http://hdl.handle.net/2440/300 Wed, 19 Jun 2013 21:02:39 GMT 2013-06-19T21:02:39Z http://hdl.handle.net/2440/78371 Title: Mutations of the ca²⁺-sensing stromal interaction molecule stim1 regulate ca²⁺ influx by altered oligomerization of stim1 and by destabilization of the ca²⁺ channel orai1 Author: Kilch, Tatiana; Alansary, Dalia; Peglow, Martin; Dorr, Kathrin; Rychkov, Grigori; Rieger, Heiko; Peinelt, Christine; Niemeyer, Barbara A. Abstract: A drop of endoplasmic reticulum Ca²⁺ concentration triggers its Ca²⁺ ssensor protein stromal interaction molecule 1 (STIM1) to oligomerize and accumulate within endoplasmic reticulum-plasma membrane junctions where it activates Orai1 channels, providing store-operated Ca²⁺ entry. To elucidate the functional significance of N-glycosylation sites of STIM1, we created different mutations of asparagine-131 and asparagine-171. STIM1 NN/DQ resulted in a strong gain of function. Patch clamp, Total Internal Reflection Fluorescent (TIRF) microscopy, and fluorescence recovery after photobleaching (FRAP) analyses revealed that expression of STIM1 DQ mutants increases the number of active Orai1 channels and the rate of STIM1 translocation to endoplasmic reticulum-plasma membrane junctions with a decrease in current latency. Surprisingly, co-expression of STIM1 DQ decreased Orai1 protein, altering the STIM1:Orai1 stoichiometry. We describe a novel mathematical tool to delineate the effects of altered STIM1 or Orai1 diffusion parameters from stoichiometrical changes. The mutant uncovers a novel mechanism whereby “superactive” STIM1 DQ leads to altered oligomerization rate constants and to degradation of Orai1 with a change in stoichiometry of activator (STIM1) to effector (Orai1) ratio leading to altered Ca²⁺ homeostasis. Mon, 31 Dec 2012 13:30:00 GMT http://hdl.handle.net/2440/78371 2012-12-31T13:30:00Z http://hdl.handle.net/2440/78351 Title: Site of isolation determines biofilm formation and virulence phenotypes of streptococcus pneumoniae serotype 3 clinical isolates Author: Trappetti, Claudia; van der Maten, Erika; Amin, Zarina; Potter, Adam Joseph; Chen, Austen Yannis; van Mourik, Paula M.; Lawrence, Andrew J.; Paton, Adrienne Webster; Paton, James Cleland Abstract: Streptococcus pneumoniae is a diverse species causing invasive as well as localized infections that result in massive global morbidity and mortality. Strains vary markedly in pathogenic potential, but the molecular basis is obscured by the diversity and plasticity of the pneumococcal genome. In the present study, S. pneumoniae serotype 3 blood (n = 12) or ear (n = 13) isolates were multilocus sequence typed (MLST) and assessed for biofilm formation and virulence phenotype. Blood and ear isolates exhibited similar MLST distributions but differed markedly in phenotype. Blood isolates formed robust biofilms only at pH 7.4, which were enhanced in Fe(III)-supplemented medium. Conversely, ear isolates formed biofilms only at pH 6.8, and Fe(III) was inhibitory. Biofilm formation paralleled luxS expression and genetic competence. In a mouse intranasal challenge model, blood isolates did not stably colonize the nasopharynx but spread to the blood; none spread to the ear. Ear isolates colonized the nasopharynx at higher levels and also spread to the ear compartment in a significant proportion of animals; none caused bacteremia. Thus, pneumococci of the same serotype and MLST exhibit distinct phenotypes in accordance with clinical site of isolation, indicative of stable niche adaptation within a clonal lineage. Mon, 31 Dec 2012 13:30:00 GMT http://hdl.handle.net/2440/78351 2012-12-31T13:30:00Z http://hdl.handle.net/2440/78326 Title: Evolutionary conservation and functional roles of ncRNA Author: Qu, Zhipeng; Adelson, David Louis Abstract: Non-coding RNAs(ncRNAs)are a class of transcribed RNA molecules without protein-coding potential. They were regarded as transcriptional noise,or the by product of genetic information flow from DNA to protein for a long time. However, in recent years, a number of studies have shown that ncRNAs are pervasively transcribed, and most of them show evidence of evolutionary conservation, although less conserved than protein-coding genes. More importantly, many ncRNAs have been confirmed as playing crucial regulatory roles in diverse biological processes and tumorigenesis. Here we summarize the functional significance of this class of “darkmatter” in terms its genomic organization,evolutionary conservation, and broad functional classes. Description: Extent: 11 p. Sat, 31 Dec 2011 13:30:00 GMT http://hdl.handle.net/2440/78326 2011-12-31T13:30:00Z http://hdl.handle.net/2440/78255 Title: Antiviral resistance and direct-acting antiviral agents for HCV Author: Aloia, Amanda Louise; Locarnini, Stephen; Beard, Michael Robert Abstract: Direct-acting antiviral (DAA) agents specifically target viral proteins. Two DAAs have been already been approved for the treatment of HCV infection and many more are in development. DAA treatment of HCV infection, however, leads to the selection of viral variants (produced by the error-prone HCV polymerase) that are resistant to the DAA agent in use. The selection of DAA-resistant HCV variants has been studied extensively in vitro and in vivo. Common amino acid substitution sites in each of the non-structural proteins are associated with DAA-resistance: D168, A155, A156 and V36 in NS3 protease; L31 and Y93 in NS5A; S282, S96, P495, M423, M414 and C316 in NS5B. In this review we cover the basic principles of DAA resistance, summarise the available resistance data for the various classes of DAAs and discuss the potential of DAA combination therapy for overcoming DAA-resistance, resulting in major advances in the treatment of HCV. Sat, 31 Dec 2011 13:30:00 GMT http://hdl.handle.net/2440/78255 2011-12-31T13:30:00Z