Conventional and unconventional interactions of the transcription factor FOXL2 uncovered by a proteome-wide analysis.

Beyond the study of its transcriptional target genes, the identification of the various interactors of a transcription factor (TF) is crucial to understand its diverse cellular roles. We focused on FOXL2, a winged-helix forkhead TF important for ovarian development and maintenance. FOXL2 has been implicated in diverse cellular processes, including apoptosis, the control of cell cycle or the regulation of steroid hormone synthesis. To reliably identify partners of endogenous FOXL2, we performed a proteome-wide analysis using co-immunoprecipitation in the murine granulosa cell-derived AT29c and the pituitary-derived alpha-T3 cell lines, using three antibodies targeting different parts of the protein. Following a stringent selection of mass spectrometry data on the basis of identification reliability and protein enrichment, we identified a core set of 255 partners common to both cell lines. Their analysis showed that we could co-precipitate several complexes involved in mRNA processing, chromatin remodeling and DNA replication and repair. We further validated (direct and/or indirect) interactions with selected partners, suggesting an unexpected role for FOXL2 in those processes. Overall, this comprehensive analysis of the endogenous FOXL2 interactome sheds light on its numerous and diverse interactors and unconventional cellular roles.

Learning From eHealth Implementations Through "Implementomics": A Multidimensional Annotation Model Applied to eHealth Projects of the RAFT Network.

The implementation of digital health technologies has increased globally, producing substantial amounts of information and knowledge. While there are still areas in digital health that are understudied, concurrently there is an exponential increase in published articles, guidelines, methods, projects, and experiences, many of which fail to reach critical mass (pilotitis). Semantically describing and documenting this implementation knowledge and the effectiveness of these tools will help to avoid the duplication of efforts, to reduce preventable implementation obstacles, and to assure that investments are targeted to the most important technological innovations. The RAFT annotation model, presented in this paper, enables to semantically describe all elements of various outputs and implementation projects that were developed, are used, or are part of the RAFT network. This model was initially developed to annotate various implementations and outputs of the RAFT network to facilitate knowledge documentation and sharing, and to be used as a proof of concept for the Implementome. The Implementome will be an interconnected knowledge system that enables the user to navigate on multiple dimensions through metadata annotated projects, people, and information, and can serve as base for consensus building, best practices and guidelines. The RAFT annotation model can be further developed to enable the annotation of outputs, implementations, people, initiatives, and projects of the digital health domain in general.

Systematic review to identify proxy indicators to quantify the impact of eHealth tools on maternal and neonatal health outcomes in low-income and middle-income countries including Delphi consensus.

OBJECTIVE: To identify interventions that could serve as reliable proxy indicators to measure eHealth impact on maternal and neonatal outcomes. DESIGN: Systematic review and Delphi study. METHODS: We searched PubMed, Embase and Cochrane from January 1990 to May 2016 for studies and reviews that evaluated interventions aimed at improving maternal/neonatal health and reducing mortality. Interventions that are not low-income and middle-income context appropriate and that cannot currently be diagnosed, managed or impacted by eHealth (eg, via telemedicine distance diagnostic or e-learning) were excluded. We used the Cochrane risk of bias, Risk Of Bias In Non- randomised Studies - of Interventions and ROBIS tool to assess the risk of bias. A three-step modified Delphi method was added to identify additional proxy indicators and prioritise the results, involving a panel of 13 experts from different regions, representing obstetricians and neonatologists. RESULTS: We included 44 studies and reviews, identifying 40 potential proxy indicators with a positive impact on maternal/neonatal outcomes. The Delphi experts completed and prioritised these, resulting in a list of 77 potential proxy indicators. CONCLUSIONS: The proxy indicators propose relevant outcome measures to evaluate if eHealth tools directly affect maternal/neonatal outcomes. Some proxy indicators require mapping to the local context, practices and available resources. The local mapping facilitates the utilisation of the proxy indicators in various contexts while allowing the systematic collection of data from different projects and programmes. Based on the mapping, the same proxy indicator can be used for different contexts, allowing it to measure what is locally and temporally relevant, making the proxy indicator sustainable. PROSPERO REGISTRATION NUMBER: CRD42015027351.

