GBS adherence to hBMEC is mediated by the interaction of Srr1 and fibrinogen.

<p>(A) Fibrinogen on the surface of hBMEC pretreated with or without exogenous fibrinogen (Fg; 20 µg/ml). Nuclei were stained with DAPI (blue) and fibrinogen was detected with anti-fibrinogen IgG, followed by Alexa Fluor 488 conjugated anti-rabbit IgG (red). (B) NCTC 10/84 (WT) or PS2645 (Δ<i>srr1</i>) incubated with hBMEC, with or without fibrinogen pretreatment (20 µg/ml). Unbound bacteria were washed out and bound bacteria were counted. Values represent percent (mean ± S.D.) of total GBS inoculum bound to the monolayers. * = P<0.01.</p

Impact of the latch-like domain on GBS virulence.

<p>Bacteria counts in the blood and brain of mice infected with GBS WT or Δl<i>atch</i> bacteria, determined 24 h post infection (A) or at the time of death (C). The horizontal lines denote the median number of bacteria in each group of 10 mice. (B) Kaplan-Meier survival curves of CD-1 male mice following i.v. infection with GBS NCTC 10/84 or Δlatch strains. (n = 10 per group). (D–F) Histopathology of representative brain tissues from mice infected with GBS<i>Δlatch</i> (D) and WT GBS (E, F). Note significant increase of meningeal thickness and neutrophil infiltration in WT infected brain tissue, but not in animals infected with the Δ<i>latch</i> variant.</p

GBS binding to fibrinogen is mediated by Srr1.

<p>(A) GBS strains COH31 (left) and NCTC 10/84 (right) were compared with their Δ<i>srr1</i> variants (Δ<i>srr1</i>) for binding to the wells pretreated with fibronectin or fibrinogen (0.1 µM per well). (B) Inhibition of GBS binding to fibrinogen by anti-Srr1 IgG. COH31 strain was co-incubated with rabbit anti-Srr1 IgG or normal rabbit IgG, and relative binding to immobilized with fibrinogen. Values are mean ± S.D. of relative binding, normalized for WT levels of binding to fibrinogen. * = P<0.01.</p

GBS binding to fibrinogen Aα variants.

<p>GBS strains NCTC 10/84 (A) and COH31 (B) were compared with their Δ<i>srr1</i> variants (Δ<i>srr1</i>) for binding to MalE:Aα_198–282 or MalE:Aα_283–410. Values are mean ± S.D. percent of WT GBS binding to immobilized MalE:Aα_283–410. * = P<0.01.</p

Interaction of the binding region (BR) of Srr1 with fibrinogen.

<p>(A) Binding of purified Srr1-BR, N terminal Srr1-BR, or C-terminal Srr1-BR protein (_FLAGSrr1-BR, _FLAGN-Srr1-BR, _FLAGC-Srr1-BR) to immobilized fibrinogen (0.1 µM). Bound proteins were detected with anti-FLAG antibody. (B) Inhibition of _FLAGSrr1-BR binding to immobilized fibrinogen with anti-fibrinogen IgG. Immobilized fibrinogen was preincubated with the indicated concentration of anti-fibrinogen antibody, and then tested for binding by _FLAGSrr1-BR (5 µg/ml). Bound proteins were detected with anti-FLAG IgG. Normal rabbit IgG served as a control. (C) Inhibition of _FLAGSrr1-BR binding to fibrinogen by unlabeled Srr1-BR. Immobilized fibrinogen was coincubated with the indicated concentrations of _FLAGSrr1-BR (5 µg/ml) or unlabeled Srr1-BR. Bound proteins were detected with anti-FLAG antibody. Values represent percent of _FLAGSrr1-BR binding to the wells treated with fibrinogen. Bars indicate the means (± S.D.). * = P<0.01.</p

Schematic diagram of three staphylococcal fibrinogen binding proteins (ClfA, SdrG and ClfB) and the serine rich repeat proteins Srr1 and GspB.

<p>Level of identity (%) between regions is indicated. *S: signal sequence; N1, N2, and N3: DEv-IgG domains; B1 and B2, repeats of unknown function; SD: serine and aspartic acid rich region; SRR1 and SRR2: serine rich regions; CNA: IgG fold domain; Siglec: sialic acid binding domain; LPxTG: cell wall anchoring motif.</p

Impact of the Srr1 latch-like domain on GBS binding.

