Development is a multi-faceted process; achieving development goals thus requires a multi-sectoral approach. For over two decades, our research group of economists and nutritionists has designed and implemented randomized trials to assess the effectiveness of multisectoral programs in improving nutrition, food security, and other measures of well-being, largely at the request of developing country governments, development partners, and non-governmental organizations. Our approach addresses three perceived pitfalls of RCTs: the “black box” nature of RCTs, limited external validity, and challenges in translation of results to impacts at scale. We address these concerns by identifying and assessing programmatic pathways to impact with quantitative and qualitative methods; studying similar programs implemented by different organizations across various settings; and working closely with implementing partners in the design, research, and dissemination processes to inform adaptation and scale-up of programs and policies. For over two decades, our research group of economists and nutritionists at the International Food Policy Research Institute (IFPRI) has designed and implemented randomized controlled trials (RCTs) to assess the effectiveness of multi-sectoral social protection and agricultural interventions undertaken largely at the request of governments, development partners, and non-governmental organizations. The well-deserved Nobel Prize in Economics awarded to Abhijit Banerjee, Esther Duflo, and Michael Kremer revived a heated debate regarding the power and limitations of RCTs. Our approach to evaluations of multi-sectoral development programs addresses three perceived pitfalls of RCTs: the “black box” nature of the relationship between interventions and their impacts, limited external validity of findings, and challenges in translation of results to impact at scale.2RCTs have been criticized as being a “black box”: they answer the question of whether the intervention had an impact, but do not inform on how impact was achieved. Going beyond the black box starts with developing a conceptual framework that maps hypothesized pathways of impact between the intervention and key outcomes . ProgramFor example, to understand whether – and how – nutrition- and gender-sensitive homestead food production programs with behavior change communication (BCC) improved child nutrition, we carried out a cluster-RCT to test the impacts of the program on maternal and child nutrition outcomes and used mixed methods, including process evaluations, to assess program impact pathways. We worked with Helen Keller International to develop the program’s hypothesized pathways of impact  and usedMixed-method analyses of impact pathways are also useful for unveiling unintended consequences of programming. The Transfer Modality Research Initiative (TMRI), an experimental study designed by IFPRI and implemented by the World Food Programme in Bangladesh, randomized provision of food or cash transfers to poor rural women, alongside nutrition BCC. It led to sustained post-program reductions in participant women’s experience of intimate partner violence, an unintended benefit of the program. Drawing on a conceptual framework linking cash transfers to reductions in intimate partner violence  and usin3Unlocking the black box contributes to understanding the external validity of our findings, i.e. the extent to which an intervention is expected to have similar effects in a different setting . Many ofWe also generate information on external validity by conducting RCTs of similar interventions in different contexts. We worked with HarvestPlus to study the adoption of biofortified orange sweet potato (OSP) using comparable randomized experiments in two different country contexts, Mozambique and Uganda . The proIFPRI also worked with the World Food Programme to evaluate the relative impact of cash transfers, food rations, and food vouchers on household food security and other outcomes in Ecuador, Uganda, Niger, and Yemen. RCTs were used to compare these modalities; the timing, frequency, and value of transfers were equalized to the extent possible across modalities. Cash  or vouchers (Ecuador) were more effective than food rations at improving household dietary diversity . Only inA third example was the evaluation of a food-assisted maternal and child health and nutrition program using RCTs in Guatemala and Burundi. The program targeted women and their children during the first 1000 days and included food rations, health services strengthening, and BCC. While both settings were characterized by very high levels of stunting (about 60%), food insecurity was a severe problem only in Burundi. The program reduced the prevalence of stunting in both settings, but also contributed to postpartum weight retention in the already overweight study population in Guatemala . This fi4A hallmark of our approach to evaluation is engagement with project partners and other local stakeholders throughout the research process, to ensure that our research not only produces global public goods but also helps inform better program design in the country setting itself. In our experience, early collaboration best enables the design of evaluations that prioritize stakeholders’ questions and earn their buy-in, increasing the chances that findings are taken up and used to guide scale-up of a program model. Although we take this approach with the range of methods we use for causal inference in impact evaluations, it is particularly important for RCTs. IFPRI’s evaluation of Mexico’s PROGRESA over two decades ago is an example of one of our earliest efforts to build an engaged RCT-based evaluation of a complex national program being implemented at scale . IFPRI sThe substantial presence of our research teams in offices in Sub Saharan Africa and South Asia also helps continuous engagement throughout the research process and often supports policy adoption and scale-up. An example of the impacts of our long-term presence and engagement in Bangladesh is the collaborative RCT we carried out with the Bangladesh Ministry of Agriculture (BMoA) on the Agriculture, Nutrition, and Gender Linkages (ANGeL) Project. The evaluation showed that combining agricultural production, nutrition knowledge, and gender sensitization trainings most effectively improved production and consumption diversity, farmers’ income, and women’s empowerment. Dissemination and continued engagement around these results motivated the BMoA to scale up the program nationally—the first time any government agency in Bangladesh used rigorous research-based results to justify a national program. The new program is now being rolled out as a large-scale RCT designed in collaboration with IFPRI, the first such experimental study of a national program in Bangladesh.5Our experience illustrates how well-designed pragmatic RCTs, informed by theory, process evaluations, qualitative research, and collaborative engagement between researchers and implementers from the design phase through the interpretation and utilization of results can generate evidence that helps partners design and implement successful multi-sectoral development programs at scale. This type of collaboration helps program implementers learn whether their good intentions have the envisioned benefits, while bringing together the tools of research with the voices of those whose lives are affected to advance our collective understanding of what works to improve the health, nutrition, and well-being of the poor.


Despite the increased utilization of clinical pathways (CPWs) as a strategy to improve patient and system outcomes in hospitals, there remain ongoing challenges with their conceptualization, implementation, and evaluation. Theories that explain how CPWs work in hospitals are lacking, making it difficult to identify important factors for sustaining changes arising from CPWs implemented in hospitals. The objective of this realist review is to develop a program theory for CPWs in hospitals.This is a protocol for a realist review. The review will use a six-step iterative process to develop a program theory for CPWs in hospitals: (1) development of a preliminary program theory; (2) search strategy and literature search; (3) study selection and appraisal; (4) data extraction; (5) data analysis and synthesis; and (6) stakeholder engagement. In addition to searching the gray literature and contacting authors, we will search electronic databases such as MEDLINE, NHSEED, CINAHL EBSCO, HMIC, and PsycINFO. Studies will be included based on their ability to provide data that test some aspect of the program theory. Two independent reviewers will select, screen, and extract data related to the program theory from all relevant sources. A realist logic of analysis will be used to identify all context-mechanism-outcome heuristics that explains how CPWs implemented in hospitals translates to better health system outcomes.Overall, the review aims to develop a program theory for CPWs in hospitals and to explore how, why, to what extent, and in what contexts does the implementation of CPWs in hospitals contribute to better health system outcomes. As a result, the review will provide a theoretical framework of how CPWs work in hospitals.CRD42018103220PROSPERO The online version of this article (10.1186/s13643-019-1046-0) contains supplementary material, which is available to authorized users. In this era of increasingly scarce resources, administrators and healthcare providers are tasked continually with the responsibility of providing high-quality patient-centered care , 2. HospClinical pathways (CPWs), also known as care maps, algorithms or protocols are evidence-based multidisciplinary care plans that detail a stepwise approach to the management of patients with a specific disease , 6. CPWsDespite the widespread use of CPWs in healthcare settings, evidence on their effectiveness in hospital settings has been equivocal , 12 A CoThe conceptualization, implementation, and evaluation of CPWs in healthcare settings are an on-going challenge for program managers and CPWs researchers , 15. SevThe use of theory in implementing evidence-based interventions is vital to the operationalization, adoption, and replication of new knowledge in hospitals. Additionally, interventions underpinned by the theory are more likely to provide meaningful information on the sequence of events and how they relate to the intended program outcomes . This inRealist philosophy acknowledges that the world is “real” and humans interact with reality which limits or constructs our interpretation . RealistWhile contexts are conditions that an intervention operates in (not necessarily settings), mechanisms in simple terms are “what cause things to