Reverse innovation experiences from the RAFT e-learning and telemedicine network.

Available infrastructure, resources, and provided services in low-and middle-income countries differ significantly from high-income countries. In healthcare for example, the uneven distribution of health professionals and lack of human resources are real barriers to equitable access to quality health care and services in most developing countries and particularly in Sub-Saharan Africa. As available resources are lower and infrastructure is les developed many services and tools that have been developed for a high-income context cannot be used or are not sustainably affordable in a low-income environment, which led to the development of tools and services that are affordable and appropriate for this context. This ranges from concepts of blended learning, over tools for distance education and diagnostic to hardware like affordable and robust ultrasound machines and services like mobile payment. Many of these solutions and tools also have a great potential to be utilized in a different context and some of them have been deployed in high-income countries.

The RAFT Telemedicine Network: Lessons Learnt and Perspectives from a Decade of Educational and Clinical Services in Low- and Middle-Incomes Countries.

BACKGROUND: The objectives of this paper are to (i) provide an overview of the educational and clinical experiences of the Réseau en Afrique Francophone pour la Télémédecine (RAFT) network, (ii) analyze key challenges and lessons learnt throughout a decade of activity, and (iii) draw a vision and perspectives of its sustainability. METHODS: The study was carried out following three main stages: (i) a literature review, (ii) the analysis of key documents, and (iii) discussions with key collaborators of the RAFT. RESULTS: Réseau en Afrique Francophone pour la Télémédecine has been offering an important quantity of educational, clinical, and public health activities during the last decade. The educational activities include the weekly delivery of video-lectures for continuing and post-graduate medical education, the use of virtual patients for training in clinical decision making, research training activities using ICTs and other e-learning activities. The clinical and public health activities include tele-expertise to support health professionals in the management of difficult clinical cases, the implementation of clinical information systems in African hospitals, the deployment of mHealth projects, etc. Since 2010, the RAFT has been extended to the Altiplano in Bolivia and Nepal (in progress). Lessons Learnt and Perspectives: Important lessons have been learnt from the accumulated experiences throughout these years. These lessons concern: social and organization, human resources, technologies and data security, policy and legislation, and economy and financing. Also, given the increase of the activities and the integration of eHealth and telemedicine in the health system of most of the countries, the RAFT network faces many other challenges and perspectives such as learning throughout life, recognition, and valorization of teaching or learning activities, the impact evaluation of interventions, and the scaling up and transferability out of Africa of RAFT activities. Based on the RAFT experience, effective integration and optimum use of eHealth and telemedicine in low- and middle-income countries (LMICs) health systems should take into account the context (resources, infrastructure, and funding), the needs of key stakeholders, and the results derived from theoretical and practical experience. The relevant items highlighted to illustrate the sustainability of the RAFT network and the analyses performed in this study, should serve as discussion basis for the development of eHealth and telemedicine in LMICs.

Appendix: Drug dosage chart.

For many medications, the pharmacokinetics and pharmacodynamics in pets have not been established and even where studies have been done, there is widespread species and individual variation. Practitioners should start with the lower end of the dose range and titrate up to maximum doses where there is insufficient therapeutic effect and no adverse effects or contraindications. Complete blood count, serum chemistry profile, and urinalysis should be performed before initiating the use of any medication, especially with off-label medications. Pharmacologic intervention for the treatment of behavior problems should be considered just one aspect of a comprehensive behavioral management and treatment protocol.

Enhancing research capacity of African institutions through social networking.

Traditionally, participation of African researchers in top Biomedical Informatics (BMI) scientific journals and conferences has been scarce. Looking beyond these numbers, an educational goal should be to improve overall research and, therefore, to increase the number of scientists/authors able to produce and publish high quality research. In such scenario, we are carrying out various efforts to expand the capacities of various institutions located at four African countries - Egypt, Ghana, Cameroon and Mali - in the framework of a European Commission-funded project, AFRICA BUILD. This project is currently carrying out activities such as e-learning, collaborative development of informatics tools, mobility of researchers, various pilot projects, and others. Our main objective is to create a self-sustained South-South network of BMI developers.

