Genomic diversity of citrate fermentation in Klebsiella pneumoniae

<p>Abstract</p> <p>Background</p> <p>It has long been recognized that <it>Klebsiella pneumoniae </it>can grow anaerobically on citrate. Genes responsible for citrate fermentation of <it>K. pneumoniae </it>were known to be located in a 13-kb gene cluster on the chromosome. By whole genome comparison of the available <it>K. pneumoniae </it>sequences (MGH 78578, 342, and NTUH-K2044), however, we discovered that the fermentation gene cluster was present in MGH 78578 and 342, but absent in NTUH-K2044. In the present study, the previously unknown genome diversity of citrate fermentation among <it>K. pneumoniae </it>clinical isolates was investigated.</p> <p>Results</p> <p>Using a genomic microarray containing probe sequences from multiple <it>K. pneumoniae </it>strains, we investigated genetic diversity among <it>K. pneumoniae </it>clinical isolates and found that a genomic region containing the citrate fermentation genes was not universally present in all strains. We confirmed by PCR analysis that the gene cluster was detectable in about half of the strains tested. To demonstrate the metabolic function of the genomic region, anaerobic growth of <it>K. pneumoniae </it>in artificial urine medium (AUM) was examined for ten strains with different clinical histories and genomic backgrounds, and the citrate fermentation potential was found correlated with the genomic region. PCR detection of the genomic region yielded high positive rates among a variety of clinical isolates collected from urine, blood, wound infection, and pneumonia. Conserved genetic organizations in the vicinity of the citrate fermentation gene clusters among <it>K. pneumoniae</it>, <it>Salmonella enterica</it>, and <it>Escherichia coli </it>suggest that the13-kb genomic region were not independently acquired.</p> <p>Conclusion</p> <p>Not all, but nearly half of the <it>K. pneumoniae </it>clinical isolates carry the genes responsible for anaerobic growth on citrate. Genomic variation of citrate fermentation genes in <it>K. pneumoniae </it>may contribute to metabolic diversity and adaptation to variable nutrient conditions in different environments.</p

Prediction of life stress on athletes’ burnout: the dual role of perceived stress

Although many studies adopted Smith’s (1986) cognitive–affective model of athletic burnout in examining stress–burnout relationship, very few studies examined the mediating/moderating role of perceived stress on the stress–burnout relationship. We sampled 195 college student-athletes and assessed their life stress, perceived stress, and burnout. Correlation analyses found all study variables correlated. Two separate hierarchical regression analyses found that the “distress” component of perceived stress mediated athletes’ two types of life stress–burnout relationship but “counter-stress” component of perceived stress-moderated athletes’ general-life stress–burnout relationship. We concluded that interweaving relationships among athletes’ life stress, perceived stress, and burnout are not straightforward. Future research should consider the nature of athletes life stress, and dual role of perceived stress in examining its’ association with related psychological responses in athletic settings

Detection of the Hepatitis B Virus X Protein (HBx) Antigen and Anti-HBx Antibodies in Cases of Human Hepatocellular Carcinoma

Hepatitis B virus X protein (HBx) expressed in Escherichia coli DH5α by recombinant DNA technology was purified to homogeneity by use of glutathione-Sepharose beads. Immunological characterization of the recombinant HBx protein was performed. Specific binding between the anti-HBx monoclonal antibody and HBx protein showed the specificity of the recombinant HBx protein. The intact HBx protein of the factor Xa-digested glutathione S-transferase-HBx fusion protein was further purified and was used as an antigen for screening the titers of anti-HBx antibodies in sera. Titers of anti-HBx in sera from 20 patients with hepatocellular carcinoma (HCC), 20 patients with chronic hepatitis (CH), and 20 healthy individuals were evaluated by Western blotting and a quantitative enzyme-linked immunosorbent assay. The results indicated that 70% of sera from HCC patients and 5% of sera from CH patients contained antibodies with significant binding to the HBx protein. Western blotting of HBx protein in liver extracts from 20 HCC patients was also performed by using the anti-HBx monoclonal antibody. Results showed that 85% of HCC patients' liver tissues contained a specific HBx protein with the same molecular size as the purified intact HBx. Full correlation was found between anti-HBx antibody positivity in serum and HBx protein positivity in HCC tissues. The data demonstrated that the etiology of HCC is involved with hepatitis B virus (HBV) infection and that HBx in particular plays a role in the development of HBV-related HCC