happen” , 23. A mThe objective of this realist review is to develop a program theory for CPWs work in hospitals.The primary review question is: “How, why, to what extent and in what contexts does the implementation of clinical pathways in hospitals translate to better health system outcomes?” To adequately capture the breadth of available evidence and to allow for comparison with our ongoing systematic review on the effects on CPWs in hospitals, we will focus on the following health system outcomes: length of hospital stay, costs, in-hospital mortality, adherence to recommended practice, and in-hospital complications.I.What contexts facilitate or hinder the implementation of clinical pathways in hospitals?II.What mechanisms support the implementation of clinical pathways in hospitals?III.Does the program theory for clinical pathways in hospitals fare better with some group of individuals, interpersonal relations, institutions, and infrastructures?IV.Are there differences in the understanding by different groups of what hospital clinical pathways are trying to accomplish?The secondary review questions are as follows:The present protocol has been registered with the PROSPERO database (registration number: CRD42018103220) and is being reported in accordance with the reporting guidance the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) statement   reporting guidelines for realist synthesis . For thiWe choose a realist approach to develop a program theory for CPWs in hospitals due to the heterogeneous nature of the evidence base generated by our ongoing systematic review  on the effects of clinical pathways in hospitals. The initial program theory will be derived from the preliminary findings of our on-going Cochrane systematic review update on the effects of CPWs in hospitals. International participants outside Saskatchewan will be involved throughout the theory building phase. We will identify and group the processes, strategies, resources, and outcomes resulting from the implementation of CPWs in hospitals based on the objective and theoretical accounts of the authors. The review team will construct the preliminary program theory by generating hypotheses of “how,” “to what extent,” and “in what contexts” does the implementation of clinical pathways in hospitals contribute to better health system outcomes. This process will be carried out in close consultation with a realist methodologist, Dr. Gill Westhorp. With support from the Saskatchewan Ministry of Health and the Saskatchewan Health Quality Council’s Variation and Appropriateness Working Group (VAWG) and our international stakeholders, we will consult with a group of multidisciplinary content experts including physicians, nurses, researchers, project managers, and other relevant frontline users of CPWs based in and outside Saskatchewan, to provide critical insights to refine the program theory.To develop an initial understanding of how CPWs in hospitals are intended to translate to better health system outcomes, we will conduct a focused examination of the CPWs literature. As recommended by the RAMESES guidelines, our search strategy and search terms will be guided by hypotheses developed from the preliminary program theory . We willAdditionally, we will search gray literature and websites of organizations involved with the implementation of CPWs, such as Intermountain Healthcare , EuropeaIteratively, we will use a two-step process to screen all potentially eligible retrieved studies beginning with the abstracts, followed by full-text assessment of the included abstracts. Two review members will independently screen all retrieved studies from the search process using the following inclusion criteria: (1) intervention meets the following definition for a clinical pathway (i) the intervention was a structured multidisciplinary plan of care; (ii) the intervention was used to translate guidelines or evidence into local structures; (iii) the intervention detailed the steps in a course of treatment or care in a plan, pathway, algorithm, guideline, protocol, or other ‘inventory of actions’ ; and (iv) the intervention aimed to standardize care for a specific population. An intervention meeting all four criteria will be considered a CPW); (2) study was conducted in a hospital; (3) availability of information on implementation strategy(s) employed; (4) at least one health system outcome reported such as length of hospital stay in days, hospital costs, in-hospital mortality, adherence to recommended practice, and in-hospital complications. We will exclude studies on CPWs that are not focused on hospital interventions.To ensure that the credibility and trustworthiness of the review are maintained, we will assess the rigor and relevance of each study’s potential contribution to the development of the program theory. Two independent reviewers will assess for rigor and relevance by examining whether the study inferences are based on evidence or author opinion. This reflexive process will use judgements and discussion to resolve conflicts. We will not exclude studies based on rigor. We will manage the screening and data extraction process using the Distiller SR software . Where aStudy characteristics: title, author, publication year, publication status, country, participants, study focus , hospital type, and hospital settingIntervention: relevance to the program theory, implementation strategies, and adoption supportProgram theory: explanatory accounts in CMO configuration, aspect(s) of program theory supported, refined or refuted, and other notesMethodology: relevance and study rigorTwo review authors will independently extract data from all included primary studies using a piloted draft proposition sheet to extract explanatory account propositions (EAPs) about the program theory for CPWs implementation in hospitals. The EAPs are explanatory statements that may support, refine or refute judgements about an aspect or the program theory in its entirety . Areas oy” will occur because “z mechanism” fire in context “x.” We will identify EAPs focused on the implementation and outcomes of CPWs in hospital settings. Subsequently, we will attempt to identify the CMOs from the extracted EAPs. Additionally, we will identify middle-range theories which are demi-regularities emerging from the data and are observable as a testable hypothesis to refine, support, or refute the program theory [Relevance and rigor are dimensions of ‘fitness for purpose’ in realist synthesis. EAPs will be extracted from primary studies using an adapted format (“If-then-because”) for extracting propositions in realist inquiries . If-thenm theory . Where fTwo review authors will extract the EAPs from the data extraction stage. Where necessary the team will consult with a review team to resolve any disagreement to improve the consistency and validity of the evidence generated. We will group the EAPs statements based on emerging themes, for example, “leadership support” or “staff empowerment.” Using abductive reasoning technique , we willWe will then identify the CMOs configuration existing within each thematic grouping and attempt to form CMOs configurations at the middle range theory level. Middle-range theories explain the regularities of social behavior that can be explored further by hypothesis testing . The CMOTo ensure the relevance of the program theory, we will consult with a CPW stakeholder advisory group comprising of content experts, a realist methodologist, a patient representative, policy-makers, program managers, healthcare providers, and other CPWs frontline users. Content experts may include authors of primary studies focused on CPW implementation in hospitals or researchers who have successfully implemented CPWs in hospital settings. We will present the initial revised program theory and other study findings to the advisory group for their perspectives and to confirm, refine, or refute the initial revised program theory. The stakeholder engagement sessions will be delivered via facilitated discussion in a semi-structured format comprising of face-to-face and remote sessions to accommodate international participants. The engagement sessions will follow the guiding principles described by Manzano 2016  for condPrior to consulting with stakeholders to help refine the draft program theory, ethical approval will be sought through the University of Saskatchewan Research Ethics Boards. All stakeholders will be consulted for consent to participate and publish.Overall, the realist review aims to assess “how, why, to what extent and in what contexts does the effective implementation of clinical pathways in hospitals contribute to better health system outcomes?” As such, the review will create an evidence and theoretical base to support key decision-makers involved with the implementation of CPWs in hospitals. Building on the successful factors and key mechanisms of the final program theory with a detailed knowledge translation plan, we anticipate an improvement in the uptake of CPWs in hospitals. However, it is important to note that the steps described in this protocol will be conducted in an iterative manner, as opposed to a linear process which does not capture the complexities and iterations associated with realist reviews and complex interventions. The review will be disseminated via peer-reviewed journals, academic conferences, and as part of a graduate thesis. Finally, we anticipate an increase in beneficial effects from the future implementation of CPWs in hospitals, particularly those focused on the outcomes of interest in this review; length of hospital stay, costs, in-hospital mortality, adherence to recommended practice, and in-hospital complications.Additional file 1:PRISMA-P 2015 Checklist. (DOCX 31 kb)Additional file 2:Part A: MEDLINE Search Strategy. Search strategy conducted for on-going update of systematic review on the effects of clinical pathways in hospitals. Part B: draft data extraction sheet for realist review. (DOCX 39 kb)