Continuing distance education: a capacity-building tool for the de-isolation of care professionals and researchers.

BACKGROUND: There are large disparities in access to health-care professionals (HCP) in low-income African countries, leading to imbalanced and suboptimal health delivery. Part of the difficulty is recruiting and retaining care professionals to work in isolated settings. AIM: To evaluate the impact of distance continuing education as a way to build capacity, increase satisfaction and enhance the performance of care professionals in these isolated health-care facilities. SETTING: Care professionals using RAFT (Telemedicine Network in French-speaking Africa) in isolated care facilities. PROGRAM DESCRIPTION: Within RAFT, an organizational framework and computer-based tools have been developed and evolved to provide useful, qualitative, applicable training material. PROGRAM EVALUATION: The activity, satisfaction, perceptions and impact of RAFT on remote health-care workers are being monitored. RAFT's potential to improve the recruitment, satisfaction and retention of care professionals in remote settings is widely recognized; however, the actual impact on the performance and quality of care remains to be demonstrated.

Precocious detection on amphibian oocyte lampbrush chromosomes of subtle changes in the cellular localisation of the Ro52 protein induced by in vitro culture.

Subterminal lampbrush loops of one of the 12 bivalents of the oocyte karyotype of Pleurodeles waltl (Amphibian, Urodele) underwent prominent morphological changes upon in vitro culture. These loops exhibited a fine ribonucleoprotein (RNP) granular matrix, which evolved during culture into huge structures that we have named 'chaussons' (slippers). This phenomenon involved progressive accumulation of proteins in the RNP matrix without protein neosynthesis. One of these proteins, which translocated into the nucleus during the culture, was identified as a homolog of the human Ro52 E3 ubiquitin ligase. RNA polymerase III was also found to accumulate on the same loops. These results suggest that the subterminal loops of bivalent XII act as a storage site for the components of a nuclear machinery involved in the quality control of RNA synthesis and maturation in response to cellular stress. They also emphasise the considerable value of the lampbrush chromosome system for a direct visualisation of modifications in gene expression and open the question of a nuclear accumulation of Ro52 in human or animal oocytes cultured in vitro for assisted reproductive technologies (ART).

Differential RNA-binding activity of the hnRNP G protein correlated with the sex genotype in the amphibian oocyte.

A proteomic approach has enabled the identification of an orthologue of the splicing factor hnRNP G in the amphibians Xenopus tropicalis, Ambystoma mexicanum, Notophthalmus viridescens and Pleurodeles walt, which shows a specific RNA-binding affinity similar to that of the human hnRN G protein. Three isoforms of this protein with a differential binding affinity for a specific RNA probe were identified in the P. walt oocyte. In situ hybridization to lampbrush chromosomes of P. waltl revealed the presence of a family of hnRNP G genes, which were mapped on the Z and W chromosomes and one autosome. This indicates that the isoforms identified in this study are possibly encoded by a gene family linked to the evolution of sex chromosomes similarly to the hnRNP G/RBMX gene family in mammals.

Tips and tricks for preparing lampbrush chromosome spreads from Xenopus tropicalis oocytes.

Due to their large size and fine organization, lampbrush chromosomes (LBCs) of amphibian oocytes have been for decades one of the favorite tools of biologists for the analysis of transcriptional and post-transcriptional processes at the cytological level. The emergence of the diploid Xenopus tropicalis amphibian as a model organism for vertebrate developmental genetics and the accumulation of sequence data made available by its recent genomic sequencing, strongly revive the interest of LBCs as a powerful tool to study genes expressed during oogenesis. We describe here a detailed protocol for preparing LBCs from X. tropicalis oocyte and give practical advice to encourage a large number of researchers to become familiar with these chromosomes.

Novel domains in the hnRNP G/RBMX protein with distinct roles in RNA binding and targeting nascent transcripts.

