of whether these studies used univariate or multivariate analyses.Evaluability was not associated with positivity in the systematic review. The rate of significant results was 60.4% for evaluable trials (32/53) compared with 60.0% (3/5) for non-evaluable trials . As for the global score, no significant difference was found between the evaluable and the non-evaluable trials (P = 0.71). Similarly, no statistically significant difference was shown between the significant trials and the non-significant trials in univariate (P = 0.39) or multivariate analysis for OS (P = 0.49) (As shown in  = 0.49) .P<0.0001)  I2 = 66.9%, P = 0.001). In multivariate analyses, patients with LVI were 1.73 times more likely to relapse compared with those without LVI   I2 = 52.0%, P = 0.027)  .P<0.0001) by univariate analysis with a significant heterogeneity . Our results also showed a risk was 59% higher for mortality  by multivariate analysis in LVI-positive patients compared with LVI-negative patients. Significant heterogeneity was not found among these studies   = 0.063) .P<0.0001) and 1.91 , respectively (P<0.0001) and multivariate analysis   .P<0.0001) and multivariate  analysis. We also found a significant higher unadjusted  and adjusted  risk for mortality of LVI-positive patients than that of LVI-negative patients. In these analyses, there was not still evidence of statistical heterogeneity  in studies evaluating LVI with H&E alone. By comparison, the combined HR for RFS by univariate analysis was 2.26  in studies evaluating LVI by elastic staining with or without immunohistochemistry, and 2.69  in those evaluating LVI by immunohistochemistry alone. Furthermore analyses found that the report by Kawata et al. as a source of heterogeneity P<0.001; heterogeneity test, Q = 11.98; I2 = 33.2%, P = 0.152). However, we obtained a borderline multivariate HR for RFS  in studies that utilized elastic staining or immunehistochemistry in defining LVI. Furthermore analysis showed the pooled multivariate HR estimate for RFS was 2.37  in studies evaluating LVI by immunohistochemistry.A high level of heterogeneity occurred when performing meta-analyses. Firstly, we conducted the subgroup analyses stratified by ethnicity or method for LVI evaluation. Although significant univariate risk for RFS in Asians and non-Asian populations was similar with the overall results, there was still evidence of statistical heterogeneity. Subgroup analyses by methods of LVI evaluation demonstrated that the combined HR for RFS by univariate analysis was 2.78  and 1.47 , respectively. When the meta-analysis was restricted into studies that utilized immunohistochemistry in defining LVI, the pooled HR estimate for OS was 2.22  by univariate analysis and 2.22  by multivariate analysis. So subgroup analyses by methods of LVI evaluation did effectively decreased or removed the heterogeneity in univariate analysis for OS.In univariate and multivariate analysis for OS in studies where LVI was investigated by elastic stains with or without immunohistochemistry, the summary HR estimates were 1.82  or for multivariate analysis of RFS (P = 0.14).Publication bias statistics were determined using the methods of Egger Microscopic metastasis begins with the local invasion by tumor cells into host stroma within or surrounding the primary tumor. When tumor cells penetrate a blood vessel or a peripheral lymphatic, they can detach, disseminate and arrest in the microvasculature through the circulation et al. found that the combination of the lymph endothelium-specific marker D2-40 and the panendothelial marker CD34 might be of value in detecting and distinguishing between LVI and BVI in breast cancer specimens LVI is significantly associated with lymph node metastasis and aggressive tumor behavior in NSCLC, indicating that it is a critical step in lymphogenous metastasis. The present analysis demonstrate that lymphatic invasion is present in 29.1% of overall patients and 26.1% of stage I patients. LVI is defined by the identification of tumor cells in the lumen of lymphatic vessels, which are often covered by endothelial cells and contained few lymphocytes. A pathological examination by H&E stain is helpful in recognizing LVI, but this method is usually impossible to distinguish between BVI and LVI, especially intratumoral areas. Lymphatic vessels do not contain elastic fibers, so they can not be confirmed by staining for elastic fibers which have been used as a routine pathological examination of BVI and pleural invasion. Elastic stains are less useful in excluding capillaries and are not useful in making the distinction between artifacts/stromal retraction and true lymphatic spaces. In fact, evaluation of LVI is relatively difficult using conventional H&E staining that showed a false-negative rate of 13.8 to18% and a false-positive rate of 4 to11.1% et al. found that lymphatic vessels and blood vessels are widely interconnected in the peritumorous region as spreading routes for the cancer cells et al. analyzed intratumoral or extratumoral LVI separately and found that patients with extratumoral lymphatic invasion were more likely to relapse or develop a distant metastasis than those with intratumoral lymphatic invasion and without lymphatic invasion et al. reported intratumoral lymphatic permeation and extratumoral lymphatic permeation were found to be 152 and 92 cases, and the 5-year OS rates were 64.1% and 32.7%, respectively LVI can occur in intratumoral or extratumoral region. Hanagiri RASSF1A methylation and BVI status in patients with NSCLC yielded significant results. Data from published trials rather than individual patient data were used in the systematic review. In addition, in most of meta-analyses, there was evidence of significant heterogeneity although the random-effect model based on Mantel–Haenszel method rather than the fixed-effect model was applied. The wide heterogeneity in results could been associated with differences in some baseline characteristics of their designs, including population sample size, the duration of follow-up, the adjuvant treatment they might have received, year of publication, staining techniques, and different criteria for positive findings. For example, different methods for LVI evaluation such as H&E or combination with elastic-van Gieson or Victoria blue-van Gieson staining were used. In fact, LVI status ranged from 0 et al., we used a methodology assessment on the treatment of lung cancer reported. However, this approach does not fully protect from potential bias because we could not take all the studies into account. These studies were finally maintained in the meta-analyses