Protein disulfide-isomerase interacts with a substrate protein at all stages along its folding pathway

In contrast to molecular chaperones that couple protein folding to ATP hydrolysis, protein disulfide-isomerase (PDI) catalyzes protein folding coupled to formation of disulfide bonds (oxidative folding). However, we do not know how PDI distinguishes folded, partly-folded and unfolded protein substrates. As a model intermediate in an oxidative folding pathway, we prepared a two-disulfide mutant of basic pancreatic trypsin inhibitor (BPTI) and showed by NMR that it is partly-folded and highly dynamic. NMR studies show that it binds to PDI at the same site that binds peptide ligands, with rapid binding and dissociation kinetics; surface plasmon resonance shows its interaction with PDI has a Kd of ca. 10−5 M. For comparison, we characterized the interactions of PDI with native BPTI and fully-unfolded BPTI. Interestingly, PDI does bind native BPTI, but binding is quantitatively weaker than with partly-folded and unfolded BPTI. Hence PDI recognizes and binds substrates via permanently or transiently unfolded regions. This is the first study of PDI's interaction with a partly-folded protein, and the first to analyze this folding catalyst's changing interactions with substrates along an oxidative folding pathway. We have identified key features that make PDI an effective catalyst of oxidative protein folding – differential affinity, rapid ligand exchange and conformational flexibility

To what extent do site-based training, mentoring, and operational research improve district health system management and leadership in low- and middle-income countries: a systematic review protocol

District health managers play a key role in the effectiveness of decentralized health systems in low- and middle-income countries. Inadequate management and leadership skills often hamper their ability to improve quality of care and effectiveness of health service delivery. Nevertheless, significant investments have been made in capacity-building programmes based on site-based training, mentoring, and operational research. This systematic review aims to review the effectiveness of site-based training, mentoring, and operational research (or action research) on the improvement of district health system management and leadership. Our secondary objectives are to assess whether variations in composition or intensity of the intervention influence its effectiveness and to identify enabling and constraining contexts and underlying mechanisms

Effectiveness of Provider and Community Interventions to Improve Treatment of Uncomplicated Malaria in Nigeria: A Cluster Randomized Controlled Trial

The World Health Organization recommends that malaria be confirmed by parasitological diagnosis before treatment using Artemisinin-based Combination Therapy (ACT). Despite this, many health workers in malaria endemic countries continue to diagnose malaria based on symptoms alone. This study evaluates interventions to help bridge this gap between guidelines and provider practice. A stratified cluster-randomized trial in 42 communities in Enugu state compared 3 scenarios: Rapid Diagnostic Tests (RDTs) with basic instruction (control); RDTs with provider training (provider arm); and RDTs with provider training plus a school-based community intervention (provider-school arm). The primary outcome was the proportion of patients treated according to guidelines, a composite indicator requiring patients to be tested for malaria and given treatment consistent with the test result. The primary outcome was evaluated among 4946 (93%) of the 5311 patients invited to participate. A total of 40 communities (12 in control, 14 per intervention arm) were included in the analysis. There was no evidence of differences between the three arms in terms of our composite indicator (p = 0.36): stratified risk difference was 14% (95% CI -8.3%, 35.8%; p = 0.26) in the provider arm and 1% (95% CI -21.1%, 22.9%; p = 0.19) in the provider-school arm, compared with control. The level of testing was low across all arms (34% in control; 48% provider arm; 37% provider-school arm; p = 0.47). Presumptive treatment of uncomplicated malaria remains an ingrained behaviour that is difficult to change. With or without extensive supporting interventions, levels of testing in this study remained critically low. Governments and researchers must continue to explore alternative ways of encouraging providers to deliver appropriate treatment and avoid the misuse of valuable medicines

Dynamic changes in eIF4F-mRNA interactions revealed by global analyses of environmental stress responses

BACKGROUND: Translation factors eIF4E and eIF4G form eIF4F, which interacts with the messenger RNA (mRNA) 5' cap to promote ribosome recruitment and translation initiation. Variations in the association of eIF4F with individual mRNAs likely contribute to differences in translation initiation frequencies between mRNAs. As translation initiation is globally reprogrammed by environmental stresses, we were interested in determining whether eIF4F interactions with individual mRNAs are reprogrammed and how this may contribute to global environmental stress responses. RESULTS: Using a tagged-factor protein capture and RNA-sequencing (RNA-seq) approach, we have assessed how mRNA associations with eIF4E, eIF4G1 and eIF4G2 change globally in response to three defined stresses that each cause a rapid attenuation of protein synthesis: oxidative stress induced by hydrogen peroxide and nutrient stresses caused by amino acid or glucose withdrawal. We find that acute stress leads to dynamic and unexpected changes in eIF4F-mRNA interactions that are shared among each factor and across the stresses imposed. eIF4F-mRNA interactions stabilised by stress are predominantly associated with translational repression, while more actively initiating mRNAs become relatively depleted for eIF4F. Simultaneously, other mRNAs are insulated from these stress-induced changes in eIF4F association. CONCLUSION: Dynamic eIF4F-mRNA interaction changes are part of a coordinated early translational control response shared across environmental stresses. Our data are compatible with a model where multiple mRNA closed-loop complexes form with differing stability. Hence, unexpectedly, in the absence of other stabilising factors, rapid translation initiation on mRNAs correlates with less stable eIF4F interactions

