Molecular characterization of the PhoPQ-PmrD-PmrAB mediated pathway regulating polymyxin B resistance in Klebsiella pneumoniae CG43

<p>Abstract</p> <p>Background</p> <p>The cationic peptide antibiotic polymyxin has recently been reevaluated in the treatment of severe infections caused by gram negative bacteria.</p> <p>Methods</p> <p>In this study, the genetic determinants for capsular polysaccharide level and lipopolysaccharide modification involved in polymyxin B resistance of the opportunistic pathogen <it>Klebsiella pneumoniae </it>were characterized. The expressional control of the genes responsible for the resistance was assessed by a LacZ reporter system. The PmrD connector-mediated regulation for the expression of <it>pmr </it>genes involved in polymyxin B resistance was also demonstrated by DNA EMSA, two-hybrid analysis and <it>in vitro </it>phosphor-transfer assay.</p> <p>Results</p> <p>Deletion of the <it>rcsB</it>, which encoded an activator for the production of capsular polysaccharide, had a minor effect on <it>K. pneumoniae </it>resistance to polymyxin B. On the other hand, deletion of <it>ugd </it>or <it>pmrF </it>gene resulted in a drastic reduction of the resistance. The polymyxin B resistance was shown to be regulated by the two-component response regulators PhoP and PmrA at low magnesium and high iron, respectively. Similar to the control identified in <it>Salmonella</it>, expression of <it>pmrD </it>in <it>K. pneumoniae </it>was dependent on PhoP, the activated PmrD would then bind to PmrA to prolong the phosphorylation state of the PmrA, and eventually turn on the expression of <it>pmr </it>for the resistance to polymyxin B.</p> <p>Conclusions</p> <p>The study reports a role of the capsular polysaccharide level and the <it>pmr </it>genes for <it>K. pneumoniae </it>resistance to polymyxin B. The PmrD connector-mediated pathway in governing the regulation of <it>pmr </it>expression was demonstrated. In comparison to the <it>pmr </it>regulation in <it>Salmonella</it>, PhoP in <it>K. pneumoniae </it>plays a major regulatory role in polymyxin B resistance.</p

Improving Antigenicity of the Recombinant Hepatitis C Virus Core Protein via Random Mutagenesis

In order to enhance the sensitivity of diagnosis, a recombinant clone containing domain I of HCV core (amino acid residues 1 to 123) was subjected to random mutagenesis. Five mutants with higher sensitivity were obtained by colony screening of 616 mutants using reverse ELISA. Sequence analysis of these mutants revealed alterations focusing on W84, P95, P110, or V129. The inclusion bodies of these recombinant proteins overexpressed in E. coli BL21(DE3) were subsequently dissolved using 6 M urea and then refolded by stepwise dialysis. Compared to the unfolded wild-type antigen, the refolded M3b antigen (W84S, P110S and V129L) exhibited an increase of 66% antigenicity with binding capacity of 0.96 and affinity of 113 μM−1. Moreover, the 33% decrease of the production demand suggests that M3b is a potential substitute for anti-HCV antibody detection

A unique case of spontaneous regression of metastatic papillary renal cell carcinoma: a case report

Spontaneous regression of cancer is a rare, but well documented, phenomenon. We present a unique case of an 82 year old Chinese male who experienced spontaneous regression of histologically-verified metastatic type II papillary renal cell carcinoma in the absence of intervening systemic therapy or surgery. This is the first reported case of spontaneous regression of papillary renal cell carcinoma. The mechanism of spontaneous regression remains unknown, and represents a challenge for existing oncology paradigms

Local Ca2+ Entry Via Orai1 Regulates Plasma Membrane Recruitment of TRPC1 and Controls Cytosolic Ca2+ Signals Required for Specific Cell Functions

Store-operated Ca2+ entry (SOCE) has been associated with two types of channels: CRAC channels that require Orai1 and STIM1 and SOC channels that involve TRPC1, Orai1, and STIM1. While TRPC1 significantly contributes to SOCE and SOC channel activity, abrogation of Orai1 function eliminates SOCE and activation of TRPC1. The critical role of Orai1 in activation of TRPC1-SOC channels following Ca2+ store depletion has not yet been established. Herein we report that TRPC1 and Orai1 are components of distinct channels. We show that TRPC1/Orai1/STIM1-dependent ISOC, activated in response to Ca2+ store depletion, is composed of TRPC1/STIM1-mediated non-selective cation current and Orai1/STIM1-mediated ICRAC; the latter is detected when TRPC1 function is suppressed by expression of shTRPC1 or a STIM1 mutant that lacks TRPC1 gating, STIM1(684EE685). In addition to gating TRPC1 and Orai1, STIM1 mediates the recruitment and association of the channels within ER/PM junctional domains, a critical step in TRPC1 activation. Importantly, we show that Ca2+ entry via Orai1 triggers plasma membrane insertion of TRPC1, which is prevented by blocking SOCE with 1 µM Gd3+, removal of extracellular Ca2+, knockdown of Orai1, or expression of dominant negative mutant Orai1 lacking a functional pore, Orai1-E106Q. In cells expressing another pore mutant of Orai1, Orai1-E106D, TRPC1 trafficking is supported in Ca2+-containing, but not Ca2+-free, medium. Consistent with this, ICRAC is activated in cells pretreated with thapsigargin in Ca2+-free medium while ISOC is activated in cells pretreated in Ca2+-containing medium. Significantly, TRPC1 function is required for sustained KCa activity and contributes to NFκB activation while Orai1 is sufficient for NFAT activation. Together, these findings reveal an as-yet unidentified function for Orai1 that explains the critical requirement of the channel in the activation of TRPC1 following Ca2+ store depletion. We suggest that coordinated regulation of the surface expression of TRPC1 by Orai1 and gating by STIM1 provides a mechanism for rapidly modulating and maintaining SOCE-generated Ca2+ signals. By recruiting ion channels and other signaling pathways, Orai1 and STIM1 concertedly impact a variety of critical cell functions that are initiated by SOCE

Improving Antigenicity of the Recombinant Hepatitis C Virus Core Protein via Random Mutagenesis

In order to enhance the sensitivity of diagnosis, a recombinant clone containing domain I of HCV core (amino acid residues 1 to 123) was subjected to random mutagenesis. Five mutants with higher sensitivity were obtained by colony screening of 616 mutants using reverse ELISA. Sequence analysis of these mutants revealed alterations focusing on W 84 , P 95 , P 110 , or V 129 . The inclusion bodies of these recombinant proteins overexpressed in E. coli BL21(DE3) were subsequently dissolved using 6 M urea and then refolded by stepwise dialysis. Compared to the unfolded wild-type antigen, the refolded M3b antigen (W 84 S, P 110 S and V 129 L) exhibited an increase of 66% antigenicity with binding capacity of 0.96 and affinity of 113 μM −1 . Moreover, the 33% decrease of the production demand suggests that M3b is a potential substitute for anti-HCV antibody detection

A study on the sustainability of family businesses in Singapore.

Family businesses are important to Singapore’s economy, contributing much to the GNP and employment levels. However, their survival rates are worryingly low: a minority survives the first generation and few, beyond that. Our research examines how local family businesses manage to hand-over successfully and common factors linking sustained family businesses