Using whole-genome sequencing, we identified a missense variant in the first exon of MFSD12, a gene recently identified to be involved in human, mouse and horse pigmentation. We confirmed the role of this variant in phaeomelanin dilution of numerous canine breeds, and the conserved role of MFSD12 in mammalian pigmentation.White coat color in mammals has been selected several times during the domestication process. Numerous dog breeds are fixed for one form of white coat color that involves darkly pigmented skin. The genetic basis of this color, due to the absence of pigment in the hairs, was suggested to correspond to extreme dilution of the phaeomelanin, by both the expression of only phaeomelanin (locus E) and its extreme dilution (locus I). To go further, we performed genome-wide association studies (GWAS) using a multiple breed approach. The first GWAS, using 34 white dogs and 128 non-white dogs, including White Shepherds, Poodles, Cotons de Tulear and Bichons allowed us to identify two significantly associated loci on the locus E and a novel locus on chromosome 20. A second GWAS using 15 other breeds presenting extreme phaeomelanin dilution confirmed the position of locus I on the chromosome 20 (position 55 Mb  SLC45A2 or OCA2 mutations [MITF or KIT gene [MC1R gene (e/e genotype) and only express phaeomelanin [White coat color is a common color in dogs caused by at least two underlying distinct genetic mechanisms: the absence of melanocytes in the skin/hair or the absence of pigment in the melanocytes or hairs. In dogs, true albinism is rare and is due to utations ,2,3,4. TKIT gene ,7,8. On KIT gene . FurtherKIT gene . This hyomelanin . Howeverhttp://dog-genetics.genouest.org) and DNA/RNA were extracted as previously described [Blood and tissue biopsy samples from dogs and wolves were collected by a network of veterinarians through the Cani-DNA BRC  and Affymetrix, Inc.  respectively. We used mixed linear model analyses, taking into account population structure and kinship (Eigenstrat), removing SNVs and individuals with a call rate below 95%.For imputation, the MACH software  was usedMFSD12 locus, long read sequences were produced through MinION (Oxford Nanopore Technologies) according to manufacturer instructions. Reads were mapped to the dog genome with minimap2 [To fill the gap  containing the first exon 1 of the minimap2  using deminimap2  (File S1HPRT gene (ENSCAFG00000018870) in skin, for 6 homozygous mutant dogs, 6 heterozygous dogs and 3 homozygous wild type dogs. Primers are available in Sanger sequencing of DNA canine samples was performed as previously described . The qPCMC1R gene , and a novel locus on chromosome 20   and 128 non-white dogs (24 German Shepherd Dogs and 104 Poodles), we first performed a genome-wide association study (GWAS) correcting for population stratification and cryptic relatedness. We detected two significant associations with solid white: one on chromosome 5, 28 kb upstream from the  × 10−8) .MC1R locus was hypothesized to be due to the e allele and the exclusive production of phaeomelanin in solid white dogs, as previously shown [MC1R:c.916C > T responsible for the e allele in the 577 dog whole-genome sequences from the Dog Biomedical Variant Database Consortium (DBVDC) [e/e for the MC1R allele . Thus, solid white dogs do not express eumelanin but only phaeomelanin, and we hypothesized that this phaeomelanin is diluted by a variant allele located on the novel chromosome 20 locus.The association with the ly shown . To conf (DBVDC)  and confe/e at MC1R) and the extreme phaeomelanin dilution (new locus on chromosome 20), we decided to complete our GWAS panel by adding breeds presenting with fixed diluted or undiluted phaeomelanin. Combining markers from the Illumina SNV chip and SNVs extracted from the 577 dog whole-genome sequences (DBVDC) [p-value; the best SNV being located ~5 kb upstream of MFSD12 gene   , we perf× 10−13) . Variant× 10−13) . Since tpothesis .MFSD12 candidate gene (MFSD12 (MFSD12: p.(Arg51Cys). This dog variant is exactly orthologous to the human variant rs751585493 . which is rare in humans with an allele frequency of 1.38390e-05 across all populations in gnomAD exome database, and only found in South Asian populations. A damaging effect of this variant (C > T) is predicted by most prediction tools, with scores such as 0.02 for SIFT (Damaging) and 0.806 for Polyphen (Probably damaging) [To identify the causal variant, we further searched for gaps of the CanFam3 genome assembly in this region and identified a gap harboring the first exon of the ate gene . Long re . We confirmed that the variant perfectly segregates with breeds presenting diluted phaeomelanin but not with breeds presenting the fixed red coat color, using Irish Setter dogs as controls. For the following breeds thought to be fixed for the phaeomelanin dilution, out of eight tested dogs, the variant was very frequent: Schnauzer (94.4%), Husky (93.8%), Samoyed (87.5%), Akita (56.3%), Coton de Tulear (50.0%). We also tested Poodles, German Shepherds, and Leonbergers, using dogs presenting various shades of phaeomelanin dilution. We confirmed the enrichment of the MFSD12 variant in dogs presenting an extreme dilution of phaeomelanin to white, with 64 cases and 235 controls  , Puli (3 dogs), Saluki (4 dogs) and Afghan Hound (3 dogs)  . None of(3 dogs) . For exanbergers . The solnbergers .MFSD12 variant appears to be ancient since it was found in nine wolves (arctic wolves and grey wolves from Canada or Poland) of the 31 tested wolves . Wolves do not carry an e/e genotype, but instead are wild type (E/E) at MC1R. In these wolves, the eumelanin is still present in their coat as intermingled black hairs or black-tipped hairs, but the phaeomelanin is diluted to cream when the MFSD12 allele is homozygous.This e/e at the MC1R locus) and an extreme dilution of phaeomelanin, confirming the hypothesis of Sponenberg and Rothschild on the existence of an I locus and later, the work of Schmutz and Berryere [e/e genotype at MC1R and an i/i genotype at MFSD12 causes cream to white in many dog breeds, it is probable that other modifier alleles exist and explain the cream to white dilution in dogs. Benefiting from the fixed coat colors in many canine breeds, a multiple breed mapping approach allowed us to identify the gene and variant underlying the I locus, by the discovery of a variant significantly linked to the dilution of phaeomelanin. After the identification of a strongly significant association between chromosome 20 and the fixed white coat color in specific breeds, we used publicly available dog genomes (DBVDC resource) [MFSD12 linked to phaeomelanin dilution. The finding of only one coding variant in the MFSD12 sequence and the fact that this variant is predicted as deleterious by most prediction tools are strong arguments to point out this variant as causal. In addition, we checked if the mRNA expression of MSD12 was decreased in dogs presenting the variant compared to wild type dogs and did not find any significant change, suggesting that the variant affecting MSFD12 is probably not a regulatory variant. All these arguments suggest that the coding variant in the first exon of MFSD12 causes the phaeomelanin dilution. However, considering the high linkage disequilibrium (LD) at the locus, one should be careful not to rule out the involvement of another SNV (in LD with the best GWAS SNV) in phaeomelanin dilution. Although we did not observe any significant difference between the expression levels of wild-type versus mutated MFSD12 mRNAs, we cannot rule out other posttranscriptional or epigenetic alterations leading to non-functional MFSD12 protein. Thus, further functional studies are needed to confirm the role of this coding variant in dogs and humans. Finally, this variant is probably ancient since it was detected in many genetically diverse breeds, old breeds, and even wolves. Further studies are needed to see if this variant is under natural selection in wolves and correlated with latitude.Thanks to a multiple step GWAS approach, we confirmed that solid white coat color in dogs may result from the absence of eumelanin   and targMFSD12 was recently associated with diluted skin pigmentation [MFSD12 (rs751585493) in the human database, in South Asian populations we anticipate that this SNV is also associated with phaeomelanin dilution in humans, enriching the list of variants linked to skin pigmentation in mammals. The MFSD12 gene was, very recently, also associated with the Mushroom coat color in horses [MFSD12 plays a conserved role in phaeomelanin intensity and vertebrate pigmentation.Looking at human pigmentation data, entation ,17 and sn horses  and withn horses ,17, answ


Lipoate is an essential cofactor for a small number of enzymes that are important for central metabolism. Malaria parasites require lipoate scavenged from the human host for growth and survival; however, it is not known why this cofactor is so important. To address this question, we designed a probe of lipoate activity based on the bacterial enzyme lipoamidase (Lpa). Expression of this probe in different subcellular locations allowed us to define the mitochondrion as the compartment housing essential lipoate metabolism. To gain further insight into the specific uses of lipoate in the mitochondrion, we designed a series of catalytically attenuated probes and employed the probes in conjunction with a chemical bypass system. These studies suggest that two lipoylated proteins are required for parasite survival. We were able to express Lpa with different catalytic abilities in different subcellular compartments and driven by different promoters, demonstrating the versatility of this tool and suggesting that it can be used as a probe of lipoate metabolism in other organisms. Plasmodium falciparum is auxotrophic for lipoate during the intraerythrocytic stages, but it is not known whether lipoate attachment to protein is required or whether attachment is required in a specific subcellular compartment of the parasite. To address these questions, we used an enzyme called lipoamidase (Lpa) as a probe of lipoate metabolism. Lpa was first described in Enterococcus faecalis, and it specifically cleaves protein-bound lipoate, inactivating enzymes requiring this cofactor. Enzymatically active Lpa could be expressed in the cytosol of P. falciparum without any effect on protein lipoylation or parasite growth. Similarly, Lpa could be expressed in the apicoplast, and although protein lipoylation was reduced, parasite growth was not inhibited. By contrast, while an inactive mutant of Lpa could be expressed in the mitochondrion, the active enzyme could not. We designed an attenuated mutant of Lpa and found that this enzyme could be expressed in the parasite mitochondrion, but only in conjunction with a chemical bypass system. These studies suggest that acetyl-CoA production and a cryptic function of the H protein are both required for parasite survival. Our study validates Lpa as a novel probe of metabolism that can be used in other systems and provides new insight into key aspects of mitochondrial metabolism that are responsible for lipoate auxotrophy in malaria parasites.Lipoate is a redox active cofactor that is covalently bound to key enzymes of oxidative metabolism.  