Cross-case analyses of factors that influence the process and implementation quality of the Tier 1 Program of the Project P.A.T.H.S. based on seven cases were carried out. Systematic and integrative analyses revealed several conclusions. First, several factors related to policy, people, program, process, and place (5 “P”s) were conducive to the successful implementation of the Tier 1 Program in the schools. Second, there were obstacles and difficulties with reference to the 5 “P”s that impeded the quality of implementation. Third, policy support and people  are two main groups of factors that influence the quality of program implementation. Fourth, although there were different arrangements for program implementation, incorporation of the Tier 1 Program into the formal curriculum was a sound and viable strategy. Fifth, implementation of the Tier 1 Program in schools that admitted students with high or low academic achievement was viable. Sixth, the program was generally perceived positively by the program participants and implementers. Finally, the program implementers perceived the program to be beneficial to the program participants.


Zambia is facing a chronic shortage of health care workers. The paper aimed at understanding how the Medical Education Partnership Initiative (MEPI) program facilitated strengthening and expanding of the national capacity and quality of medical education as well as processes for retaining faculty in Zambia.Data generated through documentary review, key informant interviews and observations were analyzed using a thematic approach.The MEPI program triggered the development of new postgraduate programs thereby increasing student enrollment. This was achieved by leveraging of existing and new partnerships with other universities and differentiating the old Master in Public Health into specialized curriculum. Furthermore, the MEPI program improved the capacity and quality of training by facilitating installation and integration of new technology such as the eGranary digital library, E-learning methods and clinical skills laboratory into the Schools. This technology enabled easy access to relevant data or information, quicker turn around of experiments and enhanced data recording, display and analysis features for experiments. The program also facilitated transforming of the academic environment into a more conducive work place through strengthening the Staff Development program and support towards research activities. These activities stimulated work motivation and interest in research by faculty. Meanwhile, these processes were inhibited by the inability to upload all courses on to Moodle as well as inadequate operating procedures and feedback mechanisms for the Moodle.Expansion and improvement in training processes for health care workers requires targeted investment within medical institutions and strengthening local and international partnerships. The limited number and mix of human resources for health (HRH) has contributed to poor health systems performance in many low and middle income countries (LMICs) –3. Of thKey strategies for addressing the HRH crisis as inscribed in the Zambia National Health Strategic Plan (2011-2015) include scaling up the recruitment as well as improving distribution and retention. Improving the capacity of training and education institutions is vital in increasing HRH . As a waThe study site: The study was conducted at UNZA, CBU, LAMU, CU, the Universities that are implementing the MEPI program. UNZA, LAMU and CU are located in Lusaka city  while CBU is located in Kitwe district in the Copperbelt province.The study design: A qualitative case study design was used. The case study design is an empirical study design that investigates contemporary phenomena within a real-life context, where the boundaries between phenomena and context are not clearly evident and in which multiple sources of evidence are used [are used . The casData collection: The data reported in this paper is from October 2010, the time implementation of the MEPI program started, to June 2013. Data were collected through review of documents, interviews with program staff and observations. The first (CM), second (JMZ) and third (MS) authors reviewed the documents, conducted the interviews and observations.Documentary review : The program documents primarily written as part of the MEPI program design and/or implementation process were reviewed. Documents included program implementation progress reports for the period of two and half years, program monitoring and evaluation frameworks, site visit and coordinating program reports, and MEPI annual survey documents, standard operation procedures for the skills laboratory, Moodle platform and course evaluation or assessment reports.Key informant interviews: Interviews were conducted with program managers at the Universities, coordinators of the new programs namely the skills laboratory, medical education (eLearning), the new master programs and the monitoring and evaluation manager. The interviews were recorded and later transcribed.Observations: Observations aimed at documenting visible MEPI implementation-related activities such as infrastructure, asset registers and classes for the new master programs were conducted. These observations were made to validate information from the review of documents and key informant interviews.Data analysis: Data analysis followed thematic analysis which is a method for identifying, analysing and reporting patterns (themes) within data. It organizes and describes data set in detail and goes further to interpret various aspects of the research topic [ch topic . The firch topic  . Verbal consent was sought from all study informants. Further, detailed explanation of the research objectives was done to each study participant. Confidentiality during and after study was assured to the study participants through withholding informants' personal details.In this section, we present the processes and systems for enhancing the capacity and quality of health care worker training and retention of faculty in Zambia which emerged following the implementation of the MEPI program. The findings have been organized around three broad themes namely: training more health care workers, improving the quality of medical training, and processes for retaining faculty.Training more health care workers: Implementation of the MEPI program resulted into improved systems and increased opportunities for training health care workers in Zambia. Below the strategies that facilitated enhancement of training processes are outlined.Developing new programs: Before the implementation of the MEPI program, the SOM had very few new postgraduate programs. However, following the implementation of MEPI, 14 new postgraduate programs were developed. The development of the new programs would potentially help address the huge HRH gap by increasing both numbers and skills of health care workers trained. The programs adopted a multi-disciplinary approach to health service provision by emphasizing both clinical as well as basic science courses. Leveraging on existing opportunities was the main strategy which facilitated the increase in master programs.Leveraging existing opportunities: One opportunity which was leveraged on when developing the master programs was pre-existing partnerships that had been developed for medical education capacity building. For example Vanderbilt University, through the AIDS International Training and Research Program partnered with UNZA beginning in 2008 to develop new curricula for the M.Med clinical residency degrees and to strengthen the research training and HIV-related research projects. Similarly, the University of Maryland and the Tropical Health and Education Trust helped establish new postgraduate programs, namely Masters of Science in HIV Medicine as well as the three Masters of Medicine in Pathology, Psychiatry, and Anesthesiology. Other newly developed programs included six Masters of Science , five for Masters of Medicine , as well as dedicated Masters programmes for Clinical Pharmacology, Physiotherapy, Biomedical Science, and Public Health. These programs increased enrollment rates at postgraduate level. Following the implementation of MEPI in 2013 postgraduate enrolment increased by 241 students. Whereas another 250 students were enrolled in the new distance learning degree program for nurses. Another form of leveraging on existing opportunities took the form of differentiating the old Master in Public Health into specialized curriculum. MEPI has also helped enhance the training processes through facilitating the revision of the existing Masters in Public Health into specialized tracks. The program was reviewed and differentiated into five specialized curricula namely Population Studies, Environmental Health, Epidemiology/Biostatistics, Health Promotion as well as Health Policy and Management. This helped to increase enrolment more than threefold from 15 candidates per year to 50 .Enhancing staff development fellowship: Staff shortage at the UNZA SOM has been a long standing challenge. The following percentages show some of vacant positions at the UNZA: 87% for professor, 94% for associate professor, 82% for senior lecturer, and 52% for lecturer II. The MEPI program established and funded Staff Development Fellowships (SDFs) for graduate students training in areas of current teaching shortages who are committed to become faculty at UNZA or the new medical schools. These Staff Development Fellows are obligated to join faculty following their graduation for a minimum period equal to the duration of their graduate program. To this effect a total of 44 SDFs were supported by MEPI. SDF distribution by program is listed in Development of new collaborations: MEPI further contributed towards the expansion of the capacity of health worker education through facilitating the development of additional collaborations with the recently introduced schools of medicine in order to support the SDF program in the other schools. From 1972 to 2008, UNZA School of Medicine was the only medical school in Zambia. Since then, three new medical schools have been independently established in the country, namely the Copperbelt University School of Medicine which is a public institution and two private medical schools, Lusaka Apex Medical University and Cavendish University Zambia. While expanding the national capacity for training medical students, the introduction of these new schools also exacerbated the shortage of medical faculty, particularly in the basic sciences. To mitigate this shortage, UNZA SOM integrated and supported these new schools into the MEPI partnership locally. More importantly was the provision of support to the SDF program which aimed at sustaining faculty recruitment and retention for all the medical schools. It was stated that the Ministries of Health and Education supported these partnerships.Improving the quality of medical training: Apart from improving the institutional capacity to train more health care workers, the MEPI program also facilitated processes for strengthening the quality of the training for health workers. This has been done through advancing technology in the medical laboratory and medical education sector.Integration of technology for medical education: Data analysis indicated that following the implementation of the MEPI program, the SOM had new technologies installed which facilitated improvement in training process. Below the new technology which was installed is discussed in detail.The eGranary digital library: The eGranary digital library is an offline repository of internet content initially developed by the University of Iowa for low-resource settings with inadequate internet access. It provides learning resources that includes video, audio, books, journals, and web sites. With help from the University of Alabama, the Medical schools in Zambia installed eGranary units at all their campuses. To improve easy access to relevant materials by members of