The heterogenous nuclear ribonucleoprotein G (hnRNP G) controls the alternative splicing of several pre-mRNas. While hnRNP G displays an amino terminal RNA recognition motif (RRM), we find that this motif is paradoxically not implicated in the recruitment of hnRNP G to nascent transcripts in amphibian oocytes. In fact, a deletion analysis revealed that targeting of hnRNP G to active transcription units depends on another domain, centrally positioned, and consisting of residues 186-236. We show that this domain acts autonomously and thus is named NTD for nascent transcripts targeting domain. Furthermore, using an RNA probe previously characterized in vitro as an RNA that interacts specifically with hnRNP G, we demonstrate a new auxiliary RNA binding domain (RBD). It corresponds to a short region of 58 residues positioned at the carboxyl terminal end of the protein, which recognizes an RNA motif predicted to adopt an hairpin structure. The fact that the NTD acts independently from both the RRM and the RBD strongly suggests that the initial recruitment of hnRNP G to nascent pre-mRNAs is independent of its sequence-specific RNA binding properties. Together, these findings highlight the modular organization of hnRNP G and offer new insights into its multifunctional roles.

Working map of the lampbrush chromosomes of Xenopus tropicalis: a new tool for cytogenetic analyses.

The amphibian Xenopus tropicalis, whose genome has been recently sequenced, has become an important model organism for vertebrate developmental genetics. The development of cytogenetic tools in this new model organism should contribute to an understanding of the organization of the amphibian genome and the mapping of a variety of loci of interest. In this respect, oocyte lampbrush chromosomes are particularly useful for the localization of genomic sequences expressed during oogenesis. We have constructed a working map of X. tropicalis lampbrush chromosomes, which allows the 10 bivalents of the oocyte karyotype to be readily identified by distinctive combinations of specific landmark structures composed of lateral loops, spheres, and granules. We have also established the patterns of RNA Pol III sites over the chromosomes by immunofluorescence using antibodies directed against two Pol III subunits. Specific staining patterns were found for each chromosome, which constitute a supplementary tool for their identification. Developmental Dynamics 238:1492-1501, 2009. (c) 2009 Wiley-Liss, Inc.

Increased superoxide anion production is associated with early atherosclerosis and cardiovascular dysfunctions in a rabbit model.

OBJECTIVE: Hypercholesterolemia (HC) has been associated with impairment of vascular and myocardial functions. As HC could generate an alteration in the oxidative status, we studied the effects of a 1-month cholesterol diet on cardiovascular oxidative stress. METHODS AND RESULTS: New Zealand rabbits received cholesterol (1%) or normal chow for 1 month. At 30 days, superoxide anion levels, assessed by ESR spectroscopy, NAD(P)H oxidase (NOX) activity, and dihydroethidium (DHE) staining of aortas were higher in the cholesterol-fed (CF) group compared with control (respectively, 4.0 +/- 0.6 Arbitrary Units/mg (AU/mg) vs. 2.6 +/- 0.3, p < 0.05; 4231 +/- 433 vs. 2931 +/- 373 AU/mg, p<0.05; 21.4 +/- 1.2 vs. 12.9 +/- 1.7% fluorescence/mm2, p < 0.001). NOX gp91 phox and p67 phox expression in the aortas were higher in the CF group vs. control (1.5 +/- 0.2 vs. 0.5 +/- 0.2, p < 0.001; 0.9 +/- 0.2 vs. 0.3 +/- 0.2, p<0.05). The endothelium-dependent relaxation evaluated on the iliac arteries was higher in control than in the CF group (64.8 +/- 10.1 vs. 13.1 +/- 3.70%, p<0.001). The cardiac diastolic pressure estimated on isolated hearts was higher in the CF group than in control (21.1 +/- 4.1 vs. 10.3 +/- 1.4 mmHg, p<0.05) after 60 min of ischemia. CONCLUSIONS: Hypercholesterolemia induced increased levels of superoxide in the aortas and a higher expression of NOX subunits, associated with altered vasorelaxation. The increased diastolic pressure observed in hearts, consistent with a post-ischemic contractile dysfunction might be mediated by the production of superoxide.