because the overall designs of studies were similar to those used in the other studies. TNM status remains the most important and differences in stage usually lead to heterogeneous results. However, heterogeneity was absent when the analysis was limited to studies of stage I or adenocarcinoma. Sensitive analysis showed individual studies contributed to significant heterogeneity. We concluded that heterogeneity probably come from differences of histological types and disease stages. Nonetheless, the precise reasons of heterogeneity remain unknown. Our results need to be substantiated by further prospective studies.Our meta-analyses had some limitations. The meta-analysis is based on retrospective data and the level of evidence is lower than that obtained by randomized controlled trials. However, similar attempts to examined prognostic influences of p53 expression, Usually, publication and reporting bias also has to be considered in meta-analyses. We did not look for unpublished papers, reviews or abstracts because the required data were usually available only in full publications. Positive results but not negative results tend to be accepted and published by journals. Another potential source of bias is related to the method used to extrapolate the HR. If the HR was not reported by author, it was calculated from the data included in the article or extrapolated from the survival curves, which involves making assumptions. In addition, each study adjusted for different covariates and only the studies that found significant results in univariate analysis performed multivariate analysis; thus, pooling the results may have produced bias. Nevertheless, no publication bias was detected using Egger’s test, suggesting that the summary statistics approximate the actual results.In conclusion, our systematic and meta-analysis of the association between LVI and the risk of recurrence and death for NSCLC patients suggests that tumors with LVI, compared to those without LVI, may be significantly associated with a higher recurrence and mortality risk. In line with our previous report about the prognostic role of BVI, LVI appears to predictive of poor outcome among patients with NSCLC including early-stage diseases and aednocarcinomas. Based on the present findings, surgically treated NSCLC patients including stage I disease with LVI might benefit most from adjunct systematic chemotherapy. Vessel invasion, including LVI and BVI, might be useful in defining individual patients’ risk after radical surgery and should be incorporated in the new edition of the TNM classification. However, large, well-designed prospective studies with clinically relevant modeling and standard methodology to assess LVI are required in defining the management of patients with NSCLC.Figure S1The flow of the included studies.(DOC)Click here for additional data file.Table S1Characteristics of literatures excluded in this systematic review.(DOC)Click here for additional data file.Table S2Data source for the estimating of HR form included studies evaluating lymphatic vessel invasion and outcome.(DOC)Click here for additional data file.Table S3Other clinicopathological variables in multivariate analysis of LVI and OS.(DOC)Click here for additional data file.Table S4Main characteristics and results of eligible studies evaluating LVI and RFS or OS in patients with NSCLC.(DOC)Click here for additional data file.Checklist S1PRISMA checklist.(DOC)Click here for additional data file.


Recently, diffuse-large-B-cell lymphoma (DLBCL) associated with serum IgM monoclonal component (MC) has been shown to be a very poor prognostic subset although, detailed pathological and molecular data are still lacking. In the present study, the clinicopathological features and survival of IgM-secreting DLBCL were analyzed and compared to non-secreting cases in a series of 151 conventional DLBCL treated with R-CHOP. IgM MC was detected in 19 (12.5%) out of 151 patients at disease onset. In 17 of these cases secretion was likely due to the neoplastic clone, as suggested by the expression of heavy chain IgM protein in the cytoplasm of tumor cells. In IgM-secreting cases immunoblastic features (p<.0001), non-GCB-type (p = .002) stage III-IV(p = .003), ≥2 extra nodal sites (p<.0001), bone-marrow (p = .002), central-nervous-system (CNS) involvement at disease onset or relapse (p<.0001), IPI-score 3–5 (p = .009) and failure to achieve complete remission (p = .005), were significantly more frequent. FISH analyses for BCL2, BCL6 and MYC gene rearrangements detected only two cases harboring BCL2 gene translocation and in one case a concomitant BCL6 gene translocation was also observed. None of the IgM-secreting DLBCL was found to have L265P mutation of MYD88 gene. Thirty-six month event-free (11.8% vs 66.4% p<.0001), progression-free  and overall  survivals were significantly worse in the IgM-secreting group. In multivariate analysis IgM-secreting  and IPI-score 3–5  were the only significant factors for progression-free-survival. Notably, four relapsed patients, who were treated with salvage immmunochemotherapy combined with bortezomib or lenalidomide, achieved lasting remission. Our data suggests that IgM-secreting cases are a distinct subset of DLBCL, originating from activated-B-cells with terminally differentiated features, prevalent extra nodal dissemination and at high risk of CNS involvement. Diffuse-Large-B-cell Lymphoma (DLBCL) is a biologically heterogeneous entity de novo DLBCL This is a retrospective study evaluating the incidence of IgM-secreting DLBCL and comparing this subset with a non-secreting control group for clinicopathological features and survival. The study was approved by our Institutional Review Board  and was conducted in accordance with the regulations of health information protection policies. Patients were asked to sign a written consent at disease onset in order to collect their data on an electronic database and to allow further pathological characterization of biological material harvested for diagnostic purposes. Clinical data, including HCV and HBV markers screening, were prospectively collected and obtained from corresponding medical records. A hundred and fifty-one patients  diagnosed with conventional High risk patients younger than 61 years were treated with R-CHOP every 14 days Before chemotherapy all patients were tested for hepatitis B surface antigen and its antibody , antibodies to the core antigen , and for hepatitis C virus (HCV) antibodies. Commercially available enzyme immunoassays were used for HBV and HCV determinations . All cases were tested for hepatitis B virus deoxyribonucleic