staff as well as students, it was reported that the library was transformed from a manual card cataloguing system to an electronic cataloguing system that is linked online to the main library. In addition, satellite libraries within the school's academic departments were established.Moodle course management software: The Moodle course management software has been installed where course materials developed by the faculty were supposed to be uploaded onto the platform. More than 24 staff had been trained on how to use it. It was reported that the platform had enhanced student access to research and training materials. Furthermore, this had also helped promote access and use of materials amongst faculty.REDCap: REDCap, the Research Electronic Data Capture system, is a user-friendly database system developed by the Vanderbilt University and licensed free of charge to participating institutions of higher learning (Citation: http://project-redcap.org). UNZA MEPI facilitated the installation of REDCap on servers at UNZA SOM, where it was being piloted for future wide-scale use by students, faculty, and research staff. This installation enhanced research activities by improving the scope of readily available data.Virtual microscopy: Another important innovation is the setting up of the Copperbelt University Medical School's virtual microscopy which is being used for histology and partially for histopathology. Virtual microscopyis a computer based method of training, in which optical microscopy images can be viewed as computer images. All manipulations, which can be done on a Microscopy can be done on the virtual microscopy images. The virtual microscopy had been used in the two undergraduate programs currently offered in the school, which are the Bachelors in Dental Surgery (BDS) and the Bachelor in Medicine and Surgery (MBChB). A total of 126 students have been taught Histology and Pathology using this program. Over the last 2 years in which the virtual microscopy has been used, the students had given virtual microscopy a favorable review. Documentary review further indicated that the students' performance in Histology was 10-20% better than their performance in Gross Anatomy and they attributed this performance partly to the new innovation.PowerLab and skills laboratory: Strengthening of the quality of training also involved the installation of the PowerLab Data Acquisition Systems in the Physiology and Pharmacology Laboratories at UNZA SOM. Five laboratory technicians were trained on its use in 2012. All students in undergraduate laboratory based courses at UNZA, LAMU and Cavendish begun utilizing the Powerlab. It was reported that the Powerlab was effective as it offered students the versatile and comprehensive data recording, display and analysis features for life science experiments in human Physiology, Pharmacology and other Biological courses as well as allowed quicker turnaround of experiments and shorter lab sessions. This enabled more lab sessions to be carried out in a single day than previously and further allowed the UNZA SOM physiology laboratory to be utilized by partner schools for their students. In addition, there were early successes in the management of the clinical skills simulation laboratory, where clinical skills were learned in a safe, standardized, and controlled environment.Improved processes for retaining faculty: The MEPI program has further contributed towards expansion of health care worker education in Zambia by putting in place approaches for improving retention of faculty. Compared to the situation before the MEPI program, it was reported that there was improvement in the following sectors following the implementation of the program: the academic environment, research support and offering more support for doctoral and postdoctoral levels.Strengthening academic environment: Strategies for transforming the academic environment into a conducive place included leveraging other support mechanisms in the school such the Welcome Trust supported research Support Centre activities on professional development and grant management training to faculty and support staff. This included training in medical education courses on curricula development, monitoring and implementation as well as courses on pedagogical approaches in medical education. Faculty were also given course and program coordination roles. All of these helped in stimulating interest in faculty to stay and work harder.Research support: Another activity for facilitating retention of faculty was research support. This was done by setting up analytical support processes  as well as research administration trainings. The archival and documentation system for the Research Ethics Committee were also strengthened. Training reports indicated that staff appreciated the training as they noted that the trainings had motivated them to engage in research activities.Doctoral and post-doctoral training support: A doctoral training support was started in the 2012 targeting faculty in PhD training. Support for this largely comprised travel and data analysis related support. This support has been integrated with similar efforts aimed at developing training capacity based consortia such as the Southern African Consortium for Research Excellence which focuses on doctoral and post-doctoral training, and is supported by the Wellcome Trust. It was reported that this integration process was vital as it may trigger improvement in training processes, commitment and retention of faculty.Implementation process limitations. Increased workload among faculty members: Although the MEPI program triggered some positive developments, interviews showed that in the short term, implementation of the new technology and increased student enrollment amplified workload among faculty members. Due to increased workload, a lot of courses were not fully developed for uploading on to Moodle. In addition, standard operating procedures and feedback mechanisms for Moodle had not been developed.In this paper, we have described the processes for strengthening and expanding the national capacity, quality for training health care workers, and retaining faculty through implementing the MEPI program in Zambia. Strategies that facilitated such improvements included the installation of new technology and the enhanced academic and research environment as well as leveraging of existing and new partnerships with other universities. Improved capacity by learning institutions to enroll more students is important as this addresses one of the major limitations to the contribution of most African medical schools towards addressing the HRH crisis which is the limited capacity by Universities to enroll a reasonable number of students –18. StudMeanwhile for technology to be effective, there is need to improve ICT infrastructure . In addiStudy limitations and strengths: Not being able to interview the students who are currently using the new technology that has been put in place by the MEPI program was one of the study limitations. This denied the study additional perspectives on the compatibility of the technology. To mitigate this limitation, the study included data from student course assessment reports. Although generalizability was not the intention, the rich description of processes for strengthening the capacity for health worker education and retaining faculty as well as our multiple methods of data collection helped in developing an account that we believe provides a valuable contribution to the knowledge base on this subject that can provide useful lessons in other LMICs.The implementation of MEPI program has resulted into processes and systems for improving the capacity and quality of training processes in medical schools as well as strengthened systems for retaining faculty. The capacity to enroll more students has been made possible through the development of new programs and differentiating old Master in Public Health into specialized curriculum. Whereas the quality of training was enhanced through integration of technology in training processes and library. This new technology enhanced access to relevant data for training and research as well as improved the features of science experiments. Improving the academic work environment, increased support towards research and staff development program has the potential of improving retention of faculty as these stimulated work motivation and interest in research among faculty.The situation of human resource for health (HRH) crisis in Zambia and other low and middle income countries;Causes of HRH crisis;Effects of HRH crisis on health service delivery.The role of local and international partnerships in improving the quality of medical education in a low-income setting;Experience of integrating new technology in medical education in a low-income setting;Relevance of integrating new technology in medical education in a low-income setting.The authors declare no competing interest.


It consists of heavy and light chains assembled into a complex oligomer. In mouse serum there are two forms of IgM, a full-length and a truncated one. The latter contains μ’ chain, which lacks a variable region. Although μ’ chain was discovered many years ago, its origin has not yet been elucidated. Our results indicate that μ’ chain is generated from a full-length heavy chain by non-enzymatic cleavage of the protein backbone. The cleavage occurred specifically after Asn209 and is prevented by mutating this residue into any other amino acid. The process requires the presence of other proteins, preferentially with an acidic isoelectric point, and is facilitated by neutral or alkaline pH. This unique characteristic of the investigated phenomenon distinguishes it from other, already described, Asn-dependent protein reactions. A single IgM molecule is able to bind up to 12 epitopes via its antigen binding fragments (Fabs). The cleavage at Asn209 generates truncated IgM molecules and free Fabs, resulting in a reduced IgM valence and probably affecting IgM functionality  IgM oligomers are covalently linked via disulfide bonds and their structures resemble snowflakes. J-chain is a 15 kDa polypeptide crucial for IgM secretion into mucosa2.IgMs are the first antibodies produced by B-cells during immune response. They are very large proteins with a molecular mass (Mw) of about 1 MDa. A complete IgM molecule consists of three types of polypeptides: heavy chains, light chains and J-chain. N-terminal parts of each heavy and light chains consist of unique variable domains that form an antigen binding site. The remaining parts of the heavy and light chains are constant regions shared by all IgMs. The heavy chain of IgM has four constant domains, called CH1-CH4, and the light chain has one, CL. Two heavy chains and two light chains associate into a monomeric IgM. CH4 domain has an intrinsic ability to oligomerize4, are considered as promising therapeutic agents with strong cytotoxic potential. The framework of IgM may be very useful in therapies due to its high avidity and fast pharmacokinetics. However, the clinical application of IgMs is challenging because of many problems with production and downstream processing6.Mouse IgMs are widely used in research and diagnostics, especially in blood typing. Moreover, IgMs, including those of mouse origin7. In the case of mouse IgMs we observed additional phenomenon adversely affecting their activity: the heavy chain N-terminus trimming, which yields 55 kDa μ’ chain7.High Mw, heavy glycosylation, dozens of disulfide bonds and a complex oligomeric structure make IgMs very difficult to express, purify and formulate. IgMs activity is rapidly lost through aggregation that is predominantly disulfide driven8. Although the authors proved that μ’ chain is a fragment of a heavy chain lacking a variable region, the origin and exact sequence of μ’ chain were unknown.The first description of μ’ chain was given by Marks and Bosma in 19859 resulting in the heavy chains that lack this domain. Alternative splicing was