Yanchun Wang

Association of NOD1 and NOD2 genes polymorphisms with Helicobacter pylori related gastric cancer in a Chinese population

AIMnTo investigate the association between the tag single nucleotide polymorphisms (TagSNPs) of NOD1 and NOD2 and the risk of developing gastric cancer.nnnMETHODSnWe conducted a hospital-based case-control study including 296 incident gastric cancer patients and 160 gastritis controls. Eight TagSNPs in the NOD1 and NOD2 genes were selected from the Hapmap database using the haploview software and genotyped by the Sequenom MassArray system. The serum levels of anti-Helicobacter pylori (H. pylori) IgG were measured by enzyme-linked immunosorbent assay to indicate H. pylori infection. The odds ratios (OR) and 95% confidence intervals (CI) were calculated by unconditional logistic regression, including sex and age as confounding factors.nnnRESULTSnThe NOD1 rs2907749 GG genotype showed a decreased risk for gastric cancer (OR 0.50, 95% CI: 0.26-0.95, P = 0.04) while the rs7789045 TT genotype showed an increased risk (OR 2.14, 95% CI: 1.20-3.82, P = 0.01). An elevated susceptibility to gastric cancer was observed in the subjects with H. pylori infection and the NaOD1 rs7789045 TT genotype (OR 2.05, 95% CI: 1.07-3.94, P = 0.03) or the NOD2 rs7205423 GC genotype (OR 2.52, 95% CI: 1.05-6.04, P = 0.04). Haplotype analysis suggested that the distribution of AGT (rs2907749, rs2075820 and rs7789045) in NOD1 between the cases and control groups was significantly different (P corrected: 0.04), and the diplotype AGT/AGT was associated with an elevated gastric cancer risk (OR 1.98, 95% CI: 1.04-3.79, P = 0.04). The association of the NOD1 rs7789045 TT genotype and the diplotype AGT/AGT was significant with H. pylori-related diffuse-type gastric cancer (OR 3.00, 95% CI: 1.38-6.53, P = 0.01; OR 4.02, 95% CI: 1.61-10.05, P < 0.01, respectively).nnnCONCLUSIONnGenetic polymorphisms in NOD1 and NOD2 may interact with H. pylori infection and may play important roles in promoting the development of gastric cancer in the Chinese population.

Identification of B-cell epitopes in urease B subunit of Helicobacter pylori bound by neutralizing antibodies

To identify linear B-cell epitopes of urease B (UreB), a series of 19 partially overlapping fragments of the UreB gene were expressed. Three MAbs against UreB of Helicobacter pylori (H. pylori), A1H10, A3C10, and B3D9, were tested for their reactivity to the truncated proteins by Western blot and enzyme-linked immunosorbent assay (ELISA). Three linear B-cell epitopes were identified covering a stretch of 15 amino acid (aa) residues and localized in the aa regions 158-172, 181-195, and 349-363 of UreB. ELISA also showed that the three synthetic peptides containing epitope sequences (UP32: GGGTGPADGTNATTI, UP35: WMLRAAEEYSMNLGF, and UP38: TLHDMGIFSITSSDS) were recognized by the corresponding MAbs and H. pylori positive sera from H. pylori infected patients. Mice immunized with glutathione S-transferase (GST) fusion peptides showed that epitope-specific antibodies were capable of inhibiting urease enzymatic activity. These results should be useful in clinical applications and highlight the potential importance of these epitopes as the targets for development of epitope-based vaccines against H. pylori.

The Upregulation of TRAF1 Induced by Helicobacter pylori Plays an Antiapoptotic Effect on the Infected Cells.

Tumor necrosis factor receptor‐associated factor 1 (TRAF1) is a member of the TRAF family and is dysregulated in diseases, such as atheroma, lymphoma, and solid tumors, but the role of TRAF1 in gastric cancer remains unknown. This study was aimed to investigate the role of TRAF1 in Helicobacter pylori (H. pylori)‐related cell apoptosis and gastric carcinogenesis.

Bacillus anthracis S-layer protein BslA binds to extracellular matrix by interacting with laminin.