Metabolism of fatty acid in yeast: characterisation of beta-oxidation and ultrastructural changes in the genus Sporidiobolus sp. cultivated on ricinoleic acid methyl ester.

Cell structure modifications and beta-oxidation induction were monitored in two strains of Sporidiobolus, Sp. Ruinenii and Sp. pararoseus after cultivation on ricinoleic acid methyl ester. Ultrastructural observations of the yeast before and after cultivation on fatty acid esters did not reveal major modifications in Sp. ruinenii. Unexpectedly, in Sp. pararoseus a proliferation of the mitochondrion was observed. After induction, Sp. ruinenii principally exhibited an increase in the activities of acyl-CoA oxidase (ACO), hydroxyacyl-CoA deshydrogenase (HAD), thiolase and catalase. In contrast, Sp. pararoseus lacked ACO and catalase activities, but an increase in acyl-CoA deshydrogenase (ACDH) and enoyl-CoA hydratase (ECH) activity was observed. These data suggest that in Sp. ruinenii, beta-oxidation is preferentially localized in the microbody, whereas in Sp. pararoseus it might be localized in the mitochondria.

Direct demonstration of nitric oxide formation in organs of rabbits treated by transdermal glyceryl trinitrate using an in vivo spin trapping technique.

Glyceryl trinitrate (GTN) is commonly delivered by a patch for the treatment of angina pectoris. The idea is now generally accepted that GTN requires a biotransformation process that activates the drug, in particular through nitric oxide (NO) generation. However, the pharmacokinetics of NO delivery from GTN still remains obscure. The objective of this study was to assess GTN-derived NO formation in vascular tissues and organs in rabbit given GTN patches. NO levels were evaluated in rabbits after 3 h of treatment with a 10 mg GTN patch (GTN group; n = 7) or a placebo patch (CTL; n = 7). Nitrosylhaemoglobin (HbNO) was evaluated by electron spin resonance (ESR) spectroscopy in red cell suspension. In vivo spin trapping technique using FeMGD as a spin trap, associated with ESR was used to quantify NO in tissues. The NO-spin trap complex, which is a relatively stable product, has been measured in several tissues. The ESR spectrum corresponding to HbNO was not found in red cell of GTN or CTL rabbits. The spectrum corresponding to the NO-spin trap complex was observed in all analysed tissues of CTL rabbits. The signal was significantly increased in liver, renal medulla, heart left ventricle and spleen of GTN-treated rabbits, and to a lesser extent in right ventricle and lung. No difference was shown between NO-spin trap levels measured in aorta or inferior vena cava from GTN or CTL rabbits. These data suggest that GTN patch treatment induced NO release, and that tissue-specific differences in transdermal GTN-derived NO exist. The GTN-NO pathway appears to be largely involved in organs such as the liver, kidney and heart.

In vitro antioxidant properties of calpain inhibitors: leupeptin and calpain inhibitor-1.

The aim of this study was 1/ to assess the efficacy of calpain inhibitors: leupeptin and calpain inhibitor-1, to inhibit calpain activity in vitro, 2/ to measure the scavenging abilities of these compounds against free radicals. The efficacy of calpain inhibitors to block calpain activity was tested with azocasein as a substrate for calpain. Leupeptin and calpain inhibitor-1 inhibited calpain activity in the same range of concentrations, the IC50 being 0.14 microM and 0.09 microM, respectively. We measured the antioxidant properties of leupeptin and calpain inhibitor-1 using the allophycocyans assay after identification offree radical species produced by the complex H2O2 + Cu(++). Electron paramagnetic resonance (EPR) spin-trapping studies performed by using DMPO showed that a quartet signal (hyperfine couplings aN = aH = 14.9 G) arisen from DMPO-OH was formed. We found a correlation between leupeptin concentration and oxygen radical absorbance capacity (ORAC) values (r2 = 0.975) indicating an in vivo antioxidant capacity. In contrast, calpain inhibitor-1 showed no protection. In conclusion, our findings indicate that leupeptin and calpain inhibitor-1 are equipotent inhibitors on calpain activity but exhibit diffrent antioxidant efficacy.