also postulated as a mechanism of μ’ chain production in mouse cells8 but this hypothesis failed to be confirmed9.To date, apart from the mouse μ’ chain, at least four examples of a truncated IgM heavy chain have been reported. In two of them the shortened variants are produced via alternative splicing. Each domain of the IgM heavy chain is encoded by a separate exon. In human B-cells the exon coding for a variable domain may be omitted during pro-mRNA splicing10.Another example is an IgM variant produced by bony fish (teleosts). The variant is shortened on C-terminus; it lacks CH4 domain and is a part of the B-cell receptor (BCR) on fish lymphocytes. Bony fish have an exceptional IgM pre-mRNA splicing pattern unique among vertebrates11. Genes coding for immunoglobulins are formed during B-cell development in a multi-step process called genomic rearrangement. The rearrangement may be stopped by an incorrect assembly, which may lead to production of a heavy chain shortened on N-terminus.The other two examples concern the generation of a shortened mouse IgM heavy chain during B-cell differentiation. This kind of molecule may result from a defective genomic rearrangement12. The proteolysis was observed intracellularly at an early stage of B-cell development, before the cell started to produce a light chain. Mw of the heavy chain fragment generated by the action of a putative intracellular protease was approximately 48 kDa, which is different from the estimated Mw of μ’ chain (55 kDa).Also, IgM heavy chain that does not associate with a light chain may be structurally labile and prone to proteolytic truncation7. Our results indicated that μ’ chain is generated extracellularly from full-length IgM in the presence of serum or in the presence of bovine serum albumin (BSA) preparation, which, apart from albumin, contains numerous other proteins. We have observed that the accumulation of μ’ chain adversely affected the activity of the IgM reagents. Substantial amount of μ’ chain was detected also in normal mouse serum8, but its influence on immune response and IgM activity in vivo is unknown.We have previously reported that the quantity of μ’ chain increases during storage of IgM-based blood typing reagentsOur research aimed to identify the factors involved in μ’ chain generation. We scrutinized the biochemical properties of μ’ chain and determined its sequence. The specific cleavage site, Asn209 in CH1 domain, suggested an enzyme-mediated process. However, our thorough search for the enzyme responsible for IgM truncation led us to the conclusion that the generation of μ’ chain is a non-enzymatic process. In the course of our work we also developed a set of IgM muteins that are resistant to truncation.i) the molecule is devoid of both N- and C-termini; the variable and CH4 domains are cut off from the full-length heavy chain or (ii) truncation occurs only on N-terminus and variable and CH1 domains are removed.We began our research from biochemical analyses of μ’ chain. Firstly, we removed potential N-glycans from three different IgMs: MM-30, O10, and Q6, using PNGase F. The experiment revealed that μ’ chain is a glycosylated protein consisting of a polypeptide with Mw of about 40 kDa and several N-glycans, which total Mw is approximately 15 kDa Fig. . AssuminTo verify which domains are present in μ’ chain we performed polypeptide sequencing using mass spectrometry (MS). The μ’ chains derived from O10 and Q6 antibodies were fragmented with trypsin or V8 protease. Figure 13; Asn209 corresponds to Asn96 in the UniProtKB record). The scissile peptide bond between Asn209 and Arg210 is localized within a solvent accessible loop . To answer the question whether the sequence surrounding the scissile bond or other parts of the molecule are responsible for mouse IgM susceptibility to truncation, we generated a chimeric molecule: mouse IgM with the linker between CH1 and CH2 domains along with the preceding loop substituted for the human IgM-derived sequence. The obtained mouse/human IgM was resistant to fragmentation in the presence of serum Fig. , indicat7. We believe that the obtained muteins resistant to the truncation of a heavy chain may address flaws of IgMs application in diagnostics or basic research7. Importantly, as presented in Fig. Trimming of IgM correlates with loss of IgM activityWe concluded from this part of the study that trimming of the mouse IgM heavy chain results from the exceptional sequence of the C-terminal fragment of its CH1. We also proved that cleavage could be prevented by mutating Asn209.7. Moreover, we traced FBS as a source of the cleaving factor in the case of IgMs secreted by hybridoma cells. The truncation was also observed in the presence of BSA obtained from bovine serum by heat fractionation. The BSA preparation contained numerous other proteins, thus we initially assumed that the cleaving factor co-purifies with BSA7.We have previously reported that IgM truncation occurs in mouse serum16 for proteases specific to Asn at P1. Among few hits there was only one mammalian enzyme – legumain – a lysosomal cysteine protease17 that is also present in serum18. We hypothesized that legumain is the factor cleaving IgMs.High specificity of the cleavage pointed to an enzyme-mediated process. We searched MEROPS databaseTo verify this hypothesis, we inhibited legumain present in serum using alkylating agents Fig.  and then7. Alike, in the present experiment the diminished accumulation of μ’ chain in samples containing the cocktail was accompanied by increased levels of aggregates and decreased levels of a full-length heavy chain  and a panel of protease inhibitors Fig. . We obseain Fig. . TherefoFirstly, we analyzed the influence of pH on IgM cleavage Fig. . IgMs weThen, we analyzed whether the cleaving factor is secreted or is an intracellular protein occasionally released from dying cells. Most of the serum proteins are produced by hepatocytes. Thus we verified if the Hep G2 human hepatoma cell line secretes the cleaving factor. IgM trimming occurred during incubation of the antibody both in Hep G2 conditioned medium Fig.  and in H19. The results indicated that the resin retained the cleaving factor, because the flow-through fraction did not induce IgM truncation  and, for the experimental convenience, the total protein concentration was about 20 times lower than that in normal mouse serum.To gain insight into IgM stability in the presence of other proteins we incubated IgM with an excess of BSA or other purified proteins that were readily available in our laboratory. The physiological serum level of albumin is about 20 mg/mlThis experiment revealed that IgM cleavage was induced, beyond BSA preparation, also by other acidic proteins: protein A, infrared fluorescent protein (IRFP) and soybean trypsin inhibitor Fig. .We also incubated IgMs with three basic proteins: salmon protamine, hen egg lysozyme and mature vascular endothelial growth factor C (VEGF-C). In the case of protamine we observed precipitates, which quantity correlated with a concentration of protamine. Non-precipitated samples with the lowest protamine concentration (0.1 mg/ml) did not contain increased amount of truncated IgM Fig. . LysozymPresented results indicate that although extracellular truncation of mouse IgMs is a non-enzymatic process it depends on the presence of proteins with not yet fully defined characteristics. Our further studies aim to elucidate the mechanism of this cleavage.24. Asn is prone to non-enzymatic deamidation. Mechanism of the reaction is dependent on pH [reviewed in23]. Under acidic conditions, the side-chain amide bond is attacked by a water molecule, yielding ammonia and aspartic acid. At neutral or basic pH, two reactions are possible: (i) deamidation or (ii) cleavage. (i) The nitrogen in the peptide bond on the carboxyl side of Asn attacks the carbon in the amide group of Asn resulting in ammonia and a cyclic succinimide intermediate. The cyclic imide undergoes subsequent reactions involving isomerization or transpeptidation. (ii) Alternatively, the nitrogen in the amide group of Asn attacks the carbonyl in the following peptide bond. The reaction cleaves the protein backbone and yields two polypeptides, one of which ends with succinimide. The reactions (i) and (ii) compete at neutral or alkaline pH, but deamidation is favored over cleavage25 due to lower activation energy26. Deamidation at neutral or basic pH starts from deprotonation of the attacking nitrogen of the peptide bond. Cleavage occurs only if nitrogen deprotonation is hampered by e.g. Pro or a bulky side chain on the carboxyl side of Asn27. There is also a report suggesting that the protein tertiary structure may promote cleavage26.Asn is a highly reactive residue that frequently contributes to inactivation and degradation of many proteins, including antibodiesHere we report non-enzymatic cleavage of the mouse IgM heavy chain after Asn209. Initially, we assumed that IgM truncation is catalyzed by a protease. However, we excluded involvement of legumain, the only known mammalian Asn (P1)-specific protease and we found that none of the inhibitors from the set that covered all protease classes was able to limit IgM trimming.29, Arg210 may have two effects: a steric hindrance and base catalysis. Arg has a large side chain which may interfere with nitrogen deprotonation in a preceding peptide bond. A positive charge of Arg210 may also enhance nucleophilicity of Asn209 amide group, thus promoting the side-chain amid nitrogen attack on a carbonyl group in the peptide bond. Asn209 in folded IgM is surrounded by four basic residues: Lys201, His204, Lys208 and Arg210, which probably strongly influence its reactivity. Importance of basic amino acids in Asn-mediated peptide bond cleavage is described for inteins [reviewed in30]. Inteins are self-splicing proteins, which in their multi-step self-processing employ peptide bond cleavage after Asn by the mechanism described above as (ii). Inteins catalyze Asn-dependent scission through a unique three-dimensional structure preventing deamidation. The basic residues near Asn209 in IgM may promote cleavage in a similar way.Observed dependence on neutral or alkaline pH suggests that IgM truncation may involve direct Asn-mediated cleavage or cyclic imide formation followed by other reaction(s) resulting in the peptide bond scission. The scissile bond in the IgM molecule is between Asn209 and Arg210. According to reports evaluating the influence of amino acids on the carboxyl side of Asn on its reactivity22. Conversely, IgM truncation was observed only in the presence of other proteins. Purified IgMs even at a concentration of 250 μg/ml were not truncated unless incubated with serum, BSA7 or certain other proteins. We compared IgM trimming in the presence of BSA, IRFP, protein A and soybean trypsin inhibitor. Although all these proteins induced IgM cleavage, their efficacy was diverse. IgM trimming was induced most efficiently by BSA obtained from serum using heat treatment. The heat fractionation results in many impurities in the final preparation. Thus, we previously hypothesized that IgM cleavage is catalyzed by a protease co-precipitating with BSA7. However, the results obtained with IRFP, protein A and soybean trypsin inhibitor suggest that many non-enzymatic proteins may induce IgM trimming.However, deamidation of Asn and Asn-dependent cleavage of a protein backbone are intramolecular processes, which do not require other proteins and do not depend on protein concentrationThe analyzed proteins were acidic, with pI equal to or lower than 5.7 . We also tried to use basic proteins: protamine (pI ~13), lysozyme (pI 11.4), and VEGF-C (pI 8.3). At high concentrations (>1 mg/ml) protamine precipitated during incubation with IgM, but at lower concentrations it neither influenced IgM quantity nor stimulated its trimming. Lysozyme also did not induce IgM trimming. In the presence of VEGF-C we observed a substantial loss of IgM. Because we did not observe precipitates in the samples