Automated High-Throughput RNAi Screening in Human Cells Combined with Reporter mRNA Transfection to Identify Novel Regulators of Translation

Proteins that promote angiogenesis, such as vascular endothelial growth factor (VEGF), are major targets for cancer therapy. Accordingly, proteins that specifically activate expression of factors like VEGF are potential alternative therapeutic targets and may help to combat evasive resistance to angiogenesis inhibitors. VEGF mRNA contains two internal ribosome entry sites (IRESs) that enable selective activation of VEGF protein synthesis under hypoxic conditions that trigger angiogenesis. To identify novel regulators of VEGF IRES-driven translation in human cells, we have developed a high-throughput screening approach that combines siRNA treatment with transfection of a VEGF-IRES reporter mRNA. We identified the kinase MAPK3 as a novel positive regulator of VEGF IRES-driven translation and have validated its regulatory effect on endogenous VEGF. Our automated method is scalable and readily adapted for use with other mRNA regulatory elements. Consequently, it should be a generally useful approach for high-throughput identification of novel regulators of mRNA translation.     

Out but Not In: The Large Transmembrane β-Barrel Protein FhuA Unfolds but Cannot Refold via β-Hairpins

1. Download : Download high-res image (119KB)
2. Download : Download full-size image

Highlights? Mechanical un- and refolding studies of single large transmembrane β-barrel proteins ? FhuA unfolds via highly reproducible steps formed by single β-hairpins ? Once unfolded, FhuA folds into highly irreproducible structures ? To assist folding into the membrane, FhuA may require cofactors     

Quantitative image analysis identifies pVHL as a key regulator of microtubule dynamic instability

Von Hippel-Lindau (VHL) tumor suppressor gene mutations predispose carriers to kidney cancer. The protein pVHL has been shown to interact with microtubules (MTs), which is critical to cilia maintenance and mitotic spindle orientation. However, the function for pVHL in the regulation of MT dynamics is unknown. We tracked MT growth via the plus end marker EB3 (end-binding protein 3)-GFP and inferred additional parameters of MT dynamics indirectly by spatiotemporal grouping of growth tracks from live cell imaging. Our data establish pVHL as a near-optimal MT-stabilizing protein: it attenuates tubulin turnover, both during MT growth and shrinkage, inhibits catastrophe, and enhances rescue frequencies. These functions are mediated, in part, by inhibition of tubulin guanosine triphosphatase activity in vitro and at MT plus ends and along the MT lattice in vivo. Mutants connected to the VHL cancer syndrome are differentially compromised in these activities. Thus, single cell–level analysis of pVHL MT regulatory function allows new predictions for genotype to phenotype associations that deviate from the coarser clinically defined mutant classifications.     

Structural Properties of the Human Protease-Activated Receptor 1 Changing by a Strong Antagonist

1. Download high-res image (226KB)
2. Download full-size image

     

Environmental Sustainability of Fluid Milk Delivery Systems in the United States

Beverage producers in the United States choose packaging based on cost and consumer preference. Monolayer high‐density polyethylene (HDPE) and gable‐top carton containers have long dominated the U.S. fluid milk market, but pressure for more sustainable packaging is increasing. We present a broad discussion on environmental sustainability of 18 fluid milk containers through life cycle assessment. Because different container types require unique milk processing, distribution, and disposal and incur or avoid milk losses, fluid milk delivery systems (FMDSs) are evaluated, rather than containers in isolation. By assessing FMDSs, a complete measure of containers’ environmental sustainability was obtained. Despite conservative assumptions about milk losses, differences in container size, milk processing, distribution, and container recycling, pair‐wise cradle‐to‐grave comparisons of FMDSs show there are no superior FMDSs. But, 500‐ to 1,000‐milliliter FMDSs are potentially superior to ≥half gallon if they prevent milk losses. Thus, the future of FMDSs in the United States depends on the industry's ability to prevent distribution (12%) and consumption milk losses (20% to 35%). Farm‐gate‐to‐grave comparisons showed that chilled HDPE FMDSs are superior to other plastic and chilled paperboard FMDSs for climate‐change impact, but the result is inconclusive for chilled HDPE to ambient (unrefrigerated) paperboard or plastic pouch FMDS comparisons. Plastic pouch FMDSs show potential to reduce nonrenewable fossil energy, but need to be recyclable. Ambient FMDSs are superior to chilled FMDSs for water depletion. Eight‐ounce paperboard FMDSs are superior to 8‐ounce plastic FMDSs. Thus, alternative FMDSs may improve environmental sustainability of the U.S. postfarm fluid milk supply chain.     

Evolutionary relationships among American mud crabs (Crustacea: Decapoda: Brachyura: Xanthoidea) inferred from nuclear and mitochondrial markers,with comments on adult morphology

Members of the brachyuran crab superfamily Xanthoidea sensu Ng, Guinot & Davie (2008) are a morphologically and ecologically diverse assemblage encompassing more than 780 nominal species. On the basis of morphology, Xanthoidea is presently regarded to represent three families: Xanthidae, Pseudorhombilidae, and Panopeidae. However, few studies have examined this superfamily using modern phylogenetic methods, despite the ecological and economic importance of this large, poorly understood group. In this study we examine phylogenetic relationships within the superfamily Xanthoidea using three mitochondrial markers, 12S rRNA, 16S rRNA, and cytochrome oxidase I (COI), and three nuclear markers, 18S rRNA, enolase (ENO) and histone H3 (H3). Bayesian and maximum‐likelihood analyses indicate that the superfamily Xanthoidea is monophyletic; however, the families Xanthidae, Panopeidae, and Pseudorhombilidae, as defined by Ng et al., are not, and their representative memberships must be redefined. To this end, some relevant morphological characters are discussed. © 2013 The Linnean Society of London     

Über die lichte Weite der Placentararterien

Ohne Zusammenfassung