Zanamivir Conjugated to Poly-L-Glutamine is Much More Active Against Influenza Viruses in Mice and Ferrets Than the Drug Itself

Purpose: Previously, polymer-attached zanamivir had been found to inhibit influenza A viruses in vitro far better than did small-molecule zanamivir (1) itself. The aim of this study was to identify in vitro—using the plaque reduction assay—a highly potent 1-polymer conjugate, and subsequently test its antiviral efficacy in vivo. Methods: By examining the structure-activity relationship of 1-polymer conjugates in the plaque assay, we have determined that the most potent inhibitor against several representative influenza virus strains has a neutral high-molecular-weight backbone and a short alkyl linker. We have examined this optimal polymeric inhibitor for efficacy and immunogenicity in the mouse and ferret models of infection. Results: 1 attached to poly-L-glutamine is an effective therapeutic for established influenza infection in ferrets, reducing viral titers up to 30-fold for 6 days. There is also up to a 190-fold reduction in viral load when the drug is used as a combined prophylactic/therapeutic in mice. Additionally, we see no evidence that the drug conjugate stimulates an immune response in mice upon repeat administration. Conclusions: 1 attached to a neutral high-molecular-weight backbone through a short alkyl linker drastically reduced both in vitro and in vivo titers compared to those observed with 1 itself. Thus, further development of this polymeric zanamivir for the mitigation of influenza infection seems warranted.National Institutes of Health (U.S.) (Grant U01-AI074443

Using twins to better understand sibling relationships

We compared the nature of the sibling relationship in dyads of varying genetic relatedness, employing a behavioural genetic design to estimate the contribution that genes and the environment have on this familial bond. Two samples were used—the Sisters and Brothers Study consisted of 173 families with two target non-twin children (mean ages = 7.42 and 5.22 years respectively); and the Twins, Family and Behaviour study included 234 families with two target twin children (mean age = 4.70 years). Mothers and fathers reported on their children’s relationship with each other, via a postal questionnaire (the Sisters and Brothers Study) or a telephone interview (the Twins, Family and Behaviour study). Contrary to expectations, no mean level differences emerged when monozygotic twin pairs, dizygotic twin pairs, and non-twin pairs were compared on their sibling relationship quality. Behavioural genetic analyses also revealed that the sibling bond was modestly to moderately influenced by the genetic propensities of the children within the dyad, and moderately to substantially influenced by the shared environment common to both siblings. In addition, for sibling negativity, we found evidence of twin-specific environmental influence—dizygotic twins showed more reciprocity than did non-twins. Our findings have repercussions for the broader application of results from future twin-based investigations

Combined use of the Consolidated Framework for Implementation Research (CFIR) and the Theoretical Domains Framework (TDF): A systematic review

Background: Over 60 implementation frameworks exist. Using multiple frameworks may help researchers to address multiple study purposes, levels, and degrees of theoretical heritage and operationalizability; however, using multiple frameworks may result in unnecessary complexity and redundancy if doing so does not address study needs. The Consolidated Framework for Implementation Research (CFIR) and the Theoretical Domains Framework (TDF) are both well-operationalized, multi-level implementation determinant frameworks derived from theory. As such, the rationale for using the frameworks in combination (i.e., CFIR + TDF) is unclear. The objective of this systematic review was to elucidate the rationale for using CFIR + TDF by (1) describing studies that have used CFIR + TDF, (2) how they used CFIR + TDF, and (2) their stated rationale for using CFIR + TDF. Methods: We undertook a systematic review to identify studies that mentioned both the CFIR and the TDF, were written in English, were peer-reviewed, and reported either a protocol or results of an empirical study in MEDLINE/PubMed, PsycInfo, Web of Science, or Google Scholar. We then abstracted data into a matrix and analyzed it qualitatively, identifying salient themes. Findings: We identified five protocols and seven completed studies that used CFIR + TDF. CFIR + TDF was applied to studies in several countries, to a range of healthcare interventions, and at multiple intervention phases; used many designs, methods, and units of analysis; and assessed a variety of outcomes. Three studies indicated that using CFIR + TDF addressed multiple study purposes. Six studies indicated that using CFIR + TDF addressed multiple conceptual levels. Four studies did not explicitly state their rationale for using CFIR + TDF. Conclusions: Differences in the purposes that authors of the CFIR (e.g., comprehensive set of implementation determinants) and the TDF (e.g., intervention development) propose help to justify the use of CFIR + TDF. Given that the CFIR and the TDF are both multi-level frameworks, the rationale that using CFIR + TDF is needed to address multiple conceptual levels may reflect potentially misleading conventional wisdom. On the other hand, using CFIR + TDF may more fully define the multi-level nature of implementation. To avoid concerns about unnecessary complexity and redundancy, scholars who use CFIR + TDF and combinations of other frameworks should specify how the frameworks contribute to their study. Trial registration: PROSPERO CRD4201502761