with VEGF-C, we suppose that VEGF-C may enhance generation of IgM complexes unable to penetrate the gel.6. According to the theory of molecular crowding [reviewed in31], the structure and oligomeric state of a protein may be modified by the presence of other proteins or crowding agents. In line with that, molecular crowding in serum modulated the fate of succinimide intermediate formed during Asn deamidation in a therapeutic antibody24. It is also possible that the added protein is directly involved in non-enzymatic peptide bond cleavage via a currently unknown mechanism.What could be the mechanism of protein-induced IgM trimming? A tempting explanation is that a protein added to IgM solution may prevent Asn209 deamidation thereby shifting the balance towards cutting. Alternatively, the accompanying protein may induce a structural change in the IgM molecule which allows truncation. IgMs are complex proteins that form various types of oligomers36. In the described examples the process was slow with a half-time ranging from about 200 to more than 10,000 days15. Thus, the Asn residue acted as a molecular clock enabling modification of a protein for a long time after its translation. Because of time requirements, Asn-dependent cleavage might be important in long-living polypeptides, e.g. proteins of the eye lens. At least two of them are subject to the Asn-dependent cleavage: crystallins35 and aquaporin-032. This process modified their stability, packing and function and is believed to orchestrate lens development and participate in its ageing36.Some authors consider Asn-dependent cleavage as a post-translational modification, which relates to protein maturation rather than degradation37. Despite the short life-time an evident amount of μ’ is constantly present in mouse sera8, indicating that truncation of IgM is faster than the known Asn-dependent peptide bond cleavage in other proteins. If the IgM trimming proceeds via already known reaction (ii), our results indicate that the process is accelerated by other proteins by a yet unrecognized mechanism. We cannot exclude that the phenomenon may be related to weak, non-specific catalytic capability of one or more serum proteins. Notably, serum albumins from a number of species are known to be able to catalyze certain reactions38. It is also possible, that IgM truncation occurs via a completely different reaction depending on labile Asn209.The calculated half-life of mouse IgM in the circulation is about 2 days39. Binding sites for C1q, the first element of the complement cascade, are hidden in IgM structure and are exposed or formed only upon antigen binding39. IgM trimming resulting in removal of Fab may reduce IgM competence to activate complement. However, according to another model of complement activation by IgM40, spatial fluctuations of Fab fragments prevent C1q binding. Sequestration of Fabs by cognate antigen molecules allows interaction between C1q and IgM. Thus, a truncated IgM may be even more potent complement activator than the non-trimmed molecule, because of reduced requirements for the epitope availability.A substantial amount of μ’ chain in mouse serum suggests that IgM truncation may be physiologically relevant. The identified site of heavy chain trimming is within CH1 domain, just before the linker joining CH1 with CH2. A cleavage at this site should produce a free antigen binding fragment (Fab) and a truncated heavy chain, which still remains within the oligomeric IgM. Both products may act as negative regulators of early immune response. The Fab fragment may bind to a cognate epitope and decrease antigen availability for other immunoglobulins. Truncated IgM has reduced avidity thus its interaction with an antigen is weakened. IgMs bound to multiple surface antigens e.g. on a bacterial surface are potent activators of the complement cascade, which leads to pathogen elimination. According to the current view, the complement activation requires a change in IgM conformation induced by antigen bindingStreptococcus suis during host (swine) invasion to evade immune response42. The bacteria produce highly specific IgM protease IdeSsuis, which cleaves porcine IgM heavy chain between CH2 and CH3 domains41. The action of IdeSsuis abrogated complement activation by porcine IgMs41. The products of IgM proteolysis induced by this pathogen differ from those of non-enzymatic IgM truncation in mouse, therefore the benefits or threats for mice, in which intrinsic IgM trimming occurs, are difficult to predict.Enzymatic cleavage of IgM is used by In conclusion, the mouse IgM heavy chain contains the labile Asn209 residue and is prone to non-enzymatic cleavage between CH1 and CH2 domains. The trimming occurs in neutral or alkaline pH and is induced by other proteins. The unique characteristic of this phenomenon suggests that it proceeds via currently unknown mechanism and may have pronounced physiological implications.7 and MM-30 was purchased from Abcam Cat# ab18401, Lot# G232282-2, G232282-3. We observed differences in proportion of full-length and truncated heavy chains in various IgM preparations. Despite our efforts to use non-trimmed antibodies only, some samples contained low levels of μ’ at the beginning of the experiment, because truncation of IgMs occurs already during their production by hybridoma cells. The antibodies (6–12 μg) were digested with PNGase F (Promega) under denaturing conditions according to the manufacturer’s instruction. Legumain (Novoprotein) was activated as described by Zhang et al.43. The activity of the enzyme was verified using the fluorogenic substrate Z-Ala-Ala-Asn-AMC (Bachem). The IgMs (10 μg) were incubated with activated legumain for 24 h at 37 °C and the substrate:enzyme mass ratio was 30:1. The incubation was performed in PBS and in the buffer suitable for legumain activity assays . Samples of the IgMs treated with the enzymes were analyzed using SDS-PAGE and subsequent staining of the gels with silver nitrate or Coomassie Brilliant Blue.To provide scientific integrity, we used three IgMs with different variable regions: O10 and Q6 IgMs were produced and purified as previously described7. All cell cultures were free of Mycoplasma spp. and were routinely screened using PCR with Mycoplasma rDNA-specific GPO1 and MGSO primers44.Hep G2 cells (ATCC #HB8065) were cultured in DMEM with 1 g/l glucose (Lonza) and 10% FBS (Biowest) at 37 °C, 5% CO2 in a humidified incubator. To prepare serum-free Hep G2 culture, the cells were cultured to reach 90% confluency and then washed 5× with PBS and 2× with DMEM. DMEM with 1 g/l glucose was added to the washed cells. Conditioned media were harvested after 24 h, centrifuged and filtered using 0.22 μm filters. HEK293 cells and hybridoma clones producing O10 and Q6 IgMs were cultured as described previouslyBands were excised from SDS-PAGE gels and destained using 25% acetonitrile (ACN)/25 mM ammonium bicarbonate (ABC) and 50% ACN/25 mM ABC alternately. To reduce and alkylate proteins the samples were treated with 50 mM DTT/25 mM ABC for 45 min at 37 °C and then with 10 mg/ml iodoacetamide/25 mM ABC for 1 h at room temperature in the dark. Then, the bands were washed twice with 50% ACN/25 mM ABC, dehydrated in 100% ACN and air-dried. Gel pieces were re-swelled in 50 mM ABC containing 20 U of PNGase F (Promega) and incubated at 37 °C overnight. After that, they were extensively washed with ultrapure water, then with 50% ACN/25 mM ABC, dehydrated in 100% ACN and air dried. Gel pieces were re-swelled in 50 mM or 25 mM ABC containing Trypsin/Lys-C Mix (Promega) or V8  enzyme and digestion was performed overnight at 37 °C. Then, trifluoroacetic acid (TFA) was added, peptides were collected, vacuum-dried and suspended in loading buffer  for LC-MS/MS analysis done with Q Exactive mass spectrometer (Thermo Scientific) coupled with nano-HPLC  through a Digital PicoView 550 ion source (New Objective). Peptides were loaded onto trap column  in 2% ACN/0.05% TFA at a flow rate of 5 μl/min and then separated on analytical column  using a 90 min gradient of ACN from 2% to 40% in the presence of 0.05% formic acid at a flow rate of 300 nl/min. Top 8 method was used for MS measurement with full MS and MS/MS resolution of 70 000 and 35 000 respectively. To identify peptides, cRAP protein database containing O10 and Q6 sequences was searched by Mascot 2.5 (Matrix Science) via Proteome Discoverer 1.4 (Thermo Scientific).SQ PVDF membrane (Millipore) and stained with Coomassie Brilliant Blue.Determination of N-terminal amino acid sequence by Edman degradation was performed using PPSQ-31A automatic protein sequencer (Shimadzu). Before sequencing the proteins were separated on a SDS-PAGE gel, electrotransferred on a Immobilon P7 was cloned into pFUSEss-CHIg-mM plasmid (Invivogen) using EcoRI and NheI restriction enzymes resulting in pFUSEss-CHIg-mM_M18 plasmid. Q5-based site directed mutagenesis kit (New England Biolabs) was used to introduce mutations within the constant region of mouse IgM encoded in pFUSEss-CHIg-mM_M18. A plasmid coding for chimeric mouse/human IgM heavy chain was generated by replacing the sequence encoding the linker between CH1 and CH2 domains  for the homologous human sequence . A synthetic DNA coding for a fragment of mouse IgM with the human linker  was cloned into pFUSEss-CHIg-mM_M18 using endogenous restriction sites NcoI and BamHI. All generated plasmids were sequenced  and are available via Addgene repository (plasmid numbers 91729–91756 and 91781) along with their complete sequences.To generate cDNA coding for a full-length IgM heavy chain, cDNA coding for the heavy chain variable region of mouse M18 antibody7) using Lipofectamine2000 (Invitrogen). Media collected from cultures of the transfected cells were centrifuged at 10,000 × g, buffered with 20 mM Tris-HCl pH 7.4 or 20 mM acetate buffer pH 4.0 and then incubated for 72 h at 42 °C. The samples were immunoprecipitated using CaptureSelect IgM Affinity Matrix (Thermo Fisher Scientific). For efficient binding to the matrix, pH of the samples incubated under acidic conditions was adjusted to 7.4 with 2 M Tris. The matrix (20 μl) with bound IgM was washed 4 times with 1 ml of PBS containing 0.05% Tween-20 and then boiled with SDS-PAGE reducing loading buffer. The samples were analyzed by western blotting using HRP-labeled goat anti-mouse IgMκ antibody .All recombinant IgMs were transiently expressed using adherent HEK293 cells cultured in DMEM supplemented with 10% FBS. The plasmids encoding IgM heavy chain (pFUSEss-CHIg-mM_M18 and its derivatives) were co-transfected with the plasmid coding for the cognate kappa light chain (pFUSEss-CLIg-mK_M18(i)To inhibit activity of legumain, 50% FBS was treated overnight with 100 mM iodoacetamide (Sigma) in 250 mM carbonate buffer, pH 8.0. Alternatively, 50% FBS was incubated with 50 mM N-ethylmaleimide (Sigma) in 250 mM phosphate buffer, pH 6.5. FBS samples were then dialyzed three times against PBS. Each time the volume of dialysis buffer was fifty-fold the volume of sample. O10 or Q6 IgM (200ng) was incubated in 20 μl of PBS with 1.5 mg/ml treated or control FBS and then immunoprecipitated as described above. Legumain activity in the samples was verified using Z-Ala-Ala-Asn-AMC according to the protocol of the substrate’s manufacturer.(ii)45, which was kindly gifted by Prof. Colin Watts . Cocktails of inhibitors (Sigma or Thermo Scientific) were added to some samples in each experiment. The inhibitors were used in two concentrations: one recommended by their supplier and the second that was 2–10 times higher than the recommended one.In studies with protease inhibitors, MM-30, O10, or Q6 IgM (200 ng) were incubated in 20 μl of PBS with 5% FBS (Biowest) and one of the inhibitors: AEBSF, antipain, aprotinin, benzamidine HCl, bestatin, chymostatin, E-64, EDTA, ε-aminocaproic acid, iodoacetamide, N-ethylmaleimide, leupeptin, pepstatin A, phosphoramidon, soybean trypsin inhibitor , phenantrolin (Bioshop), batimastat (Sigma) or the legumain-specific inhibitor MV026630(iii)5 hybridoma cells secreting O10 or Q6 IgM were seeded in 1 ml of DMEM with 5% FBS. The cultures were harvested after 24 h and centrifuged at 1000 × g for 10 min. The supernatants were then buffered with 20 mM citrate buffer pH 4.0 or 5.0; 20 mM phosphate buffer pH 6.0 or 8.0; or 20 mM Tris HCl pH 10.0. The buffered samples were then incubated for 5 min or 72 h.To analyze pH-dependency of IgM cleavage, 2.5 × 10(iv)Experiments with Hep G2 cell line were performed to establish localization of the cleaving factor. 