Plasmodium spp. have four metabolic complexes that use the lipoic acid cofactor: pyruvate dehydrogenase (PDH), which resides in the apicoplast  is a metabolic cofactor required for the decarboxylation of alpha-ketoacids and the amino acid glycine in oxidative and one-carbon metabolism . Plasmodicoplast , and theicoplast , 4. Thesicoplast . Howevericoplast .Plasmodium falciparum genome that are involved in lipoic acid acquisition: two lipoate biosynthesis enzymes, lipoate synthase  and lipoate transferase , and two enzymes with homology to lipoate ligases, designated LipL1 (PF3D7_1314600) and LipL2 (PF3D7_0923600). PfLipA and PfLipB functionally complement their Escherichia coli homologs and are hypothesized to be expressed in the apicoplast, showing that P. falciparum possesses a lipoate synthesis pathway  and murine (Plasmodium berghei) malaria parasites  is an enzyme first described in Enterococcus faecalis that can cleave lipoate from lipoylated proteins , which was previously shown to direct GFP to the apicoplast (55Lpa (aLpa) and ACP55KSA (aKSA) using the strong calmodulin promoter were unsuccessful, perhaps because overexpression of apicoplast proteins by this promoter is poorly tolerated, a phenomenon that we have observed before . In repeated transfections, we were able to generate parasite lines expressing mitochondrially targeted L133KSA (mKSA); however, multiple attempts at expressing L133Lpa (mLpa) failed  failed . These r) failed  to generE. coli inactivates the lipoate-dependent PDH and KDH, significantly reducing the production of acetyl-CoA and succinyl-CoA, respectively    with 27%10.1128/mBio.01872-18.1FIG S1E. coli and prepared as cell lysates. The lysates were analyzed by anti-HA Western blotting and then diluted to normalize the amount of lipoamidase protein in each lysate . Lipoylated H protein from Bacillus subtilis was added to each lysate to serve as the substrate in a 30-min cell-free delipoylation assay. As expected, adding an equivalent volume of buffer (lane 1) produced a signal similar to the KSA mutant (lane 2), demonstrating that this mutant does not have lipoamidase activity. Serial 10-fold dilutions of Lpa and S236A show that there is roughly a 1,000-fold difference in the lipoamidase activity of Lpa compared to S236A. Download FIG S1, TIF file, 1.7 MB.Delipoylation assay showing that LpaS236A is about 1,000 times less active compared to wild-type lipoamidase. The active Lpa, inactive KSA, and mutant S236A variants of lipoamidase were expressed in lipoylation-deficient Copyright © 2018 Jhun et al.2018Jhun et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The mS236A lipoamidase variant was successfully expressed under supplementation with 5 mM acetate. As with the other constructs, PCR for plasmid integration and Western blotting were used to confirm expression . Unlike Western blotting revealed that the apicoplast PDH remained lipoylated in both the mS236A and mKSA parasite lines, consistent with these constructs having no impact on apicoplast protein lipoylation. By contrast, there was a significant reduction in lipoylation of all three lipoate-dependent proteins found in the mitochondrion compared to either the loading control or apicoplast PDH lipoylation . IncubatLipoic acid is a metabolic cofactor that is acquired in an unusual manner by the parasite: although it is synthesized in the apicoplast, it is scavenged from the host for use in the mitochondrion. Experiments with lipoate analogs indicated that lipoate scavenging is essential for parasite growth , 4, and P. falciparum parasites do not catabolize branched-chain amino acids . Stable leucine isotope labeling experiments suggest that blood-stage no acids . Consistno acids . Knockouentation . The autentation . Our resentation . Thus, lIn addition to acetate supplementation, we also tried to see whether lipoate could rescue the growth of mS236A parasites. Supplementation with 2 μM lipoate can rescue the toxicity of the lipoate analog 8-bromooctanoate , and thiP. falciparum KDH can be deleted without affecting blood-stage parasite growth  and a means of probing cells with different levels of lipoamidase activity (Lpa versus attenuated mutants such as S236A). These constructs also demonstrate that Lpa can be tagged at the N terminus and C terminus, facilitating the use of organellar trafficking peptides and epitope tags. Adding this to inducible and knockdown systems available in other organisms offers a wide array of options to probe in vivo lipoylation, especially in systems in which the full array of lipoylation machinery has not yet been discovered. Our experiments with P. falciparum demonstrate that mitochondrial lipoylation is essential and implicate the BCDH and H protein as being responsible for this phenomenon. These results explain why lipoate scavenging is essential for parasite survival and imply that the lipoate attachment enzymes LipL1 and LipL2 will also be required for blood-stage parasite survival. Overall, these findings provide new insight into key aspects of mitochondrial metabolism in malaria parasites.Lipoamidase has proven to be an effective tool to probe  E. coli  or P. famalE gene of pMAL_cHT [E. coli as previously described  was amplified from P. falciparum cDNA using the primers L133BamHI.F and L133BamHI.R. This amplicon was inserted into the BamHI site at the 5′ end of the Lpa gene in plasmid pMS011 to create plasmid pMS030, which encodes the construct L133Lpa. Apicoplast-targeted lipoamidase was produced by a similar method, except that the first 55 amino acids of the acyl carrier protein (ACP) (PF3D7_0208500) which encode the signal peptide and apicoplast transit peptide, were amplified from the plasmid pSP002 . Mutations at the described positions were made to reduce lipoamidase enzymatic activity. These mutations were designed to disrupt carbonyl stabilization of the lipoamide bond (A256G), disrupt hydrogen bonding of the catalytic lysine (S236A/C/G), and interfere with hydrophobic interaction at the dithiolan ring of lipoate (W210F and Y375F). The catalytic triad (orange) is formed from K159, S235, and S259. Download Copyright © 2018 Jhun et al.2018Jhun et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01872-18.3FIG S3n = 5). However, extending this incubation to 1 h completely eliminates lipoylation signal. This evidence demonstrates that the mS236A variant has lipoamidase activity and suggests that it is less efficient than wild-type lipoamidase, a finding consistent with FIG S3, TIF file, 1.6 MB.The mS236A lipoamidase variant is active, but not as efficient, as wild-type lipoamidase. Incubating the lysates of parasites expressing mS236A under nondenaturing conditions for 15 min (as in previous experiments) shows partial delipoylation of the PDH  cells (Invitrogen) were transformed with this plasmid, and the pRIL plasmid was isolated from BL21-CodonPlus-RIL cells (Agilent). Cells were induced at mid-log phase with 0.4 mM IPTG and harvested after growth at 20°C after 10 h. The H protein was purified by affinity chromatography and ion exchange using MBPTrap and HiTrap Q columns .The H protein from Bacillus subtilis (serving as the substrate for the reaction). The reaction mixtures were incubated at room temperature for 30 min and were stopped by the addition of an equivalent volume of 6× Laemmli buffer. These samples were heated at 95°C for 4 min and vortexed for 1 min; this was performed twice for a total of 10 min. Lipoylated H protein was detected by Western blotting probed with 1:5,000 rabbit anti-lipoate antibodies . Each reaction mixture was 60 µl in volume and contained final concentrations of 200 mM HEPES and 100 mM NaCl at pH 7.5 with 50 nM purified lipoylated H protein from tibodies  and 1:5,10.1128/mBio.01872-18.6TABLE S2B. subtilis as the substrate. Mutations at S236 appear to have been particularly effective at reducing activity, with the S236A mutations resulting in the slowest catalysis. Download Table S2, DOC file, 0.03 MB.Coarse assessment of lipoamidase mutants. Using the same assay shown in Copyright © 2018 Jhun et al.2018Jhun et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. falciparum, lipoamidase constructs were cloned into the vector pLN-ENR-GFP , pMS040 (encoding KSA), pMS026 (encoding ACP55Lpa), pMS036 (encoding L133Lpa), pMS037 (encoding L133KSA), and pLN-L133S236A (encoding L133S236A). Amplicons from plasmids pMS021 and pMS023 were also inserted into digested pRL2 to produce plasmids pMS049 and pMS050 which encode ACP55Lpa and ACP55KSA driven by the RL2 promoter.For expression in -ENR-GFP  obtained-ENR-GFP . Lpa conP. falciparum asexual blood-stage cultures were maintained at 2% hematocrit in complete medium consisting of RPMI 1640 medium with L-glutamine  supplemented with 10% human serum or 5 g/liter AlbuMax II (Thermo Fisher), 20 mM HEPES, 23 mM NaHCO3, and 0.09 mM hypoxanthine. Transfections were performed as described previously  for 30 min in fresh media prior to fixation. All cultures were fixed in 4% paraformaldehyde–0.0075% glutaraldehyde on polylysine-treated slides for 30 min. Cells were permeabilized for 10 min in 1% Triton X-100, remaining aldehydes were reduced with 0.1 mg/ml NaBHanti-ACP , and lipanti-ACP , and mea35S-labeled lipoate was prepared as previously described  primary antibodies to detect lipoamidase, 1:5,000 rabbit antilipoate (Parasite pellets as described above were resuspended in RIPA buffer (Boston Bioproducts) to determine lipoamidase activity in cell lysates, and the reaction was quenched by the addition of Laemmli loading buffer. Otherwise, pellets were resuspended in NuPAGE LDS sample buffer (Thermo Fisher) and alternatively vortexed for 1 min and boiled for 4 min, a total of two times. Proteins were resolved by SDS-PAGE on 4 to 12% gradient gels and transferred to nitrocellulose membranes. For autoradiography, membranes were dried overnight, and autoradiography was used to detect ilipoate  to detec