Kallmann syndrome: a hystorical, clinical and molecular review

Kallmann syndrome (KS), the association of hypogonadotropic hypogonadism and anosmia, was described by Maestre de San Juan in 1856 and characterized as a hereditary condition by Franz Josef Kallmann in 1944. Many aspects such as pathogeny, phenotype and genotype in KS were described in the last fifteen years. The knowledge of this condition has grown fast, making it difficult to update. Here we review historical aspects of this condition and its discoverers and describe new findings regarding the embryogenesis of the olfactory bulb and GnRH secreting neuronal tracts that are important for understanding the association of hypogonadism and anosmia. Additionally, we describe the phenotypic and genotypic heterogeneity of KS, including five related genes (KAL-1, FGFR1, PROKR2, PROK2 e NELF), and discuss the function of each codified protein in migration and maturation of the olfactory and GnRH neurons, with data from in vitro and in vivo studies. Finally we describe the clinical phenotype of patients carrying these mutations.A síndrome de Kallmann (SK) é a associação de hipogonadismo hipogonadotrófico (HH) e anosmia descrita por Maestre de San Juan, em 1856, e caracterizada como condição hereditária por Franz Josef Kallmann, em 1944. Muitos aspectos de sua patogenia, variabilidade fenotípica e genotípica foram desvendados nos últimos 15 anos. Conseqüentemente, tem sido difícil manter-se atualizado frente à rapidez que o conhecimento dessa condição é gerado. Nesta revisão, resgatamos aspectos históricos pouco conhecidos sobre a síndrome e seus descobridores; incorporamos novas descobertas relacionadas à embriogênese dos neurônios olfatórios e produtores de GnRH. Esse processo é fundamental para compreender a associação de hipogonadismo e anosmia; descrevemos a heterogeneidade fenotípica e genotípica, incluindo mutações em cinco genes (KAL-1, FGFR1, PROKR2, PROK2 e NELF). Para cada gene, discutimos a função da proteína codificada na migração e maturação dos neurônios olfatórios e GnRH a partir de estudos in vitro e modelos experimentais e descrevemos características clínicas dos portadores dessas mutações.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de MedicinaUNIFESP, EPM, Depto. de MedicinaSciEL

Demographics of extra-articular calcaneal fractures: Including a review of the literature on treatment and outcome

Introduction: Extra-articular calcaneal fractures represent 25-40% of all calcaneal fractures and an even higher percentage of up to 60% is seen in children. A disproportionately small part of the literature on calcaneal fractures involves the extra-articular type. The aim of this study was to investigate the incidence of extra-articular calcaneal fractures in a Level 1 trauma centre, define the distribution of the various types of fractures and compare patient demographics between extra- and intra-articular calcaneal fractures. In addition the literature was reviewed for the most common types of extra-articular calcaneal fractures with regard to incidence, treatment and clinical outcome. Methods: The radiological records between 2003 and 2005 were reviewed for intra- and extra-articular calcaneal fractures. Patient gender-distribution and age were compared. A literature search was conducted for the treatment of extra-articular calcaneal fractures. Results: In this 3-year study period a total of 49 patients with 50 extra-articular calcaneal fractures and 91 patients with 101 intra-articular fractures were identified. The median age for the first group was 32.7 years, and for the second group 40.3 years; P = 0.04. Male predominance was significantly less pronounced for extra-articular (63%) compared with intra-articular fractures (79%; P = 0.04). Conclusion: One-third of all calcaneal fractures are extra-articular. Significant differences exist between the intra- and extra-articular groups, in terms of lower age and male-female ratio. The literature study shows inconsistencies in treatment options, but most extra-articular fractures are well manageable conservatively