acid polymerase chain reaction . Only cases that were positive for HCV antibodies were further investigated for HCV–RNA . Either active, inactive or occult HBV carriers Classification and subtyping of all tumors followed the definitions of the 2008 WHO classification of DLBCL Since the aim of this study was to define the clinicopathologic features of the IgM-secreting subset, the analyses of recurring chromosomal translocations, MYD88 gene mutation, the BCL2/MYC immunhistochemical score, and the presence of EBV-infection were carried out only in the IgM-secreting subset and in a small control group of non-secreting DLBCL cases  in a final volume of 25 mL with 50 nM of each primer and 100 ng of DNA. Thermal cycling conditions were as follow: 2 min. at 94°C, followed by 40 cycles of 94°C for 30 s, 57° for 30 s. and a final extension at 68°C for 5 min. The amplified PCR products (159 bp) were separated on 2% agarose gel. One case of Waldenström macroglobulinemia was used as MYD88 L265P mutation positive control (Detailed methodologies are fully described in Allele-specific polymerase chain reaction (AS-PCR) was performed using two reverse primers designed to recognize the mutant and the wild-type allele of MYD88 L265P as previously described Categorical data were compared using Fisher's exact test and two-sided p-value, whereas for ordinal data, non-parametric tests were used. The definitions of complete response (CR), event free survival (EFS), progression free survival (PFS) and overall survival (OS) were the standard In 19 out of 151 (12.6%) DLBCL a serum monoclonal IgM component was detected. The serum level of monoclonal IgM at diagnosis varied from 2.5 g/dL to 0.22 g/dL . Eleven out of 19 (58%) patients were monitored by serum immunofixation and FLC k/λ ratio during the course of treatment and follow-up. After 1-3 cycles of R-CHOP the monoclonal IgM component disappeared and the FLC k/λ ratio returned to the normal range in all of these patients. Three out of four patients (75%) at tumor recurrence were negative for serum monoclonal IgM and had a normal FLC k/λ ratio. In the remaining case the reappearance of a monoclonal IgM and of FLC k/λ abnormal ratio preceded relapse. Four patients who are in continuous complete remission are persistently negative for both. Two patients with an IgM MC not related to the neoplastic clone  showed nOne-hundred and seven out of 151 (71%) DLBCL were suitable to be classified for the cell-of-origin (COO) using the Hans algorithm Molecular analyses were performed in the IgM-secreting DLBCL and in a control group of non-secreting DLBCL  were analyzed for clinical characteristics and prognostic scores . One-hunnd complete remission at +12 months after rituximab-DHAP and lenalidomide maintenance. One patient, who achieved less than partial remission after four cycles of R-CHOP, is presently in 1st CR after two cycles of RMAD Fifteen out of 17 (88.2%) IgM-secreting patients had a DLBCL-related event compared to 36 out of 107 (33.6%) control cases (p<.0001). Nine out of 17 (53%) IgM-secreting patients and 27 out of 107 (25%) control cases died (p<.0001). In the IgM-secreting group, seven patients died with primary refractory or relapsed lymphoma. Two patients died within one month from the start of treatment for toxicity: one patient was in very poor conditions with diffuse meningeal and brain involvement, while the other died of ischemic stroke during treatment. Worthy of note, seven out of 17 IgM-secreting patients (41%) had CNS localization at disease onset (2/17 cases) or during progression/relapse (5/17 cases) . One of The estimated 36-month PFS   and OS . The difThe identification of poor-prognostic subgroups correlated to defined biological tumor characteristics is the aim of modern oncological research. In the case of DLBCL this issue is presently a matter of intense development and debate. Several prognostic factors and scores have been proposed to better stratify patients who would benefit from more intensive treatment than R-CHOP DLBCL with serum IgM MC represented a sizable subset of our series. The majority of these cases were defined as IgM-secreting DLBCL since same type of heavy chain IgM and κ or λ light chains were detected in tumor cells. Most patients had advanced disease, involvement of several extra nodal sites including bone marrow and high IPI-score. Worthy of note, the incidence of CNS involvement at diagnosis or during relapse/progression was surprisingly high. Most of these poor prognostic features remained significantly more frequent in the IgM-secreting subset even when compared to the IgM+/non-secreting subset. Monitoring patients by immunofixation and FLC κ/λ ratio during and after therapy was of little value for predicting relapse. All IgM-secreting cases were classified as non-GCB-type and the great majority showed Immunoblastic morphology.L265P mutation of MYD88 gene has been reported within 6.5% and 17% of unselected DLBCL Recurring chromosomal translocations have been reported in DLBCL In 2011 Maurer et al The expression of the heavy chain IgM gene was shown in the Wright signature to be one of the most discriminating genes between GCB and ABC DLBCL subtypes Recently, it has been identified a plasmablastic subtype of DLBCL thought to derive from terminally differentiated B-cells In our series of IgM-secreting DLBCL, we found a strikingly high incidence of CNS involvement. This finding is in keeping with previous studies showing that primary lymphoma of the central nervous system expresses an IgM isotype Outcome after R-CHOP was disappointing in IgM-secreting patients with most relapses occurring within one year from diagnosis. Interestingly, four patients who relapsed without CNS involvement achieved lasting complete remission when treated with immunochemotherapy combined with biological drugs such as bortezomib or lenalidomide Finally, we speculate that IgM-secretion in DLBCL has since been underrated because it is easily missed by clinicians given the rarity of associated clinical signs, its low entity at diagnosis and rapid disappearance during treatment.The main limitations of our study pertain to its retrospective nature. IgM expression analysis and COO subtyping were not assessable in all the cases because of the lack of suitable material. Moreover, we classified tumors basing on the Hans' algorithm, which is less sensitive and specific than GEP analysis In DLBCL patients the identification of reliable prognostic markers that may guide in selecting a more specific treatment is of considerable importance File S1Supplementary Materials.(DOCX)Click here for additional data file.