BackgroundThe Bacillus anthracis S-layer protein, BslA, plays a crucial role in mammalian infection. BslA is required to mediate adherence between host cells and vegetative forms of bacteria and this interaction promotes target organs adherence and blood–brain barrier (BBB) penetration in vivo. This study attempts to identify the potential eukaryotic ligand(s) for B. anthracis BslA protein.ResultsBiochemical approaches have indicated that the putative host cell ligand(s) for BslA is a surface protein, which is independent of the sugar components for binding to Bs1A. A ligand screening using blot overlays, far Western blots and mass spectrometry analyses revealed that BslA binds to mammalian laminin. ELISA based solid-phase binding assays and surface plasmon resonance assays demonstrated that there were high affinity interactions between BslA(260–652) and laminin. The SPR results also revealed the dissociation constants values of 3.172u2009×u200910−9M for the binding of BslA(260–652) to laminin.ConclusionsThese data demonstrated that laminin is a ligand for BslA.

Production and cell surface display of recombinant anthrax protective antigen on the surface layer of attenuated Bacillus anthracis

To investigate the surface display of the anthrax protective antigen (PA) on attenuated Bacillus anthracis, a recombinant B. anthracis strain, named AP429 was constructed by integrating into the chromosome a translational fusion harboring the DNA fragments encoding the cell wall-targeting domain of the S-layer protein EA1 and the anthrax PA. Crerecombinase action at the loxP sites excised the antibiotic marker. Western blot analysis, fluorescence-activated cell sorting and immunofluorescence analysis confirmed that PA was successfully expressed on the S-layer of the recombinant antibiotic marker-free strain. Notwithstanding extensive proteolytic degradation of the hybrid protein SLHs-PA, quantitative ELISA revealed that approximately 8.1xa0×xa0106 molecules of SLHs-PA were gained from each Bacillus cell. Moreover, electron microscopy assay indicated that the typical S-layer structures could be clearly observed from the recombinant strain micrographs.

pheS* as a counter-selectable marker for marker-free genetic manipulations in Bacillus anthracis

Several genetic tools have been developed for use in Bacillus anthracis, but there is still a need for a more marker-free gene inactivation protocols. Thus, we report a method to generate unmarked mutations in B. anthracis. This approach was based on the counter-selectable pheS* gene with assistance by the I-SceI homing endonuclease. Using this strategy, the NprR gene, a transcriptional activator of B. anthracis, was deleted at an extremely high efficiency. Our study indicates that mutated pheS is a useful counter-selective marker to design a valuable genetic tool for in-frame and unmarked gene deletions of B. anthracis.

Helicobacter pylori inhibits the cleavage of TRAF1 via a CagA-dependent mechanism

AIM To study the impact on cleavage of tumor necrosis factor receptor-associated factor 1 (TRAF1) regulated by Helicobacter pylori (H. pylori). METHODS Cleavage of TRAF1 was detected by western blotting in the human gastric cancer cell line AGS following treatment with an apoptosis inducer. Cleavage of TRAF1 mediated by caspase was examined in vitro using specific caspase inhibitors. The effect of the COOH-terminal TRAF1 fragment on gastric cell apoptosis during H. pylori infection was measured using flow cytometry. The impact of H. pylori infection on TRAF1 cleavage was detected in the presence of apoptosis inducer. The roles of H. pylori virulence factors that may regulate TRAF1 cleavage were analyzed using isogenic cagA-, vacA- and cagE-null mutants. RESULTS TRAF1 was found to be cleaved in AGS cells treated with the apoptosis inducer, and caspase-8 was the major caspase involved in the cleavage of TRAF1. The COOH-terminal TRAF1 fragment significantly induced cell apoptosis (P < 0.05) as well as promoted H. pylori-induced cell apoptosis (P < 0.05). H. pylori infection was found to significantly inhibit the cleavage of TRAF1 and to inhibit the activation of caspase-8 in the presence of the apoptosis inducer at specific infection times and different cell/bacteria ratios. We also found that the effects of cagE- and cagA-null mutants on the inhibition of TRAF1 cleavage and activation of caspase-8 were significantly attenuated, compared with wild-type H. pylori, in the presence of the apoptosis inducer, showing that the virulence factor CagA was mainly involved in the inhibition of TRAF1 cleavage. CONCLUSION H. pylori infection significantly inhibits the cleavage of TRAF1 via a CagA-dependent mechanism, which would increase the relative amounts of full-length TRAF1 and exert an antiapoptotic effect on H. pylori-infected cells.