Pseudomonas aeruginosa ExoT Induces Atypical Anoikis Apoptosis in Target Host Cells by Transforming Crk Adaptor Protein into a Cytotoxin

Previously, we demonstrated that Pseudomonas aeruginosa ExoT induces potent apoptosis in host epithelial cells in a manner that primarily depends on its ADP-ribosyltransferase domain (ADPRT) activity. However, the mechanism underlying ExoT/ADPRT-induced apoptosis remains undetermined. We now report that ExoT/ADPRT disrupts focal adhesion sites, activates p38β and JNK, and interferes with integrin-mediated survival signaling; causing atypical anoikis. We show that ExoT/ADPRT-induced anoikis is mediated by the Crk adaptor protein. We found that Crk^-/- knockout cells are significantly more resistant to ExoT-induced apoptosis, while Crk^-/- cells complemented with Crk are rendered sensitive to ExoT-induced apoptosis. Moreover, a dominant negative (DN) mutant form of Crk phenocopies ExoT-induced apoptosis both kinetically and mechanistically. Crk is generally believed to be a component of focal adhesion (FA) and its role in cellular survival remains controversial in that it has been found to be either pro-survival or pro-apoptosis. Our data demonstrate that although Crk is recruited to FA sites, its function is likely not required for FA assembly or for survival per se. However, when modified by ExoT or by mutagenesis, it can be transformed into a cytotoxin that induces anoikis by disrupting FA sites and interfering with integrin survival signaling. To our knowledge, this is the first example whereby a bacterial toxin exerts its cytotoxicity by subverting the function of an innocuous host cellular protein and turning it against the host cell.     

The anti-genomic (negative) strand of Hepatitis C Virus is not targetable by shRNA

Hepatitis C Virus (HCV) and other plus-strand RNA viruses typically require the generation of a small number of negative genomes (20–100× lower than the positive genomes) for replication, making the less-abundant antigenome an attractive target for RNA interference(RNAi)-based therapy. Because of the complementarity of duplex short hairpin RNA/small interfering RNA (shRNA/siRNAs) with both genomic and anti-genomic viral RNA strands, and the potential of both shRNA strands to become part of the targeting complexes, preclinical RNAi studies cannot distinguish which viral strand is actually targeted in infected cells. Here, we addressed the question whether the negative HCV genome was bioaccessible to RNAi. We first screened for the most active shRNA molecules against the most conserved regions in the HCV genome, which were then used to generate asymmetric anti-HCV shRNAs that produce biologically active RNAi specifically directed against the genomic or antigenomic HCV sequences. Using this simple but powerful and effective method to screen for shRNA strand selectivity, we demonstrate that the antigenomic strand of HCV is not a viable RNAi target during HCV replication. These findings provide new insights into HCV biology and have important implications for the design of more effective and safer antiviral RNAi strategies seeking to target HCV and other viruses with similar replicative strategies.     

Overview of thermostable DNA polymerases for classical PCR applications: from molecular and biochemical fundamentals to commercial systems

During the genomics era, the use of thermostable DNA polymerases increased greatly. Many were identified and described—mainly of the genera Thermus, Thermococcus and Pyrococcus. Each polymerase has different features, resulting from origin and genetic modification. However, the rational choice of the adequate polymerase depends on the application itself. This review gives an overview of the most commonly used DNA polymerases used for PCR application: KOD, Pab (Isis?), Pfu, Pst (Deep Vent?), Pwo, Taq, Tbr, Tca, Tfi, Tfl, Tfu, Tgo, Tli (Vent?), Tma (UITma?), Tne, Tth and others.     

Mayday - integrative analytics for expression data

Background  

DNA Microarrays have become the standard method for large scale analyses of gene expression and epigenomics. The increasing complexity and inherent noisiness of the generated data makes visual data exploration ever more important. Fast deployment of new methods as well as a combination of predefined, easy to apply methods with programmer's access to the data are important requirements for any analysis framework. Mayday is an open source platform with emphasis on visual data exploration and analysis. Many built-in methods for clustering, machine learning and classification are provided for dissecting complex datasets. Plugins can easily be written to extend Mayday's functionality in a large number of ways. As Java program, Mayday is platform-independent and can be used as Java WebStart application without any installation. Mayday can import data from several file formats, database connectivity is included for efficient data organization. Numerous interactive visualization tools, including box plots, profile plots, principal component plots and a heatmap are available, can be enhanced with metadata and exported as publication quality vector files.