The incidence of depressive symptoms increases during adolescence, from 10.0% to 24.5% at age 11 to 15, respectively. Experiencing elevated levels of depressive symptoms increases the risk of a depressive disorder in adulthood. A universal school-based depression prevention program Op Volle Kracht (OVK) was developed, based on the Penn Resiliency Program, aimed at preventing the increase of depressive symptoms during adolescence and enhancing positive development. In this study the effectiveness of OVK will be tested and possible mediators of program effects will be focus of study as well.The effectiveness of OVK will be tested in a randomized controlled trial with two conditions, intervention (OVK) and control condition . Schools are randomly assigned to research conditions. OVK will be incorporated in the school curriculum, maximizing program attendance. OVK consists of 16 lessons of 50 min, given by trained psychologists to groups of 11-15 students. OVK contains Cognitive Behavioral Therapy, social skills training, problem solving and decision making. Outcomes are measured at 6, 12, 18 and 24 months follow up, to monitor long term program effects. Primary outcome is level of depressive symptoms, secondary outcomes are: anxiety, hopelessness, cognitive bias, substance use, truancy, life satisfaction, coping, self-efficacy, optimism, happiness, friendship, school performance and school attitude. The questionnaires for students will be administered in the school setting. Parents will complete a questionnaire at baseline only.In this paper the study into the effectiveness of the depression prevention program OVK was described. It is expected that OVK will prevent the increase in depressive symptoms during adolescence and enhance positive development in the intervention condition, compared to the control condition. If OVK will be effective, it can be implemented in the school context by which numerous adolescents can be reached.NTR2879Netherlands Trial Register (NTR):  Depression is a significant public health concern that places a heavy burden on people and society. In the Netherlands, around 700.000 adults and 37.000 adolescents 13-17 years) are diagnosed with a depressive disorder annually -5. At ag-17 yearsBesides preventing the onset of aversive outcomes, adolescent functioning and development could be enhanced by promoting positive development. The Broaden and Build Theory posits that when experiencing positive emotions, people's thoughts and subsequent actions are expanded by becoming more creative and diverse when compared to experiencing negative emotions . By meanEarly adolescence offers great opportunities for mental health promotion and the prevention of mental health problems. As the brain matures, young adolescents' social, emotional and cognitive systems are developing ,18, and Research shows that universal depression prevention programs can be effective in reducing depressive symptoms in adolescents ,23. StroThe present study will test the effectiveness of OVK in an adolescent Dutch population by means of a randomized controlled trial with follow-up assessments up to 24 months. In addition, mechanisms by which the program effects will be obtained will by studied as well. Scholars have tested the effectiveness of depression prevention programs ,23. The The present study has three main goals: 1) Testing the effects of OVK in a Dutch adolescent population in limiting the increase of depressive symptoms during adolescence, 2) identifying mediating mechanisms that can explain program effects, and 3) testing the effects of OVK on secondary outcomes. It is hypothesized that OVK will be effective in preventing the increase in depressive symptoms in adolescence in the intervention condition during the 24 months following the program, in comparison to the control condition. In relation to identifying mediating mechanisms, it is hypothesized that OVK will lead to less cognitive biases, more adequate coping, higher self-efficacy, less hopelessness, and more optimism. Each of these constructs are hypothesized to lead to less (increase in) depressive symptoms; being mediators of the program effects on depressive symptoms. Secondary outcomes include anxiety, substance use, truancy, life satisfaction, happiness, friendship, school performance and school attitude.The effectiveness of OVK will be studied by means of a randomized controlled trial with two conditions: intervention versus control (no intervention)  effectiveness of the program, the outcomes will be assessed through self-report questionnaires approximately 1 month before the start of the program (baseline: T1), directly after the program (T2), and 6 (T3), 12 (T4), 18 (T5) and 24 (T6) months after the end of the program. Adolescents in the control condition will complete the questionnaires at exactly the same time points. Parental self-reports will be administered at baseline only. Adolescents assigned to the experimental condition will receive the OVK program, and adolescents in the control condition will receive care as usual (no program). As a reward for participation in the study, the schools in the control condition will receive a training for teachers in the OVK program between T3 and T4, on the strict condition that they will not provide the program to the adolescents who participated in the study.NTR2879.OVK will be incorporated into the school curriculum; the 16-lesson program will be delivered once a week by trained psychologists to groups with a maximum of 15 adolescents. Incorporating the program in the school curriculum enables us to truly provide the program to all adolescents in a certain grade. As a result, high attendance rates will be assured and no stigmatization will occur. To keep the attrition rates at a minimum, all adolescents absent at a given measurement will be provided another possibility in the same time period to complete the questionnaire. Also, adolescents who leave school will be asked, with assistance of the school, to continue participating in the study. The trial is registered at the Dutch Trial Register: Participants will be adolescents in their second year of secondary school  . SecondaA meta-analysis on 17 controlled studies on PRP indicated the significant average effect size to be 0.20 at 12-months follow-up with the CDI > 13 score as cut-off . Power aWith support from participating schools, parents of eligible adolescents will receive a letter, in which they will be fully informed about the study aims and procedure. For schools in the intervention condition a presentation will be held to inform parents and teachers about the content of the program and the study. Given that schools incorporate the program into their curriculum, passive consent will be obtained from parents. Each wave, adolescents will fill out a questionnaire in class during school hours. The 50-minute questionnaire will either be a web-based or a paper version, depending on the school's preferences. Parents fill out a 25-minute questionnaire at baseline only, addressing parental depression, anxiety, child and parental diagnostic status, and parenting skills. The mother will be asked to fill out the web-based questionnaire. Three weeks after the first mailing, non-responders will receive a reminder with the possibility to complete the questionnaire online or on paper. When