200 ng of purified O10 IgM was incubated with 20 μl of: fresh serum-free medium (control) or tenfold concentrated serum-free Hep G2 culture medium, or Hep G2 cell lysate (10 μg of protein) obtained by mild sonication of cells suspended in PBS. The medium was concentrated by ultrafiltration using a 3 kDa cut-off membrane (Millipore). In some experiments the medium was pretreated with Cibacron Blue 3GA agarose (Sigma).(v)46 and used at a concentration of 200 μg/ml. Three different concentrations of protamine, 5, 1 and 0.1 mg/ml, were used. All solutions were prepared in PBS.Stability of IgMs in the presence of various proteins was also investigated. O10 or Q6 IgM (10 μg/ml) was incubated in 1 mg/ml solution of BSA obtained by heat-fractionation (Sigma) or highly purified IRFP , lysozyme (Sigma), salmon protamine (Sigma), protein A (Thermo Scientific), soybean trypsin inhibitor (Sigma). VEGF-C was produced as previously describedSeveral factors affecting IgM cleavage were investigated. IgMs were incubated under conditions described below at 42 °C for 72–96 h and then boiled with SDS-PAGE loading buffer. The quantities of emerging μ’ chain were estimated by western blotting using goat anti-mouse IgMκ antibody.7. The images were analyzed using Fusion Fx apparatus with the Fusion Capt Advance Fx5 program . Unless otherwise stated, the acquired images were not processed.The analyses were performed as previously describedSupplementary Figures


Background: Chief Residents must lead, manage and mentor a diverse and often large group of residents, however there is a lack of formal leadership training throughout graduate medical education.Objective: Development of a 3-part Chief Resident (CR) Program focused on leading, managing and mentoring.Design: Each participant completes an Emotional Intelligence (EI) Inventory prior to the day-long event. Participants receive their EI scores at the beginning of the program, which features interactive sessions on leadership, management, and feedback skills. The program then reinforces the application of their new knowledge about EI through a four station OSTE (Observed Structured Teaching Encounter). CRs practice feedback and coaching skills in a simulated environment where they need to provide the context of formative feedback to a standardized resident.Results: The aggregated mean pre-session EI score for all participants was 76.9 . An independent-samples t-test compared the CRs’ leadership and feedback performance on their first and second OSTE performance within a single afternoon session. There was a significant difference between the first OSTE performance  and the second OSTE performance ; t (68) = 1.99, p = 0.006. These results suggest that participating in multiple OSTEs positively reinforces the core interpersonal and communication skills discussed in the didactic and practiced in the interactive portions of the program.Conclusion: The low mean pre-session EI score achieved by our participants supports the idea that CRs enter their new roles with a level of EI that can be enhanced. CRs had an overall positive reaction to EI and its application to the core skills addressed in the program, highlighting the fact that similar programs could be used to train early career physicians to be more skilled and comfortable with leading, managing and mentoring.Abbreviations: CR: Chief resident; EI: Emotional intelligence; GME: Graduate medical education; OSTE: Objective structured teaching encounter Chief Residents (CRs) are typically chosen based on their clinical work as residents or as a set role during the final year of residency. Described as early as the 19th century, chief residents refer to senior residents that have demonstrated competence to manage and care for patients with minimal supervision . Being aGewertz and Logan state that the challenge facing physicians, including CRs, who ‘aspire to outstanding leadership is to continue to ’own‘ what makes them star performers as individuals, while shifting their focus to others’ . As CRs One of the skills found to be central to leadership, and can therefore ensure a smooth transition, is Emotional Intelligence (EI). EI is a relatively recent concept to be embraced within the organizational sphere; however, its definition can be dated back to the early 1900s . EI is aThe use of standardized learners in Objective Structured Teaching Encounters (OSTEs) has proven to be effective in assessing teaching skills of medical school faculty, residents, and medical students –16. OSTEOur program began with each participant completing an EI Inventory three weeks prior to the day-long program. The daylong experience begins with a series of didactic sessions that have a focus on leadership, managing, and core feedback skills. During this portion of the course, participants received their individual EI report, interpretation of their scores, and strategies to improve them. The third part of the program, which takes place during the second half of the day, reinforces the application of new knowledge through a four station OSTE skill building experience . This hiNorthwell Health is a large integrated health care organization consisting of 21 hospitals and over 450 ambulatory and research sites. Northwell Health is the institutional sponsor of 120 accredited and non-accredited GME training programs, with approximately 1600 residents and 100 chief residents annually. Incoming CRs from multiple GME programs  were recruited to participate in one of four one-day, nine-hour courses prior to the start of their CR year. Participant recruitment was achieved through advertising sent to program directors, coordinators, and current chief residents. We had a goal of 20–25 participants at each of the four sessions.Participants completed an online version of the TalentSmart® EI inventory assessment, that was available through our health system, approximately three weeks prior to the course. Theinventory follows the revised Goleman model of emotional intelligence , which eSenior training program educators  lead sessions emphasizing leadership responsibilities applicable to the role of a CR, with a focus on skills necessary to deliver difficult feedback and strategies to assist with interpersonal conflict resolution among resident teams. The didactic session ended with a panel of current CRs joining the group to share their year-long experiences, thoughts on lessons learned as a CR, and best practices in leadership, managing, and mentoring.The CRs were then divided into pairs to participate in OSTEs based on de-identified authentic incidents reported by previous CRs and collected anonymously using an electronic survey. In an effort to allow for more honest critique and feedback, the pairs purposely came from two different residency programs and individuals most often did not know their partners. The OSTE component occurred in a clinical skills center consisting of fourteen separate clinical encounter rooms. The standardized case script simulated clinical education situations in which the CRs needed to rely on EI to interact with and provide collaborative feedback to junior residents who presented difficult interpersonal and professionalism situations. Script content specifically addressed professionalism, confidentiality, issues of escalation, and teamwork . Actors The program had 20 chief residents each day, to total 80 residents across 15 clinical disciplines and included all health system hospitals that had GME training programs.The aggregated mean pre-session EI score for all participants was 76.9. The EI score can range from 0–100, and research has shown that strong leaders have a mean EI score of 85 or higher; less than a score of 50 is considered very undesirable [An independent-samples t-test was conducted to compare the CRs’ leadership and feedback performance as assessed by the CR, standardized learner and observer on the first and second OSTE performance. Performances were scored on a scale with a range of 0 to 100. There was a significant difference between the first OSTE performance  and the second OSTE performance ; t (68) = 1.99, p = 0.006 . These rThe data gathered from post-program evaluation suggests the program achieved success at three different levels of Kirkpatrick’s evaluation schema . ParticiThis communication showcases the efforts of one large academic healthcare organization with a large number of residencies and fellowships to centralize education programs for CRs. Our goal was to help CRs start their new role with appropriate education and be well prepared for challenges they are likely to encounter within their respective training programs, as well as perform at the level expected by their Program Directors and organizational GME leadership. Our primary focus on EI principles as a core competency is based on an extensive literature on leadership, which by extension supports EI as a tool for GME training initiatives. The pre-intervention EI scores of our participants reported are below the mean score that is expected for strong leaders . This reOSTEs designed specifically for CR physicians hold great promise for rapid and rigorous formative assessment of clinical teaching skills and support experiential curricular efforts. In addition they simulate the clinical environment where leadership and mentorship skills can be applied with individual residents and allow for standardized teaching and assessment ,19. As sAs with any educational intervention we encountered limitations. We acknowledge our study is limited to CRs within one health system, with the caveat that we have 100 chief residents across 21 hospitals in all disciplines of medicine with about 80 individual participants. Their exposure to EI training is minimal and that could be much more extensive, but the logistics of GME make this difficult earlier in their training. EI is best learned over time through consistent attention and feedback on EI principles and future follow-up sessions (‘come-back’ sessions) would reinforce skills introduced prior to beginning their CR role. Our post-OSTE debriefs were relatively unstructured; in the future we could develop a more structured format that could add to CR learning and self-reflection . If resoOur results do suggest that CRs, across multiple disciplines, who participated in a one-day combined didactic and experiential educational program can practice communication and professionalism behaviors that support core leadership and feedback skills. Optimizing these abilities are critical for success in their upcoming administrative and leadership roles. Most importantly the acquisition of this knowledge and skills can help assure the role of CR is a positive experience that may influence future career choices at academic health systems that include clinical, academic, and administrative roles. Towards this, we encouraged all CRs to return for optional ‘CR comeback’ educational sessions during their CR year. These sessions are focused on advanced learning topics and also include experiential simulation experiences. An opportunity to re-take the EI inventory prior to completion of their CR year, for additional self-awareness is also offered to all participants. An additional EI score six-months post-residency can provide further assessment of a sustained effect of this experiential program during the chief resident year.This manuscript has not been published elsewhere and has not been submitted simultaneously for publication elsewhere. All authors have read and approved the final manuscript.


