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DNA-methylation is an important epigenetic feature in health and disease. Methylated sequence capturing by Methyl Binding Domain (MBD) based enrichment followed by second-generation sequencing provides the best combination of sensitivity and cost-efficiency for genome-wide DNA-methylation profiling. However, existing implementations are numerous, and quality control and optimization require expensive external validation. Therefore, this study has two aims: 1) to identify a best performing kit for MBD-based enrichment using independent validation data, and 2) to evaluate whether quality evaluation can also be performed solely based on the characteristics of the generated sequences. Five commercially available kits for MBD enrichment were combined with Illumina GAIIx sequencing for three cell lines (HCT15, DU145, PC3). Reduced representation bisulfite sequencing data (all three cell lines) and publicly available Illumina Infinium BeadChip data (DU145 and PC3) were used for benchmarking. Consistent large-scale differences in yield, sensitivity and specificity between the different kits could be identified, with Diagenode's MethylCap kit as overall best performing kit under the tested conditions. This kit could also be identified with the Fragment CpG-plot, which summarizes the CpG content of the captured fragments, implying that the latter can be used as a tool to monitor data quality. In conclusion, there are major quality differences between kits for MBD-based capturing of methylated DNA, with the MethylCap kit performing best under the used settings. The Fragment CpG-plot is able to monitor data quality based on inherent sequence data characteristics, and is therefore a cost-efficient tool for experimental optimization, but also to monitor quality throughout routine applications.

Background Hereditary hearing loss (HL) can originate from mutations in one of many genes involved in the complex process of hearing. Identification of the genetic defects in patients is currently labor intensive and expensive. While screening with Sanger sequencing for GJB2 mutations is common, this is not the case for the other known deafness genes (> 60). Next generation sequencing technology (NGS) has the potential to be much more cost efficient. Published methods mainly use hybridization based target enrichment procedures that are time saving and efficient, but lead to loss in sensitivity. In this study we used a semi-automated PCR amplification and NGS in order to combine high sensitivity, speed and cost efficiency. Results In this proof of concept study, we screened 15 autosomal recessive deafness genes in 5 patients with congenital genetic deafness. 646 specific primer pairs for all exons and most of the UTR of the 15 selected genes were designed using primerXL. Using patient specific identifiers, all amplicons were pooled and analyzed using the Roche 454 NGS technology. Three of these patients are members of families in which a region of interest has previously been characterized by linkage studies. In these, we were able to identify two new mutations in CDH23 and OTOF . For another patient, the etiology of deafness was unclear, and no causal mutation was found. In a fifth patient, included as a positive control, we could confirm a known mutation in TMC1. Conclusions We have developed an assay that holds great promise as a tool for screening patients with familial autosomal recessive nonsyndromal hearing loss (ARNSHL). For the first time, an efficient, reliable and cost effective genetic test, based on PCR enrichment, for newborns with undiagnosed deafness is available.

Un vade-mecum à l'usage des gérants de SPRL, pour répondre à toutes leurs questions. Depuis 1935, date de son entrée dans notre Code des sociétés, la SPRL a toujours été, et de loin, la forme de société la plus utilisée en Belgique. En 2009, alors que notre pays était frappé par une crise économique et financière exceptionnelle, plus de 20 000 SPRL furent constituées, ce qui représente plus des 2/3 des sociétés nouvelles en Belgique. C''est la preuve éclatante que la SPRL est le véhicule idéal pour la PME qui forme notre tissu économique. Entre le commerce exercé à titre personnel avec ses risques et la société anonyme et ses lourdeurs, la SPRL est l''habit parfait pour l''entreprise familiale ou unipersonnelle. Ce livre est conçu comme le « livre de poche » de ces « hommes orchestres » que sont les gérants de SPRL, qui sont, plus encore que les administrateurs de sociétés anonymes, dans l''impossibilité de suivre l''évolution législative galopante et donc les obligations légales qui pèsent sur eux. L''éditeur de ce vade-mecum a voulu qu''il soit jalonné de questions que se posent le plus souvent les gérants. Une liste de ces questions se trouve d'ailleurs en début d''ouvrage pour aider le lecteur à trouver plus facilement leur réponse. Un guide pratique pour comprendre le droit des sociétés qui entoure les SPRL belges. À PROPOS DE L'AUTEUR Jean-Pierre Renard est avocat aux Barreaux de Bruxelles et de Nivelles, où il est également juge suppléant au tribunal de commerce. Il est spécialisé en droit commercial (notamment en droit des sociétés et en restructurations d'entreprises) et en droit pénal financier.Il intervient comme conférencier sur ces sujets et est auteur de nombreux livres et articles. Il a notamment écrit chez le même éditeur le Guide pratique du conseil d'administration et de l'assemblée général e.