not responding within 2 weeks, these parents will be contacted by phone. At any time, parents are free to withdraw themselves, and/or their child from the study. The Ethical Committee (ECG) of the Faculty of Social Sciences of the Radboud University Nijmegen has approved the study design and data collection.OVK is based on Beck's Cognitive Theory -37, the OVK consists of 16 lessons of 50 minutes. The first 8 lessons are devoted to explaining and practicing the CBT derived principles. Lessons 9 to 16 are directed at social and coping skills, self-esteem, problem solving, and decision making. During every lesson, the theory behind the techniques is shortly explained followed by actively practicing the skills by students. Several means are used for teaching, such as discussions, role-plays, and skits. Group leaders are responsible for group atmosphere and cohesion, and they will guide discussions and role-plays. Each lesson includes homework for the next lesson. Practicing skills facilitates internalization of these skills, which results in better program effects .In lesson 1 to 8, students are familiarized with the CBT-principles; the relation between an event, thought, feeling, and behavior. First, students are introduced to the program, whereby building group cohesion, discussing confidentiality, and setting group rules are central. The students are told they will learn to deal with daily situations they feel or think pessimistically about. Afterwards, the link between a situation, thought, feeling and behavior is outlined. Inaccurate negative automatic thoughts  are discussed. Challenging mind traps by means of formulating alternative realistic-optimistic thoughts on the spot, and searching for proof of your thoughts, is practiced. Lessons 9 to 16 are devoted to interpersonal problem-solving whereby the CBT-principles taught in the first half of the program are applied to the interpersonal domain. In sum, students learn about: (1) The link between situations, thoughts, feelings, and behaviors, 2) cognitive biases, including pessimistic versus optimistic explanatory styles, 3) cognitive restructuring skills, and 4) students learn a variety of techniques for coping and problem-solving, including assertiveness, negotiation, decision making, and relaxation.The program will span approximately 5 months. Classes will be split up in two groups, thus consisting of 12-15 students, because this appeared to be the ideal class size for teaching PRP . Gender The primary outcome will be the level of depressive symptoms at 6, 12, 18 and 24 months follow up measured with the Child Depression Inventory (CDI) , which iThe 'negative' secondary outcomes will be the level of anxiety, truancy and substance use. Anxiety will be assessed with the Revised Children's Manifest Anxiety Scale (RCMAS) . The RCMThe parent questionnaire will include measures of socio-demographics of both parents and the participating child: Information about (prior) diagnosis and treatment, marital status, parental SES indicators such as education, employment, working hours, and salary. In addition, parents' own characteristics and parenting skills will be included . All instruments are widely used and have satisfactory psychometric properties. The Beck Depression Inventory  will be Data will be analyzed in accordance with the intent-to-treat principle and for the completers only. Multiple imputations will be used for missing observations at follow-ups. The hypotheses will be tested with regression analyses for dichotomous outcome measures in MPLUS 5.1 ,66. AddiN = 1324) by means of a randomized controlled trial with an intervention and control condition. Follow-up measures will be conducted up to 24 months to capture long-term program effects. It is hypothesized that adolescents in the intervention condition will display less depressive symptoms during the follow-up measures, compared to adolescents in the control condition. It is expected that cognitive bias, problem and engagement-focused coping, self-efficacy, hopelessness, and optimism mediate the intervention effect on depression. Lastly, the present study will test whether OVK has effects on secondary 'negative' outcomes: Anxiety, substance use, and truancy, as well as on positive outcomes: Life satisfaction, happiness, friendship, and school performance and attitude.In the present study the effectiveness of Op Volle Kracht, a universal school-based depression prevention program for adolescents, in preventing the increase in depressive symptoms during adolescence will be tested in a large sample (Several strengths of the present study are identified. First, both prevention of aversive outcomes and the promotion of positive development are central in OVK. Therefore, program effects on depression, anxiety, and hopelessness, but also on optimism, happiness and life-satisfaction will be examined. Second, program effects will be tested in randomized controlled study and a large sample. Third, trained psychologists will administer the program ,23. FourLimitations of the present study are present as well. No placebo treatment will be included in the design, limiting the extent to which lower depressive symptoms in the intervention condition can be uniquely ascribed to the program. Another limitation of the study will be the single use of self-reports; interviews by trained professionals would be more sensitive in identifying depressive symptoms . At the If OVK will found to be effective in limiting the increase in depressive symptoms during adolescence over the 2-year follow-up period and by enhancing positive development, this will have positive consequences for the adolescents themselves, their family, friends, and for society. The program can be implemented in schools whereby the lives of adolescents can be enhanced and the negative consequences of experiencing depressive symptoms can be minimized. The needs expressed by school teachers and professionals to stimulate social and emotional development of adolescents at school are thereby addressed. Moreover, OVK has the potential to reach all adolescents without stigmatizing groups and without the risk of missing the ones who need the skills thought in OVK the most. In addition, the present study could increase our knowledge on the factors that contribute and foster intervention effects, thereby enabling us to understand the prevention of aversive outcomes and promotion of positive outcomes in adolescence, which could result in further improvement of universal depression prevention programs.The present paper described a Dutch adolescent school-based depression prevention program OVK and the study design into its effectiveness in preventing the onset of depressive symptoms and promoting positive outcomes over the 24 follow-up period. By means of this study a first attempt will be made to shed light on the mechanisms by which program effects will be obtained.The authors declare that they have no competing interests.YR is responsible for data collection, analysis, and reporting of study results. All others authors are supervisors and grant applicators. All authors read and approved the final protocol.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/12/21/prepub


