A Unique Automation Platform for Measuring Low Level Radioactivity in Metabolite Identification Studies

Generation and interpretation of biotransformation data on drugs, i.e. identification of physiologically relevant metabolites, defining metabolic pathways and elucidation of metabolite structures, have become increasingly important to the drug development process. Profiling using 14C or 3H radiolabel is defined as the chromatographic separation and quantification of drug-related material in a given biological sample derived from an in vitro, preclinical in vivo or clinical study. Metabolite profiling is a very time intensive activity, particularly for preclinical in vivo or clinical studies which have defined limitations on radiation burden and exposure levels. A clear gap exists for certain studies which do not require specialized high volume automation technologies, yet these studies would still clearly benefit from automation. Use of radiolabeled compounds in preclinical and clinical ADME studies, specifically for metabolite profiling and identification are a very good example. The current lack of automation for measuring low level radioactivity in metabolite profiling requires substantial capacity, personal attention and resources from laboratory scientists. To help address these challenges and improve efficiency, we have innovated, developed and implemented a novel and flexible automation platform that integrates a robotic plate handling platform, HPLC or UPLC system, mass spectrometer and an automated fraction collector

Incomplete posttranslational prohormone modifications in hyperactive neuroendocrine cells

Contains fulltext : 76028.pdf (publisher's version ) (Open Access)8 p

A novel method for standardized application of fungal spore coatings for mosquito exposure bioassays

<p>Abstract</p> <p>Background</p> <p>Interest in the use of fungal entomopathogens against malaria vectors is growing. Fungal spores infect insects via the cuticle and can be applied directly on the insect to evaluate infectivity. For flying insects such as mosquitoes, however, application of fungal suspensions on resting surfaces is more realistic and representative of field settings. For this type of exposure, it is essential to apply specific amounts of fungal spores homogeneously over a surface for testing the effects of fungal dose and exposure time. Contemporary methods such as spraying or brushing spore suspensions onto substrates do not produce the uniformity and consistency that standardized laboratory assays require. Two novel fungus application methods using equipment developed in the paint industry are presented and compared.</p> <p>Methods</p> <p>Wired, stainless steel K-bars were tested and optimized for coating fungal spore suspensions onto paper substrates. Different solvents and substrates were evaluated. Two types of coating techniques were compared, i.e. manual and automated coating. A standardized bioassay set-up was designed for testing coated spores against malaria mosquitoes.</p> <p>Results</p> <p>K-bar coating provided consistent applications of spore layers onto paper substrates. Viscous Ondina oil formulations were not suitable and significantly reduced spore infectivity. Evaporative Shellsol T solvent dried quickly and resulted in high spore infectivity to mosquitoes. Smooth proofing papers were the most effective substrate and showed higher infectivity than cardboard substrates. Manually and mechanically applied spore coatings showed similar and reproducible effects on mosquito survival. The standardized mosquito exposure bioassay was effective and consistent in measuring effects of fungal dose and exposure time.</p> <p>Conclusions</p> <p>K-bar coating is a simple and consistent method for applying fungal spore suspensions onto paper substrates and can produce coating layers with accurate effective spore concentrations. The mosquito bioassay was suitable for evaluating fungal infectivity and virulence, allowing optimizations of spore dose and exposure time. Use of this standardized application method will help achieve reliable results that are exchangeable between different laboratories.</p

Global Protection and the Health Impact of Migration Interception

In the fourth article in a six-part PLoS Medicine series on Migration & Health, Zachary Steel and colleagues discuss the interception phase of migration and the specific health risks and policy needs associated with this phase

EROS is a selective chaperone regulating the phagocyte NADPH oxidase and purinergic signalling

EROS (essential for reactive oxygen species) protein is indispensable for expression of gp91phox, the catalytic core of the phagocyte NADPH oxidase. EROS deficiency in humans is a novel cause of the severe immunodeficiency, chronic granulomatous disease, but its mechanism of action was unknown until now. We elucidate the role of EROS, showing it acts at the earliest stages of gp91phox maturation. It binds the immature 58 kDa gp91phox directly, preventing gp91phox degradation and allowing glycosylation via the oligosaccharyltransferase machinery and the incorporation of the heme prosthetic groups essential for catalysis. EROS also regulates the purine receptors P2X7 and P2X1 through direct interactions, and P2X7 is almost absent in EROS-deficient mouse and human primary cells. Accordingly, lack of murine EROS results in markedly abnormal P2X7 signalling, inflammasome activation, and T cell responses. The loss of both ROS and P2X7 signalling leads to resistance to influenza infection in mice. Our work identifies EROS as a highly selective chaperone for key proteins in innate and adaptive immunity and a rheostat for immunity to infection. It has profound implications for our understanding of immune physiology, ROS dysregulation, and possibly gene therapy.</jats:p

An Introduction to RNA Databases

We present an introduction to RNA databases. The history and technology behind RNA databases is briefly discussed. We examine differing methods of data collection and curation, and discuss their impact on both the scope and accuracy of the resulting databases. Finally, we demonstrate these principals through detailed examination of four leading RNA databases: Noncode, miRBase, Rfam, and SILVA.Comment: 27 pages, 10 figures, 1 tables. Submitted as a chapter for "An introduction to RNA bioinformatics" to be published by "Methods in Molecular Biology

Analysis of the Human Kinome Using Methods Including Fold Recognition Reveals Two Novel Kinases

Background: Protein sequence similarity is a commonly used criterion for inferring the unknown function of a protein from a protein of known function. However, proteins can diverge significantly over time such that sequence similarity is difficult, if not impossible, to find. In some cases, a structural similarity remains over long evolutionary time scales and once detected can be used to predict function. Methodology/Principal Findings: Here we employed a high-throughput approach to assign structural and functional annotation to the human proteome, focusing on the collection of human protein kinases, the human kinome. We compared human protein sequences to a library of domains from known structures using WU-BLAST, PSI-BLAST, and 123D. This approach utilized both sequence comparison and fold recognition methods. The resulting set of potential protein kinases was cross-checked against previously identified human protein kinases, and analyzed for conserved kinase motifs. Conclusions/Significance: We demonstrate that our structure-based method can be used to identify both typical and atypical human protein kinases. We also identify two potentially novel kinases that contain an interesting combination o