Recent evidence shows that high glucose levels recruit carbohydrate response element-binding protein, which binds the promoter of thioredoxin-interacting protein （txnip）, thereby regulating its expression in β-cells. Overexpression of txnip not only induces β-cell apoptosis but also reduces insulin production. Thus, the discovery of compounds that either inhibit TXNIP activity or suppress its expression was the focus of the present study. INS-IE cells stably transfected with either a txnip proximal glucose response element connected to a luciferase reporter plasmid （BG73） or full-length txnip promoter connected to a luciferase reporter plasmid （CL108） were used in primary and secondary high-throughput screening campaigns, respectively. From 256 000 synthetic compounds, a small molecule compound, W2476 [9-（（1-（4-acetyl-phenyloxy）-ethyl）-2-）adenine], was identified as a modulator of the TXNIP-regulated signaling pathway following the screening and characterized using a battery of bioassays. The preventive and therapeutic properties of W2476 were further examined in streptozotocin-induced diabetic and diet-induced obese mice. Treatment with W2476 （1, 5, and 15 pmol/L） dose-dependently inhibited high glucose-induced TXNIP expression at the mRNA and protein levels in INS-1E cells and rat pancreatic islets. Furthermore, W2476 treatment prevented INS-IE cells from apoptosis induced by chronic exposure of high glucose and enhanced insulin production in vitro. Oral administration of W2476 （200 mg-kg-1.d-1） rescued streptozotocin-induced diabetic mice by promoting β-cell survival and enhancing insulin secretion. This therapeutic property of W2476 was further demonstrated by its ability to improve glucose homeostasis and insulin sensitivity in diet-induced obese mice. Thus, chemical intervention of the TXNIP- regulated signaling pathway might present a viable approach to manage diabetes.

DNA-methylation is an important epigenetic feature in health and disease. Methylated sequence capturing by Methyl Binding Domain (MBD) based enrichment followed by second-generation sequencing provides the best combination of sensitivity and cost-efficiency for genome-wide DNA-methylation profiling. However, existing implementations are numerous, and quality control and optimization require expensive external validation. Therefore, this study has two aims: 1) to identify a best performing kit for MBD-based enrichment using independent validation data, and 2) to evaluate whether quality evaluation can also be performed solely based on the characteristics of the generated sequences. Five commercially available kits for MBD enrichment were combined with Illumina GAIIx sequencing for three cell lines (HCT15, DU145, PC3). Reduced representation bisulfite sequencing data (all three cell lines) and publicly available Illumina Infinium BeadChip data (DU145 and PC3) were used for benchmarking. Consistent large-scale differences in yield, sensitivity and specificity between the different kits could be identified, with Diagenode's MethylCap kit as overall best performing kit under the tested conditions. This kit could also be identified with the Fragment CpG-plot, which summarizes the CpG content of the captured fragments, implying that the latter can be used as a tool to monitor data quality. In conclusion, there are major quality differences between kits for MBD-based capturing of methylated DNA, with the MethylCap kit performing best under the used settings. The Fragment CpG-plot is able to monitor data quality based on inherent sequence data characteristics, and is therefore a cost-efficient tool for experimental optimization, but also to monitor quality throughout routine applications.

Aim： To find new antagonists on human melanin-concentrating hormone receptor-1 （MCHR-1） through high-throughput screening （HTS） of a diverse compound library. Methods： MCHR-1, [^3H]SNAP7941, and FlashBlue G-proteincoupled receptor beads were used to measure the receptor-binding activities of various compounds based on scintillation proximity assay （SPA） technology. The guanosine 5＇ （γ-[^35S]thio） triphosphate （[^35S]GTPγS） binding assay was subsequently applied to functionally characterize the ＂hits＂ identified by the HTS campaign. Results： Of the 48 240 compounds screened with the SPA method, 12 hits were confirmed to possess MCHR-1 binding activities, 8 were functionally studied subsequently with the [^35S]GTPγS binding assay, and only 1 compound （NC 127816） displayed moderate human MCHR- 1 binding affinity （Ki=115.7 nmol/L） and relatively potent antagonism （KB=23.8 nmol/L）. This compound shares a novel scaffold （1-ethoxy-2H-2-aza-1-phospha-naphthalene 1-oxide） with 3 other analogs in the group. Conclusion： Considering the marked difference in molecular shape and electrostatic status between NC 127816 and the structures reported elsewhere, we anticipate that its derivatives may represent a new class of potent MCHR-1 modulators.