Understanding the linkages between ecosystem services (ES) and human well-being (HWB) is crucial to sustain the flow of ES for HWB. The Millennium Ecosystem Assessment (MA) provided a state-of-the-art synthesis of such knowledge. However, due to the complexity of the linkages between ES and HWB, there are still many knowledge gaps, and in particular a lack of quantitative indicators and integrated models based on the MA framework. To fill some of these research needs, we developed a quantitative index system to measure HWB, and assessed the impacts of an external driver – the 2008 Wenchuan Earthquake – on HWB. Our results suggest that our proposed index system of HWB is well-designed, valid and could be useful for better understanding the linkages between ES and HWB. The earthquake significantly affected households' well-being in our demonstration sites. Such impacts differed across space and across the five dimensions of the sub-index . Since the conceptual framework is based on the generalizable MA framework, our methods should also be applicable to other study areas. Understanding the linkages between ecosystem services (ES) and human well-being (HWB) is crucial to sustain the flow of ES for HWB Although the MA is a monumental work, the linkages between ES and HWB are complex and remain poorly understood So far there have been relatively few studies quantitatively integrating the four components of the MA conceptual framework to study the linkages between ES and HWB In recent years, a substantial amount of effort has been made to quantify various ES at multiple scales and assess the trade-offs and synergies that occur in both natural ecosystems and constructed/artificial ecosystems In response to some of these research needs, in this study, we attempt to (1) develop a new index system to quantify HWB based on the MA framework and (2) empirically demonstrate the application of the index through assessing the impacts of the 2008 Wenchuan Earthquake on HWB.http://www.humanresearch.msu.edu/).We obtained the permission from the Wolong Administration Bureau of Wolong Nature Reserve for conducting household surveys inside the reserve and outside the reserve at Sanjiang Township through the Sanjiang Conservation Station. Since many of our interviewees are not literate or have very low level of education, a verbal consent process was used. We first read the verbal consent script to the selected interviewees. Once they agreed, we then continued to interview them. If consent was not obtained, we did not collect any further information from that interviewee and switched to the next selected interviewee. The survey instruments, verbal consent process, and script were approved by the Institutional Review Board of Michigan State University  to construct the overall index and sub-indices. CFA addresses several problems in this type of data analysis. First, it allows us to avoid the pitfall of assuming individual items are identical with the underlying theoretical variables There are four reasons we chose the Wolong Nature Reserve (WNR) and the adjacent Sanjiang Township (SJT) in Wenchuan County of Sichuan Province, southwestern China  as our s2 with approximately 4,900 local rural residents from about 1200 households. SJT is 491 km2 of which 344 km2 is enclosed in WNR, and all local residents now live in the remaining 147 km2 zone outside WNR (WNR is approximately 2000 kmside WNR . SJT hass 8.0 or Mw 7.9) was close to our demonstration sites  after the earthquake. One major reason is undoubtedly post-earthquake road construction. Due to the implementation of the Wenchuan Earthquake Reconstruction Plan Our results also suggest that households with higher overall HWBI or less freedom of choice and action pre-earthquake were less affected than those with lower indices. These results are consistent with findings from other studies such as the Hurricane Katrina The post-earthquake reconstruction outside the reserve was almost completed by May of 2010 but is still ongoing inside the reserve with completion planned for 2015. At this time it is difficult to predict how the earthquake-induced changes in HWB may in turn affect households' socioeconomic activities and use of ES in the long run. But according to information gained from regular monitoring by the local government and our own field investigation, starting shortly after the earthquake there seemed to be dramatic increases of illegal logging and poaching outside the reserve and increases in poaching inside the reserve. This indicates that post-earthquake reconstruction must consider households' use of and dependence on ES and their interactions with HWB. Priorities should be given to helping local households to build capacity and find alternative income sources that do not harm or offset conservation efforts.We believe the HWB index systems we developed has some major advantages compared to other approaches that have been proposed. First, our index system is based on the general MA framework and explicitly considers the contribution of ES to HWB. Second, its construct validity has been confirmed empirically in our demonstration sites. Therefore, it could easily be applied to other study areas and across different scales with some modifications if necessary. Third, our index system is developed using CFA techniques that examine the relationships of multiple indicators to multiple factors  and the correlations among different dimensions. This allows a nuanced assessment of the measurement properties of the index and its components. Finally, our index system provides both a composite index and sub-indices. This allows the quantitative examination of the interwoven linkages between different types of ES and different components of HWB. The value of having both aggregate and disaggregate measures was evident when we considered the effects of the earthquake, which differed across sub-indices. However, similar to other composite indices but unlike single question indices, we acknowledge that this advantage is achieved at the costs of more data being needed and a more effort in constructing the measure.We developed an index of HWB based on the MA framework and applied it to a region in which ES are very important for HWB. Our results suggest that our HWBI system has reasonable internal and external validity. Our index was able to detect the Wenchuan earthquake's impact on household well-being, and show that the estimated impacts differed between households inside and outside the reserve as well as across the five dimensions of the sub-index.Human and natural systems are complex and coupled Our proposed HWBI system seems to be a viable approach and could be useful for further research to better understand the linkages between ES and HWB. We demonstrated the development of the HWBI system and its application in assessing the impacts of 2008 Wenchuan Earthquake on households' well-being. Since the conceptual framework is based on the generalizable MA framework, our methods should also be applicable to other study areas.Data and Instrument File S1Dataset and instrument used in this study.(ZIP)Click here for additional data file.Table S1The index system for assessing human well-being based on the Millennium Ecosystem Assessment conceptual framework.(DOC)Click here for additional data file.Table S2Standardized coefficients of the confirmatory factor analysis for Human Well-Being Index (HWBI).(DOC)Click here for additional data file.