Drosophila wing imaginal discs. We used genetic tools to ablate the wing primordium to induce regeneration, and carried out transcriptional profiling of the regeneration blastema by fluorescently labeling and sorting the blastema cells, thus identifying differentially expressed genes. Importantly, by using genetic mutants of several of these differentially expressed genes we have confirmed that they have roles in regeneration. Using this approach, we show that high expression of the gene moladietz (mol), which encodes the Duox-maturation factor NIP, is required during regeneration to produce reactive oxygen species (ROS), which in turn sustain JNK signaling during regeneration. We also show that JNK signaling upregulates mol expression, thereby activating a positive feedback signal that ensures the prolonged JNK activation required for regenerative growth. Thus, by whole-genome transcriptional profiling of regenerating tissue we have identified a positive feedback loop that regulates the extent of regenerative growth.Regenerating tissue must initiate the signaling that drives regenerative growth, and sustain that signaling long enough for regeneration to complete. How these key signals are sustained is unclear. To gain a comprehensive view of the changes in gene expression that occur during regeneration, we performed whole-genome mRNAseq of actively regenerating tissue from damaged  Drosophila imaginal discs, the epithelial structures in the larva that will form the adult animal during metamorphosis, have been an important model system for tissue repair and regeneration for over 60 years. Here we show that damage-induced JNK signaling leads to the upregulation of a gene called moladietz, which encodes a co-factor for an enzyme, NADPH dual oxidase (Duox), that generates reactive oxygen species (ROS), a key tissue-damage signal. High expression of moladietz induces continuous production of ROS in the regenerating tissue. The sustained production of ROS then continues to activate JNK signaling throughout the course of regeneration, ensuring maximal tissue regrowth.Regenerating tissue must initiate the signaling that drives regenerative growth, and then sustain that signaling long enough for regeneration to complete.  The capacity to regenerate damaged or lost organs or limbs is significantly greater in some animals than others. The use of model organisms with varying degrees of regenerative capacity, from whole-body regeneration in planaria and hydra, to limb regeneration in amphibians, organ and fin regeneration in zebrafish, and the limited tissue regeneration that occurs in mammalian models, has advanced our understanding of this process , antz (mol)  , AdoR (F0039747) , and kay0001297)   , Nlaz (FE FBgn00117 activi0003900) , and zfh0004606)  showed u (corto) . Some offeration ,73, and % were dipuckered, can be observed . Vari. VariDroresponse , during response ,85 and aresponse . After acontrols . A seconneration . Additio (Alp-4) , the 4E-4) , an89) /TM6B (gift from M. Uhlirova)[nlaZ:GFP[R2] (gift from M. Ganfornina)[w1118; P{10xStat92E-GFP}1 (BL26197)[cn1P{PZ}dve01738/CyO; ry506 (BL11073)[cn1P{PZ}sm05338/CyO; ry506 (BL11403)[y1w*; P{PTT-GB}LamCCB04957ttvCB04957/SM6a (BL51528)[y1w*; Mi{PT-GFSTF.1}AdoRMI01202-GFSTF.1/TM6C, Sb1Tb1 (BL60165)[y1w*; P{lacW}Thork13517 (BL9558)[y1w*; Mi{PT-GFSTF.0}kayMI05333-GFSTF.0 (BL63175)[w1118; PBac{corto-GFP.FPTB}VK00037 (BL42268), w1118; PBac{Hr78-GFP.FLAG}VK00037 (BL38653), w1118;PBac{NC2β-GFP.FPTB}VK00033 (BL56157), y1 w*; Mi{PT-GFSTF.0}CatMIO4522-GFSTF.0 (BL60212)[HmlΔRFP (gift from K. Bruckner)[TRE-red and gstD-GFP (gifts from D. Bohmann)[Ets21Cf0369 (BL18678)[y1w*; Mi{MIC}CG9336MI03849 (BL36397)[y1w67c23; P{lacW}Col4a1K00405/CyO (BL10479)[y1w67c23; P{lacW}vkgk00236 (BL10473)[P{PZ}Thor06270cn1/CyO; ry506 (BL11481)[w1; P{UAS-Sod1.A}B36 (BL24754), w1; P{UAS-CatA}2 (BL24621), w1; P{UAS-Sod2.M}UM83 (BL24494), w1118; PBac{RB}mole02670/CyO (BL18073)[y1sc*v1; P{TRiP.HMS02560}attP40 (UAS-molRNAi) (BL42867), y1v1; P{TRiP.GL00678}attP40 (UAS-DuoxRNAi) (BL38907), y1v1; P{TRIP.GL00678}attP40 (UAS-NoxRNAi) (BL32902), y1w*; Mi{MIC}NoxMI15634/SM6a (BL61114)[pucE69 [UAS-JNKDN [w* hepr75/FM7C (BL6761)[w*; P{neoFRT}82B p38a1 (BL8822)[Flies were reared on standard molasses medium at 25° C except during regeneration experiments. The following (BL37920), y1,w67c(BL10440), y1, w* (BL56274), P{PZ}Al(BL12285), w1118; BL11515), y1w67c(BL12173), ry506PS (BL684), w;;pBACUhlirova), nlaZ:GFnfornina), w1118; (BL26197), cn1P{P(BL11073), cn1P{PBL11403), y1w*; (BL51528), y1w*; (BL60165), y1w*; (BL63175), w1118; (BL60212), HmlΔRFP15, y1w603, y1w*(BL36397), y1w67c(BL11481), w1; P{U(BL61114), pucE69 AS-JNKDN , w* hepr (BL6761), w*; P{n (BL8822).Immunostaining was carried out as previously described .Anitbodies and dilutions used were Anti-Nubbin (1:500) (gift of S. Cohen) , mouse aAlexa Fluor (AF) secondary antibodies from Molecular Probes were AF488, AF555 and AF633 (used at 1:500). Nuclei were labeled with DAPI (Sigma)(1:5000).EdU incorporation was carried out using the click-it EdU Alexa Fluor 594 Imaging kit (Molecular Probes) as previously described . SamplesImmunostained samples were imaged on a Zeiss LSM 700 confocal microscope and images were processed using ZenLite, Adobe Photoshop, and Image J software. Bright-field imaging of adult wings was done on an Olympus SZX10 microscope using the CellSens Dimension software, and images were processed using Image J.ROS were detected in imaginal discs using Dihydroethidium (DHE)  using the protocol described in Owusu-Ansah et al. , with slgstD-GFP, whose expression was not uniform and thus was quantified by measuring GFP intensity in the entire pouch area. Average intensities of multiple wing discs were combined to calculate the final average intensity plotted in the graphs. For measuring the pouch area, a maximum projection of all the confocal slices was taken and the Nubbin-expressing area measured in Image J. Graphs were plotted in Excel, R, and GraphPad Prism 7.0.Fluorescence intensity analysis was performed using single confocal slices. Average intensity was calculated by measuring intensity values in three equal-sized boxes in the pouch region of the wing disc in Image J, except for the For imaginal disc measurements and immunofluorescence quantifications, the Welch’s t-test was performed using R and GraphPad Prism 7.0. For the adult wing size assay, chi-squared tests were performed using GraphPad online tools. Statistical analyses for adult wing measurements were performed using Welch’s t-test.nub-GFP/+; rn-Gal4, GAL80ts, UAS-rpr /+, while the mock-ablated controls had the genotype nub-GFP/+; rn-Gal4, GAL80ts/+. Cells were isolated for the transcriptional profile as previously described [The ablated regenerating discs had the genotype escribed . Brieflyescribed . RNA quaDrosophila melanogaster genome  with a maximum of 2 mismatches permitted. Intron length was set between 20 and 150,000, and a gene model was provided as GTF . FPKM estimation was done using Cufflinks (v.0.0.7) [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE101797).Fastq reads were trimmed using FASTQ Quality Trimmer (v.1.0.0) and adaptor sequences were removed using Clip (v.1.0.1) in Galaxy . Reads wv.0.0.7) , and botv.0.0.7) , geometrv.0.0.7) . All biov.0.0.7) . The datv.0.0.7)  and are S1 Fig(A) Variation in the size of the wing pouch as marked by anti-Nub immunostaining in undamaged and ablated wing discs. Note the minimal variation in pouch size at R0, indicating consistency of ablation. Undamaged n = 8, regenerating n = 28. Error bars are SEM. (B) Quantification of percentage of regenerating animals that had pupariated by each day, showing the asynchronous progression to pupariation that occurred after tissue damage. Undamaged animals were w1118;; rnGAL4, Gal80TS/+, regenerating animals were w1118;; rnGAL4, UAS-reaper, Gal80TS/+, and both experienced the 24-hour temperature shift. Three independent experiments, total undamaged n = 144 pupae, regenerating n = 176 pupae. (C) Percentage of wings of different sizes on w1118;; rnGAL4, UAS-reaper, Gal80TS/+ animals that eclosed on different days after egg laying due the asynchronous development induced by tissue damage. Note that the animals that eclosed first (day 18) had smaller wings than those that eclosed on day 19, and those that eclosed on day 20 had the largest wings. Thus, variation in wing size after disc regeneration is partly determined by length of time for regeneration. Three independent experiments, total n = 371 wings.(TIF)Click here for additional data file.S2 Fig(A) Venn diagrams showing genes at least 1.3-fold upregulated or downregulated in three transcription profiles  of r(TIF)Click here for additional data file.S3 FigAdoR-GFP MiMIC enhancer trap. (B-B’) Kayak-GFP protein trap. (C-C’) Thor-lacZ enhancer trap. (D-D’) Corto-GFP protein trap. (E-E’) NLaz-GFP MiMIC enhancer trap. (F-F’) anti-Twist. (G-G’) zfh1-lacZ enhancer trap. Blue dashed line outlines the wing primordium. Scale bars are 100 μm. (H) Quantification of upregulation of mol, Nox, and Ets21C expression using qPCR. Four biological replicates each. Error bars are SEM, *p<0.05.Undamaged (A-G) and regenerating (R24) (A’-G’) wing discs. (A-A’) (TIF)Click here for additional data file.S4 Figfru3/+ males had larger wings after regeneration than controls. Three independent experiments, w1118 n = 112 wings, fru3/+ n = 95 wings, p<0.001 by a chi-squared test. (B) Adult htlAB42/+ animals had larger wings after regeneration than controls. Three independent experiments, w1118 n = 316 wings, htlAB42/+ n = 223 wings, p<0.001 by a chi-squared test. Error bars are SEM.(A) Adult (TIF)Click here for additional data file.S5 Fig(A-D) Hemolectin-RFP (Hml-RFP) (green) showing hemocytes near undamaged  and R24  wing discs.  Anti-Nimrod (green) also showing hemocytes near an R24 wing disc, confirming the Hml-RFP results. (B), (D), and (F) are orthogonal slices with the columnar epithelium or the disc proper (DP) toward the bottom and the peripodial epithelium (PE) toward the top of the images. (G) Schematic of an undamaged and a regenerating epithelium showing the location of a hemocyte outside the PE.(TIF)Click here for additional data file.S6 FigmolRNAi caused smaller adult wings after regeneration of the imaginal discs than controls. Three independent experiments, w1118 n = 402 wings, UAS-molRNAi n = 221 wings, p<0.0001 by a chi-squared test. (B-C) Cellular debris is visually distinct from the regenerating epithelium. Regenerating w1118 (B) and mole02670/+ (C) discs at R24, expressing the nub-GFP enhancer trap and stained with DHE. The speckled, grainy GFP tissue is cellular debris, outlined with yellow. The smooth GFP tissue is the intact epithelium, outlined with blue. The side panels are zoomed-in views of debris and epithelium, to show that they are easily distinguished.  Undamaged and R24 w1118 discs stained with DAPI and the ROS detector H2DCFDA. (F-J) gstD-GFP expression in undamaged w1118 (F), and regenerating w1118  and mole02670/+  wing discs at R24  and R48 . (K-O) Anti-Myc immunostaining in w1118  and mole02670/+  regenerating wing discs at R24  and R48 . (O) Quantification of immunofluorescence in Myc staining. R24 w1118 n = 14 discs, mole02670/+ n = 12 discs. R48 w1118 n = 11 discs, mole02670/+ n = 14 discs. Scale bars are 100 μm. Error bars are SEM except where noted. *p<0.02.