In the workplace depression and suicide are serious mental health problems. A lack of knowledge and mental health skills along with the stigma toward mental health problems often results in delays in seeking professional help. Interventions targeting not only persons with mental health problems but also people around the individual are warranted in order to encourage supporting behavior within entire workplace. In the present study, we investigated the efficacy of our newly developed educational training program in the management with depression and suicidal risk in the workplace as a single-arm pilot trial.The program is a two-hour (2-h) training course for employees based on the Mental Health First Aid (MHFA) program which aims to increase public mental health literacy. We conducted this program at a company workplace among 91 employees, and ultimately 83 participants completed the self-rated questionnaires. Changes in confidence and practical skills in early intervention of depression and suicide-prevention, and stigma toward mental health problems were evaluated using self-rated questionnaires at 3 time-points; pre-program, immediately post-program, and 1 month after the program.Confidence and practical skills were significantly improved even 1 month after the program, and stigma reduced just after the program.Our pilot study suggests that the program has a positive impact on encouraging employees to support their co-workers with mental health problems, and is applicable for busy workers due to its short duration. A single-arm design, evaluation using self-rated questionnaire and short-term follow up period are the main limitations of the present study. Hence, future research is required to validate the effects of this program with control groups, and also to assess long-term effectiveness and objective changes such as absenteeism and sick leave.R000023258UMIN Clinical Trials Registry (UMIN-CTR)  Depression and suicide are serious mental health problems that greatly impact the workplace. According to the World Mental Health Japan (WMH-J) survey, the most frequent mental health problem among Japanese employees is major depressive disorder, and its 12-month prevalence is estimated to be 2.6% . On the Mental Health First Aid (MHFA) is a training program developed in Australia in 2000. The purpose of the program is to increase public mental health literacy, including recognition and knowledge of mental health problems, help-seeking behavior, and providing help to someone with a mental health problem . The staAlthough the standard MHFA program takes 12-h, shortened versions of program have been warranted to make such training more accessible to people for whom time may be a consideration. In Japan, a 2-h program was developed targeting medical staff and university administrative staff by focusing on depression and the risk of suicide and showed effectiveness in improving confidence and skills in managing persons with mental health problems, and attitudes toward psychiatric disorders . To our This study was approved by the ethics committee of Kyushu University, and was registered at the UMIN Clinical Trials Registry (UMIN-CTR) (Registration number: R000023258).91 employees working in a manufacturing company in western Japan attended a 2-h mandatory mental health literacy course during their work day. This course was held twice and employees attended either of the courses. The first course was held in June, and the second was held in July of 2016, the number of employees attending the course was 40 and 51, respectively. Employees were informed of the aims and methods of the present study and that their participation as whether or not to answer the self-rated questionnaires was completely voluntary. Participants who agreed to join this study were then registered as participants with written informed consent. 39 out of 40 employees participated in the first course, and 44 out of 51 in the second course. Finally, 83 employees participated in this study. The participant flow chart is shown in This training program was established based on the standard MHFA program which is a 12-h course consisted of lectures and workshops focusing on the following 5-step MHFA principles: 1) Approach the person, assess and assist with any crisis; 2) Listen non-judgmentally; 3) Give support and information; 4) Encourage the person to get appropriate professional help; and 5) Encourage other support . Based oThe previous lecture and role-play sessions for clinical settings was modified including audiovisual material (DVD) and role-play scenario enabling workers to learn how to approach and respond to co-workers with depression and suicidal thoughts.At the beginning of the program, a 15-min role-play session where participants were required to approach a person (co-worker) with depression without any knowledge or skills of MHFA was introduced in order for participants to recognize the difficulty in dealing with such a person. After the introductory session, topics relevant to supporting such people were briefly discussed .In the training program, we emphasized the difficulty in approaching a person with depression in the workplace which was partly due to Japanese workplace culture and/or stigma. Recent topics on mental health in the workplace were included. In particular, the Stress Check Program and modern-type depression in Japan were introduced. The Stress Check Program is an occupational policy program started in 2015, aiming at screening for workers with high psychological stress by questionnaires assessing mental health problems. The results of the questionnaire are informed to workers individually, then a physician’s interview is arranged. In addition, this program requires employers to make efforts to improve the workplace environment –21. The To evaluate the effectiveness of the training program, a questionnaire assessing respondents’ confidence and skills in dealing with people with depression and suicidal risk based on the MHFA, and stigma toward mental health problems was conducted. The same questionnaire was conducted anonymously at all 3 time points . In addition, at pre-program, participant demographic data were collected .The confidence level in management of people with depression and/or suicidal risk was evaluated by 6 questions based on the MHFA strategy; 1) approaching a person with symptoms of depression, 2) listening non-judgmentally, 3) telling the possibility of clinical depression, 4) giving support and information, 5) asking “suicidal thoughts”, 6) encouraging the person to obtain appropriate professional help. Participants were asked to answer these questions on a 5-point Likert scale, ranging from 1  to 5 (very confident).To evaluate practical skills in early intervention of depression and suicide-prevention in the workplace, original questionnaires using a case vignette was developed as shown below:Ms. M is a 30-year-old office worker. Formally, she was a bright and sociable person, over the last few weeks she seems to be feeling sadness and unhappiness in a way that she has never experienced before. She always looks tired and drowsy in the workplace. She appears not to sleep well. She has no appetite and is clearly losing weight. She can’t concentrate on her tasks at work, and she tends to make easy mistakes. Her boss considers Ms. M’s current condition as concerning.You (respondent), Ms. M’s co-worker, are concerned about Ms. M’s current condition too.Imagine you are a co-worker of Ms. M, would you deal with Ms. M in the following way or not?Participants (respondents) are asked to imagine themselves as co-worker of Ms. M, and to answer 10 questions rating the possibility of performing behaviors; each question was developed to evaluate skills for early intervention of depression and suicide-prevention based on the MHFA strategy. The possibility of each behavior was rated using 5-point Likert scale, indicating 0 (absolutely no), 1 (probably not), 2 (don’t know), 3 (probably yes), and 4 (absolutely yes).The Japanese version of Link’s Devaluation-Discrimination Scale was conducted to measure respondents’ stigma toward mental health problems, by asking respondents to appraise their feelings toward people who have experienced psychiatric treatment. This scale is consisted of 12 items with a 4-point Likert scale, ranging from 1 (strongly disagree) to 4 (strongly agree), the higher the total score, the higher the stigma the respondent feels. Reliability and validity of the Japanese version scale were confirmed . AverageAll analysis was performed using IBM SPSS 23 Advanced Statistics for Mac OS. Results of confidence level, skills, and stigma were compared at pre-program, post-program and 1 month post-program. Comparisons were performed using total participant data . Shapiro-Wilk test was used to evaluate the normality of data, and revealed non-normal distribution except for total score of Link’s Devaluation-Discrimination Scale. A one-way repeated measures analysis of variance (ANOVA) is known to be reasonably accurate even with non-normal distribution data [We conducted our newly developed program at a company workplace twice among 91 employees, and 83 participants were agreed to participate in this study. All the participants completed the self-rated questionnaires at 3 time points . The basic characteristics of participants were shown in Self-confidence in dealing with people with depression and/or the risk of suicide based on the MHFA was ascertained in 6 areas. Changes in participants’ confidence were shown in Practical skills based on the MHFA were evaluated by 10 questions using an original case vignette shown in . 6 out oStigma toward psychiatric disorders was assessed by the Japanese version of Link’s Devaluation-Discrimination Scale. The total score was shown in In the present study, we newly developed a brief educational training program for workplace settings based on the MHFA, and evaluated the effectiveness of this program. We found that the brief program was effective in improving confidence in all areas required under the MHFA 5-step principles. Regarding practical skills in management of people with depression or suicidal thoughts, we have revealed that the program has a positive impact on some skills. These skills include dealing with situations utilizing active and direct communications such as “directly ask the person about suicidal thoughts” or “directly tell the probability of depression.” In general, even if an employee experiences mental health problems, his/her co-workers tend not to recognize the occurrence of the problem or feel unwilling to offer help with the employee’s mental health problems due to inadequate knowledge . TherefoPrevious MHFA-based training courses for workplaces took 9 or 12 hours –10, 25. Besides the lack of knowledge of mental health problems, stigma is one of the most important barriers affecting the delay in seeking professional help in the workplace. For example, individuals suffering from mental health problems feel “embarrassed” when people around him/her come to know about their mental health problems specifically in young employees . FurtherPrevious systematic reviews on intervention programs targeting workplace settings pointed out the difficulty in drawing general conclusions of effectiveness of the intervention owing to the broad diversity of research , 30–33. There are some limitations in our study. First, we utilized self-rated questionnaires, which might not reflect participants’ actual attitudes and behaviors. In evaluations, assessment of objective outcomes such as reduction in absenteeism or sick leave, and increase in utilization of mental health services among employees is needed. Regarding the skill assessments using responses of the case vignette, we have evaluated step 1, 3, 4 and 5 of the MHFA five-step skills . We haveS1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.