<p>(A) Binding of _FLAGSrr1-BR and _FLAGSrr1-BRΔlatch proteins to immobilized fibrinogen. Indicated concentration of _FLAGSrr1-BR and _FLAGSrr1-BRΔlatch were added to wells coated with fibrinogen or casein blocking reagent. (B) Binding of _FLAGSrr1-BR and _FLAGSrr1-BRΔlatch proteins to hBMEC monolayers pretreated with PBS (left panels) or fibrinogen (20 µg/ml, right panels). After washing out unbound proteins, bound proteins were detected with anti-FLAG mAb, followed by Alexa Fluor 488 conjugated anti-mouse IgG (red). Nuclei were stained with DAPI (blue). (C) Expression of Srr1-WT and Srr1Δ<i>latch</i> on the cell surface. Isolated cell wall proteins were probed by Western blotting with anti-Srr1 IgG. (D) GBS NCTC 10/84 WT, Δ<i>srr1</i> and Δlatch variant binding to immobilized fibrinogen. Values represent percent of WT GBS binding to fibrinogen. (E) GBS NCTC 10/84 WT, Δ<i>srr1</i> and Δ<i>latch</i> isogenic variant adherence to hBMEC monolayers. * = P<0.01.</p

Prevalence and risk factors of hyperuricemia: results of the Kailuan cohort study

<p><b>Objective:</b> The objective of this study is to determine the serum uric acid (SUA) level and the prevalence of hyperuricemia (HUA) in Chinese population.</p> <p><b>Methods:</b> We conducted a cross-sectional study among 100,226 employees (79.9% male) of the Kailuan Group using physical examination data in 2006–2007. HUA was defined as SUA >356.9 μmol/L (6.0 mg/dL) for women and SUA >416.4 μmol/L (7.0 mg/dL) for men. We investigated crude and age adjusted HUA prevalence and compared characteristics of subjects with and without HUA in men and women using multivariate logistic regression.</p> <p><b>Results:</b> SUA levels were 244.9 ± 71.5 μmol/L in women and 302.0 ± 83.5 μmol/L in men. About 8290 (8.27%) subjects were diagnosed with HUA. Age-adjusted prevalence of HUA was 8.02% in the total sample (6.87% in women and 8.57% in men). The SUA level and HUA prevalence showed U-shaped or J-shaped associations with age. Multivariate logistic regression revealed age, waist circumference, total cholesterol, triglyceride, hypertension and non-alcoholic fatty liver disease history, prolonged sitting, alcohol consumption, and oral diuretics were independent risk factors of HUA, while long sleep duration was protective against HUA.</p> <p><b>Conclusions:</b> The prevalence of HUA is 6.87% and 8.57% in Chinese women and men. HUA is likely related with life style and metabolic disorders.</p

CiaR regulation influences GBS intracellular trafficking in brain endothelial cells.

<p>A. hBMEC monolayers were infected with WT COH1 GFP expressing GBS for 2 hours (MOI = 10) and then stained with antibodies to Rab5, Rab7, and LAMP1 following the indicated time points as described in Materials and Methods. Representative images of triplicate experiments demonstrate co-localization (yellow) of GBS (green) with each marker (red), Scale Bar, 5μm. B, C, D. Percentages of co-localization of Rab5, Rab7, and LAMP1 with GFP expressing GBS WT and mutant strains after various time points post infection during antibiotic treatment. At least 100 cells containing intracellular GBS were counted for each time point in triplicate. Statistical analysis performed was a Two-way ANOVA with a Bonferroni’s multiple comparisons test and the data represents mean ± S.D. <i>p <</i> 0.05 *, <i>p <</i> 0.0005 ***, <i>p <</i> 0.00005 ****.</p

GBS recovery from Lysosomes.

<p>A. Isolated lysosomes infected with WT COH1 GFP expressing GBS were subjected to staining with Lysotracker Red (0.5μM) and visualized using fluorescence microscopy. Scale Bar, 5 μm B. hBMEC were infected with WT or mutant GBS strains for 2 hours (MOI = 10) and subjected to lysosomal isolation by differential centrifugation after 1 or 12 hours post antibiotic treatment. Lysosomal pellets were plated to enumerate the amount of recovered viable CFU. Statistical analysis performed was a One-way ANOVA with a Tukey’s multiple comparisons test and the data represents mean ± S.D. <i>p <</i> 0.05 *, <i>p <</i> 0.005 **.</p