(A) Expression of (TIF)Click here for additional data file.S7 Figw1118 and UAS-DuoxRNAi animals. Three independent experiments. w1118 n = 390 wings, UAS-DuoxRNAi n = 200 wings, p = 0.0005 using a chi-squared test. (B) Sizes of adult wings after disc regeneration in w1118 and UAS-NoxRNAi animals. Two independent experiments, thus error bars are SD. w1118 n = 299 wings, UAS-NoxRNAi n = 257 wings, p<0.0001 using a chi-squared test. (C) Sizes of adult wings after disc regeneration in w1118 and NoxMI15634/+ animals. Three independent experiments. w1118 n = 349 wings, NoxMI15634/+ n = 180 wings, p<0.0001 using a chi-squared test.  qPCR showing effectiveness of Duox (D) and Nox (E) RNAi. The RNAi was expressed under rn-GAL4 control in the pouch of normally developing wing discs for 24 hours before collecting for qPCR. Three biological replicates each, *p<0.05. (F) Total number of mitotic cells as identified by anti-phospho-Histone H3 staining in the wing pouch as identified by anti-Nub staining in the indicated genotypes. R0 w1118 n = 14 discs, NoxMI15634/+ n = 15 discs, UAS-NoxRNAi n = 15 discs, R24 w1118 n = 12 discs, NoxMI15634/+ n = 17 discs, UAS-NoxRNAi n = 15 discs, R48 w1118 n = 11 discs, NoxMI15634/+ n = 18 discs, UAS-NoxRNAi n = 19 discs. (G) Area of the wing pouch as marked by anti-Nub staining was measured at R0, R24 and R48 for the indicated genotypes. R0 w1118 n = 14 discs, NoxMI15634/+ n = 15 discs, UAS-NoxRNAi n = 15 discs, R24 w1118 n = 12 discs, NoxMI15634/+ n = 17 discs, UAS-NoxRNAi n = 15 discs, R48 w1118 n = 11 discs, NoxMI15634/+ n = 18 discs, UAS-NoxRNAi n = 19 discs. (H) Pupariation timing for regenerating animals of the indicated gentoypes. Three independent experiments. w1118 n = 204, NoxMI15634/+ n = 113, UAS-NoxRNAi n = 206 (I) Pupariation timing for normally developing animals that did not experience the thermal shift and so did not ablate and regenerate the wing primordia. Note that because these animals did not experience the thermal shift, the pupariation timing cannot be compared directly to the pupariation timing in panel H, in which the animals did experience the thermal shift. w1118 n = 97, NoxMI15634/+ n = 49, UAS-NoxRNAi n = 122. Error bars are SEM.Regeneration assays using adult wing size to assess extent of regenerative growth in the imaginal discs. (A) Sizes of adult wings after disc regeneration in (TIF)Click here for additional data file.S8 Figmol-lacZ reporter (green) in w1118 undamaged discs (A), and in regenerating wing discs at R0 (B), R24 (C) and R48 (D). (E) Quantification of mol-lacZ expression changes. Undamaged n = 3, R0 n = 7, R24 n = 10, R48 n = 10.  Anti-β-galacosidase immunostaining showing expression of the mol-lacZ reporter (green) in w1118 (F) and p38a1/+ (G) R24 discs. (H) Quantification of the fluorescence from the immunostaining. Two independent experiments, for a total w1118 n = 10 discs, p38a1/+ n = 12 discs. Scale bars are 100 μm. Error bars are SEM.(A-E) Anti-β-galacosidase immunostaining showing expression of the (TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


There are very few opportunities for long-term, comprehensive postgraduate education in developing countries because of fiscal and human resource constraints. Therefore, physiotherapists have little opportunity following graduation to advance their skills through the improvement of clinical reasoning and treatment planning and application.To address the need for sustainable advanced instruction in physiotherapy within the country, a postgraduate Residency program was initiated in Nairobi, Kenya in 2012. The mission of the program is to graduate advanced orthopedic practitioners who can lead their communities and local profession in the advancement of clinical care and education. Since its inception, six cohorts have been initiated for a total of 90 resident participants. In addition, six program graduates are being trained to continue the Residency program and are serving as teaching assistants for the on campus modules. This training will result in a self-sustaining program by 2020.The manual therapy Residency education model allowed for advancement of the participating physiotherapists professional development utilizing evidence-based practice. This was done without altering the current education system within the country, or accessing expensive equipment.The Residency program was developed and established with the cooperation of a local education institution and a non-profit corporation in the United States. This collaboration has facilitated the advancement of orthopedic clinical standards in the country and will, hopefully, one day serve an as a template for future programs. In the year 2000, it was estimated that there were 234 million moderately or severely disabled people living in developing countries . This nuPhysiotherapists in Kenya currently have the opportunity to earn a 3-year diploma or a Bachelor of Science degree in the field of physiotherapy . In addiTo assist with the progression of clinical reasoning and skill development, an Orthopaedic Manual Therapy Residency (Residency) program was introduced in Nairobi, Kenya in 2012 . The ResMultiple steps were taken to establish a long-term educational program, including comprehensive didactic education and clinical mentoring, to improve clinical practice and health-care delivery by physiotherapists in Kenya. During the development of the program, meetings were held with key stakeholders; the Foundation, the director of the KMTC, and the head of the department of physiotherapy education. Discussions centered on a shared vision and mission for the program. Common goals for the program were agreed upon. In addition, details regarding what each stakeholder could provide to ensure the success of the program were examined. Following the development of the mission for the program and drafting of a Memorandum of Understanding, the goals and scope of the program were shared with the University of Nairobi and Kenyatta University to ensure that misunderstandings did not ensue. In addition, the program was presented to the Ministry of Health. Once the program was accepted by all parties, the development of the content was addressed by the Foundation and physiotherapy faculty at KMTC.The physiotherapists participating in the Orthopaedic Manual Therapy Residency program have a 3-year technical diploma in physiotherapy and have reported no previous access to continuing education throughout their careers. The residents complete six modules over 18 months. The online didactic portion of the program utilizes the Clinical Practice Guidelines and Current Concepts in Orthopedics, 3rd edition  as background reading and preparation for participation in onsite modules . Each moIn addition to onsite modules and online resources, residents receive clinical mentoring focused on integrating the knowledge and skills learned during the Residency program into clinical practice. Although 90% of residents work within the Nairobi area, 10% work outside of the city. To allow for mentoring of these residents, KMTC contracts with providers to allow the residents to practice as students within local facilities. To progress in the program, residents must achieve adequate performance on written and practical examinations provided at the completion of each module. Following completion of the 18-month Residency program, residents must successfully pass a comprehensive written examination and a live patient practical examination to earn the Higher Diploma.Since 2012, 51 volunteers from the US have provided instruction in the Residency program as didactic instructors and clinical mentors to the Residents. The first cohort of the program graduated in December 2014, and the second cohort graduated in December 2015. Currently, four additional cohorts are in progress of completing the Residency program for a total of 90 Residents. Although significant support has been provided through the Foundation to institute the program, a train-the-trainer program has been established. Six graduates are currently being trained to continue the Residency program and are serving as teaching assistants for the on campus modules. Mentoring is being provided to the teaching assistants to allow progression of their understanding of the content and effective delivery skills for teaching both didactic and procedural knowledge. The training of graduates to provide ongoing education will result in a self-sustaining program by 2020.The estimated cost of the Residency program over 6 years is $600,000. The Foundation has provided the costs associated with airfare from the US to Kenya for volunteers. Furthermore, the Foundation has sought professionals with specialty training in manual therapy to provide mentoring for the residents in their current clinical setting focused. This mentoring focuses on integrating the knowledge and skills learned during the Residency program into clinical practice. KMTC provides the resources and facilities needed for the onsite modules. These include a large gym space with standard treatment tables and exercise equipment and audiovisual equipment. KMTC also arranges lodging and transportation for the volunteer instructors and mentors. Residents pay $1,100 in tuition for the program  to offset some of the local costs. As a gift from the Foundation to successful graduates, a messenger bag with an inclinometer, two goniometers, pinwheel, reflex hammer, exercise band, stethoscope, pulse oximeter, and blood pressure cuff is provided at graduation. This provides the graduates with some tools for to providing treatment to patients.Even though residents verbally relayed their gratitude for the education, a formal program evaluation was initiated to begin to explore the outcomes of the Residency program and discover any facilitators and/or barriers for participation in the program. In addition, the Foundation hoped to determine obstacles to the integration the newly acquired knowledge and skills gained through the Residency into clinical practice. Outcomes were explored through a mixed methods approach with interviews and a paper survey.All 15 residents in the first (2012) cohort of the Residency program consented to participate in the outcome measurement process.Individual interviews with residents were conducted following completion of their final live patient practical examination. Interviews were audio recorded and transcribed by an independent transcriptionist to ensure accuracy. The descriptive phenomenology approach was utilized to describe the data. The information from the interviews was coded using the constant comparative method by two investigators, and general themes were identified specifically addressing facilitators and barriers for participation in the Residency program and the implementation of new skills in clinical practice. Thick descriptions and narrative of the participants were provided to inform the themes. The following themes were identified: (1) there was an initial discontent expressed by colleagues regarding participation in the Residency and the required time away from the clinic to participate in onsite modules; (2) patients’ required education regarding manual therapy treatment techniques to be comfortable with the new approach to care; (3) residents experienced a decrease in productivity as they were practicing new skills and assessment techniques in the clinic; (4) there was a positive impact on the ability to utilize clinical reasoning to determine multiple hypotheses for the patient’s diagnosis and then effectively determine a working diagnosis for which to choose evidence-based treatment techniques; and (5) mentoring in the clinic was the most effective method for integrating knowledge into practice.The primary researcher developed a 19-item written survey to determine the impact of the Residency program on professional development and career advancement. The survey was adapted from a tool that had been used previously in studies related to outcomes of residency training in the US , 8. The Residency programs emphasizing clinical reasoning and manual therapy could provide a means to optimize the effects of physiotherapy  without the need for or access to expensive equipment. The Residency format provided in Kenya could allow physiotherapists with technical degrees access to specialty training and ongoing mentorship without a substantial change to educational infrastructure for entry-level education currently provided at higher education institutions within developing countries. The outcomes from the first cohort of the Residency program and the continued positive collaboration between institutions in the US and Kenya suggest that the program is positioned for a sustainable impact on patient care. The success of this program may provide a template for the future development of similar long-term programs in countries with limited resources.This research was approved by the Kenya Medical Training College Ethics and Research Committee and the University of Evansville Institutional Review Board. Residents were made aware of the upcoming study by the residency coordinator in Kenya through verbal communication in June 2014. In September 2014, the primary investigator traveled to Kenya. She discussed the purpose of the study, procedures associated with the study (survey completion and one-on-one interviews), and requirements for time involvement up to 50 min. Written consent was obtained. Instructors in the Residency program were not aware of which