Supplemental Digital Content is available in the text. The Accreditation Council for Graduate Medical Education describes steps to guide programs regarding self-study. The process relies heavily on obtaining stakeholder input to validate findings. Focus groups have been noted to provide input and data not elicited using other methods. Our objective was to show the value of focus groups in obtaining a clear picture of stakeholder priorities as they relate to program aims, opportunities, threats, areas for improvement, and sustainability of improvements.We conducted a series of focus groups in 2017–2018 at a pediatric residency program with residents and core faculty members. The first session for each group elicited input regarding program aims, opportunities, and threats. The second session focused on evaluating program progress over the past 10 years and identifying strengths and needed areas for improvement. We documented responses obtained during the focus groups and used inductive content analysis methods to identify major themes. We repeated the focus groups every 6 months.We identified 8 program aims and pinpointed opportunities and threats. Areas for improvement were selected and then presented back to the stakeholders who confirmed their appropriateness. Repetition of the focus groups every 6 months facilitated ongoing measurement of progress and promoted the development of multiple plan, do, study, act cycles for areas of improvement.Focus groups are an effective way to initiate the self-study process, examine the program aims, opportunities, and threats, and formulate a detailed improvement plan. They are also useful as part of continual evaluation during a dynamic change process. The Accreditation Council for Graduate Medical Education (ACGME) describes 8 steps to guide programs that are organizing their first self-study. These steps include:(1) assemble the self-study group;(2) engage program leaders and constituents in a discussion of program aims;(3) aggregate and analyze data from annual program evaluations (APEs) and the self-study to create a longitudinal assessment of program strengths and areas for improvement;(4) examine the program’s environment for opportunities and threats;(5) obtain stakeholder input on strengths, areas for improvement, and threats to prioritize actions;(6) interpret the data and aggregate the self-study findings;(7) discuss and validate the findings with stakeholders;1(8) develop a succinct self-study document for use in further program improvement as documentation for the Program’s 10-Year Site Visit.Although the steps to organizing a self-study are well defined, the process of obtaining stakeholder input, validating findings, and sustaining improvement can vary. Programs often use structured ACGME surveys or questionnaires designed internally as methods to determine the opinions of residents and faculty. Although these do provide valuable information, they require a priori identification of concerns which may not reflect current issues. ACGME allows programs significant freedom in deciding the best way to gain stakeholder input, leading to a current research gap regarding best practices.2 Our goal is to describe how we used focus groups to gather stakeholder input and how the groups helped assure we defined measurable outcomes and sustained our improvements (1) An excellent model for obtaining stakeholder input regarding program aims, opportunities, threats, and areas for improvement.  An effective approach for defining and sustaining measurable outcomes.The study was conducted in 2017–2018 at a pediatric residency program within an academic medical center. IRB approval was not required as this project was not Human Subjects Research.5 were invited by email to participate in the focus groups and all elected to do so. We conducted focus groups for residents and faculty members separately, and each group was seen twice within 1 week at the beginning of the self-study and then again for follow-up at 6 and 12 months after the initial focus group sessions. All focus groups were 1 hour in length and were facilitated by a doctoral-level psychologist who had experience with focus groups. At session one and session two, a psychology graduate student was also present to help with data collection. At each session, participants received feedback regarding prior sessions.We conducted a series of focus groups with residents and faculty members serving as focus group participants. All 18 categorical and 8 medicine/pediatric residents enrolled in the program and 20 core faculty members, as defined by ACGME standards,1 To increase the transparency of stakeholder responses, the program director was not in attendance after the presentation.The first session for each group consisted of a series of questions designed to elicit responses regarding program aims, opportunities, and threats. At the beginning of this session, the program director provided a presentation explaining the ACGME self-study process with the specific directions provided on the ACGME website for defining aims, opportunities, and threats. We explained that aims are a long-term strategic view of key expectations for the program that show what kind of physicians the program aims to produce.During the second session, each group reviewed and evaluated the past 10 years of program data, focusing on program growth and responses to areas of need. Before the second session, participants had received copies of 10 years of APEs in addition to an aggregate summary of all 10 years to facilitate discussion and to provide a historical perspective for both residents and junior faculty.To facilitate thinking more broadly about the proposed areas for improvement, we asked both resident and faculty groups to identify a “wish list” of things that would make the program better. In addition to questions regarding the growth and development of the residency program, the facilitator asked faculty about their perceptions regarding faculty-specific needs, including faculty development options.We recorded responses obtained during the focus groups in writing, and we used inductive content analysis methods to identify major themes across all questions. Inductive content analysis is a qualitative method used to identify themes and develop hypotheses/theory by repeated examination of raw data. These data can include written documents, interviews, etc. Using this method, we make no a priori hypotheses, and themes are seen as emerging from data in such a way that it tells the story of what is being studied. Data were coded based on emerging themes, and a validity check was done by having individuals not involved in the focus groups (psychology graduate students) sort the data into the identified themes/categories. Also, themes identified in the focus groups were shared with the residents and core faculty to provide closed-loop communication and to provide an additional validity check with the focus group analysis in terms of identified program aims, opportunities, threats, strengths, and areas for improvement. The focus group leader provided a written report of each session to the self-study group.Once we established areas for improvement, we developed improvement teams. Each team was led by at least one resident and faculty volunteer. Because other programs have had difficulty with measuring outcomes and maintaining improvement, team leaders were asked to oversee these issues through individual quality improvement initiatives.We conducted additional focus group sessions at 6-month intervals. The focus during these sessions was on reviewing the identified areas of improvement and identifying successes and challenges of the improvement teams and needed adjustments to the improvement plans. At each of these follow-up sessions, participants were encouraged to suggest additional measurable outcomes.Following the first follow-up session 6 months into the process, participants reported primarily positive progress and had little to say regarding challenges. At the follow-up session at 12 months, the program director made a short presentation at the beginning of both the resident and faculty groups to encourage more feedback regarding challenges and ways to measure progress.The focus groups identified 8 program aims:Provide varied learning opportunities to train individuals to be outstanding general pediatricians and to prepare those who choose fellowship training appropriately.Plan and execute educational experiences that result in the mastery of pediatric milestones and core competencies and success on the pediatric board exam.Mold a commitment to self-directed learning and participation in research.Serve the needs of underserved individuals in this part of the Appalachian region both directly and by fostering an understanding of rural populations and of the challenges of providing healthcare in small communities.Serve the needs of our infants with neonatal abstinence syndrome both directly and by teaching about the comprehensive needs of this population.Provide a “family-like” atmosphere that is positive and supportive, where physician wellness and self-care are important.Prepare residents to advocate for children, including involvement in legislative advocacy and formation of health policies.Teach residents to understand the business of medicine.1 these aims were then approved by the self-study group that included the department chair, the program director, and the associate program director. These aims were also approved at the institutional level by the graduate medical education office designated institutional officer before being submitted to ACGME. Also, the self-study group identified opportunities and threats and selected 5 areas for improvement based on themes from both the resident and core faculty focus group. We included most areas for improvement that emerged in the focus group report. The only ones that were not included were those that the self-study group determined would not be best addressed by an improvement team. For example, one area involved concern about a specific faculty member which the group determined would be appropriately addressed directly by the department chair. Due to input from the faculty focus group, we also included faculty development in the improvement plan. Finally, the self-study group added 2 more areas based on the more global program aims, as the areas identified by the focus groups tended to be quite detailed and specific. This addition resulted in a total of 8 improvement teams, 6 directly identified based on areas for improvement which were defined in Session Two, including one to improve faculty development, and two based directly on program aims from Session One. Supplemental Digital Content, Table 1, available at http://links.lww.com/PQ9/A119, lists the 8 improvement teams and details baseline data, action plans, 6- and 12-month outcomes, and next steps. Supplemental Digital Content 2, Table 2, available at Supplemental Digital Content 1, http://links.lww.com/PQ9/A106. includes additional data regarding the faculty development improvement team.As per ACGME self-study instructions,Each improvement team was assigned resident and faculty leaders who tracked and measured outcomes and reported back to the self-study group. The first 6-month follow-up showed many positive outcomes. After encouragement to do so, the second 6-month session also included more input on what was not going well and could be further improved and new ideas for measurement. This expanded input allowed for the creation of additional plan, do, study, act cycles by the improvement teams.7The definition of program aims and the elucidation of specific opportunities and threats is a natural fit for focus group methodology as it allows for an active process that can capture the nuances of a program. Although the use of questionnaires is helpful and is one of the many elements we use in our APEs, it requires to a greater extent, a priori identification of specific areas of inquiry that may or may not reflect current program norms. This factor is especially important as the ACGME encourages programs to develop their own identities. A secondary gain in the utilization of focus groups is that participants are truly “heard,” and this can allow for greater stakeholder participation and buy-in than is typical when less dynamic methods are used.8 Basch9 suggests that the data that are collected could be considered similar to formative evaluations in that they do not rate or score a program but rather identify the current state of the program and offer guidance in terms of opportunities for growth and change. Participants in this project received feedback from the previous sessions and were able to utilize that feedback as they went into the remaining sessions. This feedback allowed for the process to remain focused and to dig deeper into the areas identified as important.Focus groups are seen as allowing for greater flexibility and the ability to gather a richer set of data due to participant interactions with each other during the focus group process.For example, our second area for improvement involved the hiring of a faculty person triple-boarded in pediatrics, psychiatry, and child psychiatry. Part of the goal was that during the behavior development rotation, the pediatric resident would spend 2 mornings a week with this triple-boarded faculty member in her child psychiatry clinic. At the 12-month follow-up focus group, we realized residents were being assigned to work with this faculty member during her time seeing routine general pediatric patients for nonpsychiatric visits instead of working with her in her psychiatry clinic. Perhaps, one of the most important things the ongoing focus groups accomplished was revealing what was not working and keeping us accountable to the improvement process.Another important success was in encouraging participants to continue to develop new outcome measures. For this same improvement plan, the 12-month follow-up focus group recommended including in-training exam scoring in this specific area of mental healthcare.The major limitation of this study is that we did not have a comparison group using other methods besides focus groups. We do have some feeling for comparison as the self-study is a natural expansion of the APE. Every year, we consider data including faculty and resident ACGME surveys, internal in-house faculty and resident electronic surveys, resident progression on milestones, graduate surveys, in-training exam scores, American Board of Pediatrics first-time pass rates, and confidential resident evaluations of faculty and rotations. The resident members also communicate directly with all residents and supply further information by word of mouth. In the setting of our experience with multiple methods, we anecdotally found that focus groups were a unique approach to identifying more varied and detailed areas for improvement.Focus groups are an effective way to gather stakeholder input during the self-study process. They allow a program to examine aims, opportunities, threats, and strengths, and to formulate a detailed improvement plan. When guided to do so, they can establish measurable outcomes, and when conducted in an ongoing fashion, they facilitate staying on track during a dynamic improvement process.The authors have no financial interest to declare in relation to the content of this article.