Successful Propagation of Flavivirus Infectious cDNAs by a Novel Method To Reduce the Cryptic Bacterial Promoter Activity of Virus Genomes▿ †

Reverse genetics is a powerful tool to study single-stranded RNA viruses. Despite tremendous efforts having been made to improve the methodology for constructing flavivirus cDNAs, the cause of toxicity of flavivirus cDNAs in bacteria remains unknown. Here we performed mutational analysis studies to identify Escherichia coli promoter (ECP) sequences within nucleotides (nt) 1 to 3000 of the dengue virus type 2 (DENV2) and Japanese encephalitis virus (JEV) genomes. Eight and four active ECPs were demonstrated within nt 1 to 3000 of the DENV2 and JEV genomes, respectively, using fusion constructs containing DENV2 or JEV segments and empty vector reporter gene Renilla luciferase. Full-length DENV2 and JEV cDNAs were obtained by inserting mutations reducing their ECP activity in bacteria without altering amino acid sequences. A severe cytopathic effect occurred when BHK21 cells were transfected with in vitro-transcribed RNAs from either a DENV2 cDNA clone with multiple silent mutations within the prM-E-NS1 region of dengue genome or a JEV cDNA clone with an A-to-C mutation at nt 90 of the JEV genome. The virions derived from the DENV2 or JEV cDNA clone exhibited infectivities similar to those of their parental viruses in C6/36 and BHK21 cells. A cis-acting element essential for virus replication was revealed by introducing silent mutations into the central portion (nt 160 to 243) of the core gene of DENV2 infectious cDNA or a subgenomic DENV2 replicon clone. This novel strategy of constructing DENV2 and JEV infectious clones could be applied to other flaviviruses or pathogenic RNA viruses to facilitate research in virology, viral pathogenesis, and vaccine development

Resistance Analysis and Characterization of a Thiazole Analogue, BP008, as a Potent Hepatitis C Virus NS5A Inhibitor

Hepatitis C virus (HCV) is a global health problem, affecting approximately 3% of the world's population. The standard treatment for HCV infection is often poorly tolerated and ineffective. Therefore, the development of novel or more effective treatment strategies to treat chronic HCV infection is urgently needed. In this report, BP008, a potent small-molecule inhibitor of HCV replication, was developed from a class of compounds with thiazol core structures by means of utilizing a cell-based HCV replicon system. The compound reduced the reporter expression of the HCV1b replicon with a 50% effective concentration (EC50) and selective index value of 4.1 ± 0.7 nM and >12,195, respectively. Sequencing analyses of several individual clones derived from BP008-resistant RNAs purified from cells harboring HCV1b replicon revealed that amino acid substitutions mainly within the N-terminal region (domain I) of NS5A were associated with decreased inhibitor susceptibility. Q24L, P58S, and Y93H are the key substitutions for resistance selection; F149L and V153M play the compensatory role in the replication and drug resistance processes. Moreover, BP008 displayed synergistic effects with alpha interferon (IFN-α), NS3 protease inhibitor, and NS5B polymerase inhibitor, as well as good oral bioavailability in SD rats and favorable exposure in rat liver. In summary, our results pointed to an effective small-molecule inhibitor, BP008, that potentially targets HCV NS5A. BP008 can be considered a part of a more effective therapeutic strategy for HCV in the future

A cross-sectional study on the level of knowledge, attitude and practice among parents of children aged 18 years old and below on refractive errors in Rumah Juntan, Sibu from 26th October to 31st December 2009

A cross sectional research on the assessment of level of knowledge, attitude, and practice on refractive errors among 80 parents with children ages 18 years old and belowin Rumah Juntan, Sibu was carried out from 26th October to 31st December 2009