residents consented to participate in the study, and participation did not affect the residents performance or standing within the program.SC: developed the methodology for surveys and qualitative research performed to investigate outcomes associated with the Residency program; analyzed the results of the quantitative and qualitative data. RJ: instrumental in developing the curriculum for the Residency program in Kenya; collaborated with the Kenya Media Training College for develop and initiate the Residency program; and assisted with writing information regarding the development of the program. DM: instrumental in coordinating efforts with administrators from both the United States and Kenya; coordinated data collection in Kenya. JM: assisted with methodology development for the outcomes research associated with the Residency program.RJ is the founder of the Jackson Clinics Foundation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Soccer is one of the most widely played sports in the world. However, soccer players have an increased risk of lower limb injury. These injuries may be caused by both modifiable and non-modifiable factors, justifying the adoption of an injury prevention program such as the Fédération Internationale de Football Association (FIFA) 11+. The purpose of this study was to evaluate the efficacy of the FIFA 11+ injury prevention program for soccer players.This meta-analysis was based on the PRISMA 2015 protocol. A search using the keywords “FIFA,” “injury prevention,” and “football” found 183 articles in the PubMed, MEDLINE, LILACS, SciELO, and ScienceDirect databases. Of these, 6 studies were selected, all of which were randomized clinical trials.The sample consisted of 6,344 players, comprising 3,307 (52%) in the intervention group and 3,037 (48%) in the control group. The FIFA 11+ program reduced injuries in soccer players by 30%, with an estimated relative risk of 0.70 . In the intervention group, 779 (24%) players had injuries, while in the control group, 1,219 (40%) players had injuries. However, this pattern was not homogeneous throughout the studies because of clinical and methodological differences in the samples. This study showed no publication bias.The FIFA 11+ warm-up program reduced the risk of injury in soccer players by 30%. Soccer is the most popular sport worldwide, with approximately 400 million players in 208 countries, generating approximately 1 trillion US dollars per year . The BraIn addition to causing large financial losses for professional soccer leagues, injuries lead to player withdrawals and decreased team performance at the professional and amateur levels . Recent Epidemiological studies categorize injury severity according to a player’s period of inactivity for better understanding and classification as follows: minimal (1–3 days), medium 4–7 days), moderate 8–28 days), or severe (>28 days) –7 days, . Most in–28 days,Soccer-related injuries are associated with both non-modifiable factors, such as sex and age, and modifiable factors, such as those that can be improved through programs that influence force, balance, and flexibility. Although both sets of factors interact and are risk determinants –15, profThe FIFA 11+ injury prevention program was developed in 2006 to address this matter, under the leadership of the FIFA Medical Assessment and Research Centre and in collaboration with the Oslo Sports Trauma Research Center and the Santa Monica Orthopaedic and Sports Medicine Center. The program comprises a complete warm-up procedure aimed at injury prevention in soccer players. It includes 15 structured exercises, is available as printed material or online, and is easily executed [Program effectiveness was confirmed by various studies involving female and male players that revealed significant decreases in the incidence of non-contact injuries. The program was initially designed for amateur soccer; however, several studies demonstrated its effectiveness for other sports such as basketball .The program is composed of 3 stages, with 15 exercises following a specific sequence. It is essential that the correct techniques are used, with emphasis on appropriate posture and body control, including leg alignment, knee positioning over the foot tip, and smooth landings  [This study was conducted using the PRISMA Statement 2015 .The chosen measure of association was relative risk (RR). Analyses were conducted using a random effects model, as studies presented significant variations among ages and training frequencies in addition to sex-related differences within the selected samples. Heterogeneity was evaluated using The analysis included 6 studies performed in different countries, with 3 in Europe –25, 2 inThe total sample consisted of 6,344 players, of which 3,307 52%) belonged to the intervention group (IG) and 3,037 (48%) belonged to the control group (CG). The IG had 779 injuries, while the CG had 1,219 injuries. Therefore, we can conclude that the FIFA 11+ program is effective for preventing injuries in soccer players, as its use led to a 30% reduction in injury occurrences, with an estimated RR of 0.70  were submitted to a heterogeneity analysis using the Higgins and Thompson I2 test , 30, whiτ2 = 0.10) and a chi-square test , which also confirmed the heterogeneity among the studies and distortions in their distributions . In addition, the risk of moderate/severe injuries was analyzed in 5 studies that contained this information and revealed consistent RR results of 0.69 .Finally, this study did not show evidence of publication bias. Analysis was performed using a funnel plot Fig. .Fig. 3EvThe FIFA 11+ injury prevention program has been recommended and adopted worldwide, owing to its effectiveness and easy application. The main finding of this study was that the program reduced injury risk by 30% . This result corroborates data from other studies regarding the effectiveness of the FIFA 11+ program for injury prevention in soccer players. One example is a cohort study performed in the USA in 2013 , which eSoligard et al. (2010) obtained similar results when they evaluated the same parameters in female soccer players aged 13–17 years. The program was applied 1.3 times a week for 10 months and was associated with a 46% lower risk of injuries in the IG . In the same context, a systematic review published in 2014  analyzedOwoeye et al.  studied The literature presents a few studies that suggest the ineffectiveness of the use of the FIFA 11+ program in decreasing the injury rate, which highlights the need for an improved understanding of this subject. However, this dichotomy is possibly a result of the lack of a specific program protocol.I2 = 90%). We decided that a relevant approach would be to address this heterogeneity by identifying its main points in order to better understand factors that may interfere with the effectiveness of the program and to propose solutions.Analysis of the results of the systematic review showed increased heterogeneity , and clinical characteristics of the injuries. Furthermore, methodological heterogeneity may occur because of the lack of a protocol, type of warm-up adopted by the CG, non-blinded trainers, differences in capacity among training teams, and technical managers, as well as study frequency and duration.In this review, the sample ages typically ranged from 13 to 25 years old, though the study conducted by Hammes et al.  includedAnother important factor was BMI, which is composed of non-modifiable (height) and modifiable factors (weight). Hammes et al.  reportedSex was another relevant clinical factor. Three studies analyzed men , 27, 28,Clinical aspects were also evaluated, and important differences were observed, which may have affected the heterogeneity of the results. Initially, emphasis was placed on the intrinsic subjectivity of injury categorization, even though all studies had declarations of consensus on the injury definitions and data collection procedures used in soccer studies  for evalAnother relevant factor was the analyzed player type, as most were amateurs who are more susceptible to injuries because their technical abilities are inferior to those of professionals. In addition, professional players are more likely to adopt prevention programs . CompariSeveral studies used weekly monitoring , 27, 28,Attention to proper trainer monitoring and data recording was found to be an essential factor, as the trainers were present in some studies , 26, 27,Methodology was also associated with relevant factors. As previously mentioned, the FIFA 11+ program is easily applicable; however, effectiveness is only obtained if exercises are performed within the existing standards. On the other hand, homogeneity in program application was absent among different reference groups, which affected the results obtained through the combined analysis. A standard protocol for the warm-up program application was nonexistent; thus, application in some studies was according to FIFA recommendations, i.e., 2 or 3 times a week , 26, whiMethodological differences were also identified in terms of the capacity and randomization knowledge of the trainers. All results revealed that program assessors were not blinded; correct FIFA 11+ program application would be impossible without proper knowledge. All the studies used support materials such as DVDs, posters, and online information; only two studies used a training workshop  or a 3-hFew studies verified exercise similarities among groups , 27, whiThus, it is important to emphasize that soccer is one of the most popular sports worldwide; however, it carries a significant risk of injuries, especially in the lower limbs. These injuries are mainly related to modifiable factors, which corroborates the critical role played by warm-up programs. Accordingly, these programs should be easily applicable and involve all soccer players, which is consistent with the proposal of the FIFA 11+ program. Thus, new research on this subject is warranted and must follow the recommendations of the FIFA 11+ program to determine its effectiveness based on a specified utilization period (frequency and duration) and quality of exercise performance.The effectiveness of the program is evident in committed players who are supported by training and health teams. The ineffectiveness presented by some studies is related to the lack of commitment to a program venue and an inappropriate application period or frequency due to the lack of motivation by trainers or players involved in the intervention.The fact that the FIFA 11+ program was launched in 2006 is a limitation, as it is recent and there are few literature references. Moreover, the few studies available in the literature did not follow a specific protocol or provide a venue for the warm-up programs. These factors were also observed in references analyzed by this study, where important clinical differences were identified within the sample and were important injury-determining factors. In addition, the characteristics of the clinical studies may have generated categorization errors or modifications in the injury incidence among players. The methodological approach adopted by studies also presented limitations, which may have affected results and contributed to possible program ineffectiveness resulting from application errors.The FIFA 11+ warm-up program is effective for preventing injuries in soccer players of both sexes aged >13 years.


The Tier 2 Program of the Project P.A.T.H.S. , designed and implemented primarily by school social workers, attempts to help adolescents with greater psychosocial needs. After completion of the Tier 2 Program in the Full Implementation Phase (2006/07 school year), 10,255 Secondary 1 students in 207 schools responded to the Subjective Outcome Evaluation Form (Form C) to assess their views of the program, instructors, and perceived effectiveness of the program. Results showed that high proportions of the respondents had positive perceptions of the program and the instructors, and roughly four-fifths of the respondents regarded the program as helpful to them. Pearson correlation analyses showed that perceptions of the program and instructors were significantly correlated with perceived effectiveness of the program. Participants who joined volunteer training activities generally had higher global subjective outcome evaluation scores than did participants who attended programs without volunteer training activities.