Backgrounds: A number of genetic and biological phenomena imply that tumorigenesis of clear cell renal cell carcinoma (ccRCC) is highly correlated with hypoxia-induced factor-1a (HIF-1α). Recently, research focusing on the post-transcriptional regulation of HIF-1α has provided a new perspective for ccRCC therapy. In this study, we observed the expression pattern of the RNA-binding protein QKI, which could regulate HIF expression in ccRCC both in vitro and in vivo.Methods: Tissue microarraywas subjected to immunohistochemistry and tumour cell lines and nude mice were used for in vitro and in vivo assays. QKI overexpression or knockdown was assessed in renal cancer cells.Results: The overexpression of QKI inhibited the proliferation of the 786-0 and caki-1 cells, blocked the cells' entry into the S phase, and promoted apoptosis. In ectopic-implantation nude mice model, QKI depletion significantly increased tumor sizes and initiation rates. Tissue microarrays showed that the expression of QKI genes, and especially QKI-6, was significantly decreased in tumor tissues compared with these in normal kidney tissues. Moreover, decreased QKI expression was closely correlated with high tumor grade, poor differentiation, and poor survival.Conclusions: QKI may be useful as a novel, independent diagnostic and biological marker for ccRCC. What is more, pVHL can affect fibronectin assembly The morbidity of ccRCC has increased, which makes ccRCC one of the most notorious diseases threatening the health of the general population. Renal-cell carcinoma (RCC) accounts for 1.9 % of all cancers is one of the most common type of cancers in adults and RNA-binding proteins and microRNAs are key regulators in cell biology, by regulating the RNA stability, transcriptional efficiency, translocation and alternative splicing. Deficiency of mRNA regulation is closely related to cancer as well as many other human diseases Although it has recently been proven that QKI-5 inhibits ccRCC proliferation through the RAS-MAPK signal pathway, we have differentiated the mechanism by which QKI inhibits ccRCC from its roles in metabolism and angiogenesis. Above all, with new insights into the different functions of QKI, we conducted a series of interesting tests aiming to improve the diagnosis, prognosis and therapy of ccRCC. We tested the relationship between renal cell carcinoma and expression of QKI in the following aspects:(1) The expression of QKI in renal cell carcinoma and adjacent normal tissue;(2) The correlation of QKI with the degree of differentiation, degree of invasion, TNM grading, age, tumor size and survival rates of patients with renal cell carcinoma; andin vitro and in vivo. In view of our findings, we have come to the conclusion that QKI inhibits the development of renal cell carcinoma by decreasing the expression of HIF-1α. Moreover, QKI maybe an independent factor that influences the prognosis of renal cell carcinoma, and therefore may serve as a potential diagnostic and therapeutic target.(3) How the expression of QKI influences the biological behavior of the cells and tumors both This study was approved by the ethics committee of the Fourth Military Medical University . We used tissue microarray technology to analyze samples from 161 participants who were diagnosed with clear cell renal cell carcinoma between July 2007 and February 2008. Patients, who received treatment before surgery, including partial or radical nephrectomy, were excluded. A part of the patients (129) went through follow-up visits up to September 2012. The cancer tissues and the matched adjacent normal tissues of every participant had been reviewed and confirmed by the Department of Pathology, Xijing Hospital, China. Pathological information was collected from the clinical patient database, and the information was blinded for the study physicians who reviewed all the records of ccRCC and recorded the data into the database.2O2 in phosphate-buffered saline (PBS) for 50 min, followed by incubation in 5 % bovine serum albumin blocking buffer. The tissue sections were incubated with the primary anti-QKI antibody  for 24 h, 4 °C. Immunodetection was performed using a 3-step protocol, with a streptavidin-horseradish peroxidase complex and visualized using 3, 3-diaminobenzidine.The QKI antibody was purchased from Sigma Biotechnology, America, and the assay was conducted according to the manufacturer's instructions. Tissue array was fixed in 10% neutralized formalin and was embedded in paraffin blocks. Tissue arrays (4 μm) were prepared for H&E staining and also for immunohistochemical examination. For immunohistochemical processing, the endogenous peroxidases were blocked using 0.75% HThe final follow-up was completed in December 2012, with around 5 years of monitoring for each patient. The follow-ups were conducted from the date of surgery until death or the December 2012 deadline. The doctor responsible for recording the follow-up information did not have access to the clinical pathological database and the QKI immunohistochemistry results.2 plastic cell culture flasks (Corning), and grown at 37 °C in an atmosphere comprising 5 % CO2and 21 % O2. The cells were maintained as monolayers in 60 cm2 treated tissue culture flasks  at 37 °C in a humidified atmosphere comprising 5 % CO2. Vector transfection was conducted using the FuGENE 6 kit (Roche) in accordance with the manufacturer's instructions. For the hypoxia treatment, cells were cultured under hypoxic conditions  and harvested at 24 h after transfection.The 786-0 and caki-1cell lines were purchased from American Type Culture Collection  used to model renal cell carcinoma, and the HEK293 (ATCC) cell line was purchased from Chinese Academy of Sciences and used to model normal kidney tissue. The cells were cultured in RPMI 1640 medium  supplemented with 10% FBS and 0.1 % penicillin-streptomycin (1:1000), seeded into 25 cmRIPA buffer  was used to extract total cellular protein from the two renal cell carcinoma cell lines, renal cell carcinoma tissues and adjacent normal tissues. The BCA assay was conducted to quantify the amount of protein, and 60 μg of protein from each sample was subjected to SDS-PAGE on 10 % polyacrylamide gels, and transferred to nitrocellulose membranes. The membranes were incubated with a primary antibody raised against QKI  and other regulatory proteins  overnight at 4 °C. After the membranes were washed with TBST, they were incubated with an IgG-IRDye TM800CW  fluorescently-labeled secondary antibody (diluted 1:10000 in TBS) for 40 minutes at 37 °C. The protein bands were visualized using an Odyssey Infrared Imaging Laser scanning imaging system.2O.Cells that underwent the indicated treatments were harvested for RNA isolation using the Trizol reagent  according to manufacturer's instructions, and the RNA concentration was measured using a UV spectrophotometer. One microgram of total RNA was used as template for reverse transcription in accordance with the instructions of the M-MLV assay kit (Invitrogen). The primer sequences used in this study are shown in The amplification conditions comprised 40 cycles at 95 °C for 5 s and 65 °C for 30 s. The results were analyzed by the Bio-Rad CFX Manager software.2HPO4 and 2.5 mM sodium citrate) at 37 °C for 20 min and then re -suspended in PBS containing 40 mg/ml PI , 0.1 mg/ml RNase  and 0.1% Triton X-100  at 37°C for 30 min in the dark. Cell cycle distribution was detected by FACS on an instrument .To analyze the cell cycle distribution, cells were washed twice in PBS and fixed for at least 2 h in 300 μl PBS and 700 μl ethanol. The resulting fixed cells were spun down gently in 200 μl of extraction buffer  per well. After 24, 48, 72, 96, 118 hours of incubation, 20 μl/well of a 5 mg/ml 3--2, 5-diphenyl-tetrazolium bromide  in 200ul solution was added and incubated for 4 hours at 37 °C. Next, the culture medium was removed, and 150 μL of DMSO was added. After 15 min of gentle shaking, the absorption at 490 nm was measured on a multi-well plate reader. Cell growth curves were determined according to the average absorption values.The MTT test was used to assess cell proliferation. Briefly, cells were seeded at a density of 2 × 10 5 cells per well were seeded in 6-well plates (Corning). The culture medium was removed when the cells were 75 % confluent. One milliliter of lentiviral particles targeting sh-QKI was added. Scrambled lentiviral particles were added as controls. Afterwards, the cells were cultured at 37 °C in an atmosphere comprising 5 % CO2. Eight hours post-infection, the lentiviral particles were removed, and the cells were switched to regular culture medium for 24 hours. Subsequently, 1640 medium  with 5 µg/ml puromycin was used to culture the cells for another 24 hours. After 3 days, lentivirus-positive cells were selected based on conditions above. Western blot analysis was used to evaluate the efficiency of infection. The positive clones were selected for animal experiments after seven days.We selected the 786-0 cell line for this series of experiments. 1 × 10 2 after transfection with plasmids. We collected the supernatants after culturing in serum-free 1640 medium for 24 hours. The ELISA was conducted in accordance with the manufacturer's instructions. The absorption values were measured at 450 nm on a multi-well plate reader.The human VEGF ELISA KIT  was used to investigate the VEGF expression profile of human renal cell carcinomas. 