Halobacterium salinarum ssp. NRC-1 (NRC-1), which was used as a model halophilic protein. The effects of salt on the structure and stability of NRC-1 and of E. coli CysRS were investigated through far-UV circular dichroism (CD) spectroscopy, fluorescence spectroscopy, and thermal denaturation melts. The CD of NRC-1 CysRS was examined in different group I and group II chloride salts to examine the effects of the metal ions. Potassium was observed to have the strongest effect on NRC-1 CysRS structure, with the other group I salts having reduced strength. The group II salts had little effect on the protein. This suggests that the halophilic adaptations in this protein are mediated by potassium. CD and fluorescence spectra showed structural changes taking place in NRC-1 CysRS over the concentration range of 0–3 M KCl, while the structure of E. coli CysRS was relatively unaffected. Salt was also shown to increase the thermal stability of NRC-1 CysRS since the melt temperature of the CysRS from NRC-1 was increased in the presence of high salt, whereas the E. coli enzyme showed a decrease. By characterizing these interactions, this study not only explains the stability of halophilic proteins in extremes of salt, but also helps us to understand why and how group I salts stabilize proteins in general.Proteins from extremophiles have the ability to fold and remain stable in their extreme environment. Here, we investigate the presence of this effect in the cysteinyl-tRNA synthetase from  Understanding the effects that environmental factors have on a protein's structure is a difficult task. A protein's structure, or fold, is influenced by many interactions. The most influential is the interaction of nonpolar residues that form the hydrophobic core of the protein. Electrostatic interactions between the protein's polar residues are able to form salt bridges and hydrogen bonds that essentially lock the protein into a particular configuration. While some proteins can spontaneously fold into the correct structure, others need external cues in order to fold correctly. In the cell, chaperonins play a major role in protein folding and structure Halophiles, organisms that thrive in extreme amounts of salts, can also use the surrounding environment to their advantage. Electrostatic and hydrophobic interactions of a protein are changed when it is subjected to an extremely ionic environment. Electrostatic interactions, especially salt bridging, become unfavorable, due to their disruption by ions, while the hydrophobic effect between nonpolar residues is strengthened Haloarcula marismortui was found to have no secondary structure without salt, and to gain most of it in 2 M KCl. For this enzyme, salt-dependent folding was observed around 0.25 M KCl, where there was an increase in α-helical content Halobacterium salinarum ssp. NRC-1 revealed partial secondary structure in no salt, but the amount of secondary structure increased in higher concentrations of NaCl, MgCl2, and MnCl22How these adaptations relate to the stability of halophilic protein structure is unknown. Previous research has demonstrated the importance of salt for halophilic protein folding Halobacterium salinarum ssp. NRC-1. This enzyme catalyzes the attachment of cysteine to its cognate tRNA to generate a substrate for the ribosome. Because this is a required reaction in every cell, CysRS is highly conserved and, therefore, any evolutionary adaptations that the H. salinarium ssp. NRC-1 required for activity and stability in high salt should be more readily discernible. While adaptations to environmental extremes are not always obvious, a number of adaptations in NRC-1 CysRS to salt have been observed and characterized through sequence alignments with E. coli CysRS and homology modeling Because we are interested in how proteins maintain their structure and stability under molar concentrations of salts, a protein was chosen that contains known halophilic adaptions while still remaining similar to a non-halophilic homologue. For this reason, we chose to study the salt-dependent stability of the cysteinyl-tRNA synthetase from From past work, it was determined that NRC-1 CysRS loses half of its activity at KCl concentrations below 2 M E. coli CysRS as a control. We also investigate the different effects of group I- and some group II-containing salts on the salt-dependent structure of NRC-1 CysRS. Our data shows that this halophilic protein displays salt dependent folding and stabilization of its structure, even in concentrations of salt below NRC-1's intracellular conditions. The protein structure was also observed to be stabilized more effectively by potassium than by other group I metals. However, all group I metals had similar stabilizing effects which indicate that group I metals, in general, can improve halophilic protein stability. Group II metals were significantly less stabilizing towards the overall structure of NRC-1 CysRS. In contrast, the non-halophilic CysRS, E. coli CysRS, showed destabilization of its structure as the concentration of salt exceeded 500 mM KCl. These data show that the NRC-1 CysRS has prominent halophilic adaptations that interact best with group I metals, especially potassium. Our conclusions should help improve our overall understanding of protein stability and help to engineer proteins that are particularly salt stable for biotechnology work.In this work, we describe the effects of salts on the structure and stability of NRC-1 using far-UV circular dichroism, intrinsic fluorescence, and thermal melt experiments. We used 2, CsCl, MgCl2, CaCl2, NiCl2, and CuCl2) along with solid Tris(hydroxymethyl)aminomethane (Tris) base, sodium phosphate (monobasic and dibasic) and 2-mercaptoethanol (β-Me) were purchased from ThermoFisher Inc. Solid RbCl and imidazole were purchased from Acros Organics. All salt stock solutions (3–3.8M) were made in 50 mM Tris pH 7.5 buffer and were filtered using 0.22 µm Millipore filters. Centrifugation was done using a Beckman Model J2-21 Centrifuge and a Beckman JA-20 rotor. Chromatography was performed on a