786-0 and caki-1 cells were cultured for 24 hours at 37 °C in an atmosphere comprising 5% CO3) = width2 (mm2) × length (mm) / 2. At the indicated time points, the mice were sacrificed after barbiturate overdose by a properly trained person via cervical dislocation, and the cadavers were dissected for the evaluation of tumor size, and immunostaining.Male nude mice (4-6 weeks old) were obtained from the Experimental Animal Center of the Fourth Military Medical University . All animal experiments were performed in accordance with the Animal Care and Use Committee of the Fourth Military Medical University . Animals were looked after very well during experiment process. The 786-0 cells were suspended in sterile PBS and injected subcutaneously into the dorsal area of the nude mice. The size and incidence of subcutaneous tumors was recorded every 3 days. Tumor volumes were calculated according to the formula V , under a light microscope at 200× magnification. Tumor masses were obtained from nude mice at day 33. Frozen sections with 5 µm thickness were prepared from each specimen and were stained for CD31  to detect blood vessels. Three fields from 2 tumor masses of each mouse were analyzed.2) test was used for QKI expression measurements. The Kaplan-Meier estimator was used to assess the survival rates. Statistical significance was evaluated using the means ± SD by single-factor analysis of variance (one-way ANOVA). Differences were considered statistically significant at P < 0.05.Statistical analysis was performed using SPSS 17.0 software. Pearson's chi-square .To investigate the role of QKI in the initiation and progression of ccRCC, western blot analysis and immunohistochemistry were conducted to analyze the expression of QKI in the ccRCC tumor mass and matched adjacent normal tissues of 161 patients. As shown in Table 1, QKI expression was evident in 97.5% (157/161) of the matched adjacent kidney tissue samples, which represented a significantly higher percentage than in the QKI-positive samples of clear cell renal cell carcinoma . According to immunohistochemistry analysis, the expression of QKI in the nuclei and cytoplasm of ccRCC and matched adjacent normal tissues was completely different. The percentage of nucleus-positive samples of ccRCC was 74.5% (120/161), and the percentage of nucleus-positive samples of matched adjacent normal tissues was 75.7% (122/161). Accordingly, there was no statistical significance between ccRCC and normal kidney tissues χ2 =0.07, P > 0.05, as shown in Tables 3. By contrast, the percentage of cytoplasm-positive tissues in ccRCC was 6.2% (10/161),whereas the percentage of nuclear-positive tissues of matched adjacent normal tissues was77.6 % (125/161), which represented a highly statistically significant difference between ccRCC and normal kidney tissues , as shown in Table 3.Our initial efforts focused on investigating the relationship between QKI and tumor growth. As an RNA-binding protein, QKI may function by modulating the expression of its target mRNAs at the posttranscriptional level. We noted that HIF-1α, which is highly expressed in VHL-mutated renal cancer cells, acts as a downstream effector of QKI. Analysis of QKI expression by western blotting and RT-PCR in two ccRCC cell lines and one normal kidney cell line showed that QKI expression was significantly lower under pathological conditions than in the normal cell line . To evaluate the extent of the QKI up- and downregulation, we utilized western blotting and RT-PCR. Transfection with QKI-modulating vectors was used to both upregulate and downregulate its expression both in 786-0 and caki-1 cells. Cell proliferation curves showed that the overexpression of QKI inhibited the proliferation of 786-0 and caki-1 cells to an increasing degree over time , while the downregulation of QKI resulted in elevated proliferation of both 786-0 and caki-1 cell . FCM analysis showed that the overexpression of QKI in 786-0 and caki-1 cells created a block in the G0/G1 phase which was more prominent than in the control group . Conversely, knockdown of QKI in the 786-0 and caki-1 cells promoted their entry into the S phase of the cell cycle .In order to investigate the effects of QKI on the biological behavior of cancer cells, we overexpressed and knocked down QKI in the 786-0 and caki-1 cell lines, and verified the efficiency of the expression manipulation in the 786-0 cell line , while the percentage of apoptotic cells in the QKI knockdown group was significantly lower than in the negative control group .As cell survival relies on proliferation and apoptosis, we next explored the relationship between QKI and apoptosis. The percentage of apoptotic cells in the QKI-overexpressing group was significantly higher than in the pc(3.1+)group .Conversely, in the QKI knockdown groups of both 786-0 and caki-1 cells, the CyclinD1 expression level was upregulated, and the P27 and Ki-67 expression levels were downregulated .Our previous study demonstrated that QKI was positively correlated with cell apoptosis and negatively correlated with cell proliferation, we then examined the relationship between QKI and cell cycle markers. Western blot analysis showed that in the QKI-overexpressing 786-0 and caki-1 cells, the expression level of cyclin D1was downregulated and the expression levels of P27 and Ki-67 were up-regulated . By contrast, in the 786-0 and caki-1 cells with QKI knockdown, the HIF-1α expression level was upregulated, and similar to the tendency mentioned above, the HIF-1α expression level was even higher under hypoxic than under normoxic conditions . To investigate the regulation of the downstream targets of HIF-1α, we tested vascular endothelial growth factor (VEGF), Glucose transporter 1(GLUT-1) and phosphoglycerate kinase-1(PGK-1), which play roles in angiogenesis, glycolysis, and glucose transport, respectively. To determine the relationship between these genes and the expression of QKI in ccRCC under both normoxic and hypoxic conditions, we performed qRT-PCR analysis. The data showed that in the QKI-overexpressing ccRCC cells, the VEGF, GLUT-1, and PGK-1 expression levels were downregulated, and even more so under hypoxic conditions . Conversely, in the QKI-knockdown ccRCC cells, the expression levels of these genes were upregulated. Moreover, the VEGF, GLUT-1, and PGK-1 expression levels were even higher under hypoxic conditions .To further investigate the downstream targets of QKI in ccRCC, we scrutinized the coding and noncoding regions of transcriptional factors, which may contain potential QREs. Based on the screening results, HIF-1α was a potential candidate. Since the expression ofHIF-1α is also regulated by the oxygen concentration, we investigated normoxic and hypoxic group transfected with the QKI vectors. After 48 hours of culture, the QKI expression was upregulated and downregulated, respectively, in both the 786-0 and caki-1 cells. RT-PCR and western blot analysis showed that in the QKI-overexpressing 786-0 and caki-1cells, HIF-1α expression was downregulated, which was more obvious under hypoxic conditions  and conversely was upregulated in the knockdown groups. Again displaying a similar tendency to the one mentioned above, the VEGF expression level was even higher under hypoxic, as compared to normoxic conditions .To study whether QKI would affect angiogenesis in vivo, we injected normal caki-1 cells and QKI-knockdown caki-1 cells into the dorsal area of nude mice. We conducted HE-staining and immunofluorescence staining for CD31 in the tumor mass, which showed a significant increase of vascularization in the QKI knockdown group . Moreover, the MVC further corroborated this observation .To further explore the effects of QKI up-and downregulation on angiogenesis, we used ELISA. Firstly, si-QKI small-interfering RNA and the QKI vector were used to transfect 786-0 and caki-1 cells for 24 hours. Additionally, the serum-containing medium was replaced with serum-free medium, and the cells were cultured for another 24 hours before the supernatants were harvested. After 48 hours post-transfection, the supernatants of 786-0 and caki-1 cells were harvested for ELISA. The assay showed that VEGF expression was downregulated in the QKI-overexpressing groups . Subsequently, siRNA transfection was conducted in both 786-0 and caki-1 cells 24 hours prior to transfection with the QKI vectors. After a further 48 hours, the cells were harvested and RT-PCR analysis was conducted. As shown in Figure , when si-HIF-1α and the QKI vector were used to co-transfect the cells, them RNA expression levels of HIF-1α, as well as the bona-fide downstream genes VEGF, GLUT-1, and PGK-1 were not significantly downregulated compared to the control group. It can therefore be concluded that the mechanism by which the RNA-binding protein QKI was able to negatively regulate the expression levels of HIF-1α and its target genes VEGF, GLUT-1 and PGK-1 requires the expression of HIF-1α. The similar trend was verified in protein level in 786-0 cells by western blot assay .We next investigated if QKI directly affected the expression of VEGF, GLUT-1, and PGK-1, or of its effect was realized via HIF-1α. Firstly, two small-interfering RNAs (siRNAs) which specifically silence HIF-1α were designed and verified in 786-0 and caki-1 cells . Statistically significant correlations were not found between QKI and depth of invasion, pTNM stage, age at diagnosis, gender, primary tumor location and maximum tumor diameter (P>0.05) (Table 4).The coefficients of correlation between QKI and clinical pathological characteristics are shown in Table 5.To analyze the correlation of the QKI expression with different clinical factors, we conducted semi-quantitative analysis and divided the tissue samples into two QKI expression groups according to the QKI immunohistochemistry results: increased (> 25%) and decreased (<= 25%). The correlation of QKI expression levels in the ccRCC samples with different clinicopathologic factors is shown in Fig. 7). These results showed that decreased QKI expression was significantly correlated with poor survival. In the group showing non-decreasing QKI expression, the median survival time of clear cell renal cell carcinoma patients was undefined. However, in the decreased-QKI-expression group, the median survival time was only 68 months. The age at diagnosis, depth of invasion, TNM stage, gender, primary tumor location and maximum tumor diameter were not correlated with the survival time.The correlation of QKI expression with patient survival was determined using the Kaplan-Meier estimator  accounts the sixth place in male patient and the ninth place in female patient et al.The incidence and development of renal cell carcinoma depends upon the expression of several oncogenes, such as HIF-1α, VEGF et al. showed that in the mouse brain, all three isoforms are expressed in glial cells. Notably, QKI-5 locates in nuclear whereas QKI-6 and -7 retentate in cytoplasmic In vitro, the overexpression of QKI inhibited the proliferation of the 786-0 and caki-1 cell lines, blocked the cells' entry into the S phase, and promoted cell apoptosis. Using small interfering RNA, we confirmed the role of QKI in ccRCC. In vivo, the knockdown of QKI promoted tumor growth and upregulated the expression of HIF-1α. These data demonstrated that QKI plays a tumor-suppressive role in ccRCC.The RNA-binding protein QKI is an important factor that attracts researchers' attention for its key role in tumor cell initiation and differentiation in various studies. Our previous studies demonstrated significant role of QKI in cancers QKI regulates the mRNA expression of many target genes at the posttranscriptional level. In fact, the expressions of hundreds of genes are regulated by direct binding of QKI at their 3′UTR or CDS regions harboring QRE-binding sites with a specific AYUAAY sequence in vitro and in vivo data, our results strongly support the idea that QKI can inhibit the development of ccRCC by regulating HIF-1α and its downstream target genes, and that it requires the expression of HIF-1α. Clear cell renal cell carcinoma is associated with loss-of-function mutations in the VHL(von Hippel- Lindau) tumor suppressor gene. VEGF is an important target of HIF and a known potent mediator of angiogenesis Combining the Our previous reports have shown that QKI functions as a suppressor of carcinogenesis in diverse tumors through the coordinated targeting multiple genes cell growth and differentiation genes