GE-Healthcare Äkta-Purifier and monitored by inline UV absorbance at 280 nm.The salts used  and the fractional change in ellipticity were calculated according to Golynskiy, et al. M was calculated by solving for the temperature at which ΔG = 0 in I is ellipticity at 222 nm at temperature T, FI is the folded ellipticity at 4°C, and UI is the final unfolded ellipticity. The fractional change ellipticity (FCE) was calculated based on Circular dichroism spectra were collected from 300 to 200 nm at room temperature in a 1 cm quartz cuvette. A scanning speed of 50 nm/min was used with a 1 second averaging time and a 0.1 nm step. The signal was averaged over 10 scans. Each spectra was acquired independently three times. Protein was diluted to 1 µM in 50 mM Tris pH 7.5 and 0.5 mM β-Me with various salt solutions. Thermal denaturation experiments were conducted using 5 µM protein in a 1 mm quartz cuvette with various concentrations of potassium chloride and 5% (w/w) glycerol, which was included to prevent evaporation. The samples were heated from 20 to 90°C at a rate of 1.0°C/min, with 3 averaged CD spectra collected from 300 to 200 nm every 2°C (5 nm bandwidth and 100 nm/min scanning speed). Melting transition temperatures  which fits with its potassium rich cellular environment 2 and CaCl2 had only a fraction of the effect of group I salts, averaging 0.6×106 millidegrees, suggesting a relatively weak amount of salt-induced folding. While strontium and barium were not tested due to solubility issues, we would expect the same trend as seen with magnesium and calcium. The divalent transition metals, Zn+2, Ni+2 and Cu+2 were also tried; however, interference from NiCl2 and CuCl2 in the CD signal made testing their effect on NRC-1 CysRS structure not possible (data not shown). Zn2+ is an important metal for the active site of CysRS, where it is involved in cysteine discrimination 2, there was a denaturing effect on NRC-1 CysRS observed in the CD signal, perhaps due to disruption of the structure  (M appeared to increase slightly to 35.2°C (±0.7°C). A significant increase of the TM was observed when NRC-1 CysRS was heated in 2 M KCl to 42.8°C (±0.5°C), indicating a stabilizing effect by molar concentrations of KCl.The effect of salt on the stability of NRC-1 CysRS was examined by thermal denaturation studies of the enzyme while following the CD signal at 222 nm in 0 M, 500 mM and 2 M KCl. The stability of a protein is defined as the Gibbs free-energy change upon unfolding  to 90°C . The tra(±2.2°C) . At 500 E. coli CysRS, salt decreased the enzyme's stability. Without salt, E. coli CysRS was observed to melt with two transitions, having a calculated melting temperature at the higher transition of 66.8°C (±0.3°C)  , and preH. salinarum spp. NRC-1 and the mesophilic E. coli. The large change in fluorescence emission spectra of NRC-1 CysRS in high salt compared to the E. coli version suggests that the halophilic version is more structurally sensitive to its ionic environment  than Na+, Rb+, and Cs+. The high effect of K+, then, may be due to evolutionary selection, since it is the most abundant cation in the intracellular environment of H. salinarum ssp. NRC-1 (4.7 M) The salt-dependent structure of NRC-1 CysRS also appeared to be affected by the type of salt . K+ was E. coli and NRC-1 CysRS. In the E. coli version of the enzyme, the thermal denaturation shifted from a double-transition curve in no salt to a single transition in 500 mM KCl. The lost transition could correlate to the disruption of stabilizing electrostatic interactions which are not able to form in highly ionic environments. This is a compelling explanation for E. coli CysRS's reduced stability in high salt. In contrast, NRC-1 CysRS becomes more stable in high salt, suggesting external, stabilizing salt-protein interactions instead of intramolecular electrostatic interactions. This salt-protein interaction would most likely be due to favorable interactions between the negatively charged acidic surface residues on the halophilic protein and the positively charged metal ion of the salt.This work has shown that, like many other extremophiles, halophiles are capable of using their environment to help fold their proteins and keep their folded form stable. Here, it appears that salt-protein interactions replace the usual electrostatic interactions that proteins use to remain folded correctly. Support for this is found in the thermal denaturation experiments conducted on E. coli CysRS were consistently higher than NRC-1 CysRS for all the salt conditions tested. It is unclear whether this is an inherent difference between the two enzymes or if it is a consequence of non-physiological experiment conditions. The stability of NRC-1 CysRS would be expected to increase further past 2 M KCl, especially since the intracellular potassium concentration of H. salinarum ssp. NRC-1 is near 4 M Another observation from the thermal denaturation experiments was that the melting temperatures of H. salinarum NRC-1.Overall, our findings on the salt-dependent structure and stability of NRC-1 CysRS support previous research that showed the same effects in different halophilic proteins. This demonstrates how halophilic proteins utilize their salty environment to fold into a functional form by stabilizing protein structure in high salt concentrations. Also, NRC-1 CysRS's apparent specificity for potassium ions suggests that evolution has selected for strong protein-salt interactions with that cation, a consequence, most likely, from the prevalence of that ion in the intracellular environment of Figure S12 at 222 nm.CD signal of NRC-1 CysRS in ZnCl CD ellipticity of 1 µM NRC-1 CysRS in various concentrations of ZnCl2 at 222 nm. The standard deviation of three separate spectra is shown as error bars.(TIF)Click here for additional data file.Figure S2CD signal of E. coli CysRS in KCl at 222 nm. CD ellipticity of 1 µM E. coli CysRS in various concentrations of KCl at 222 nm. The standard deviation of three separate spectra is shown as error bars.(TIF)Click here for additional data file.