Sequence diversity in the A domain of Staphylococcus aureus fibronectin-binding protein A

<p>Abstract</p> <p>Background</p> <p>Fibronectin-binding protein A (FnBPA) mediates adhesion of <it>Staphylococcus aureus </it>to fibronectin, fibrinogen and elastin. We previously reported that <it>S. aureus </it>strain P1 encodes an FnBPA protein where the fibrinogen/elastin-binding domain (A domain) is substantially divergent in amino acid sequence from the archetypal FnBPA of <it>S. aureus </it>NCTC8325, and that these variations created differences in antigenicity. In this study strains from multilocus sequence types (MLST) that spanned the genetic diversity of <it>S.aureus </it>were examined to determine the extent of FnBPA A domain variation within the <it>S. aureus </it>population and its effect on ligand binding and immuno-crossreactivity.</p> <p>Results</p> <p>Seven different isotype forms (I – VII) of the FnBPA A domain were identified which were between 66 to 76% identical in amino acid sequence in any pair-wise alignment. The <it>fnbA </it>allelic variants in strains of different multilocus sequence type were identified by DNA hybridization using probes specific for sequences encoding the highly divergent N3 sub-domain of different isotypes. Several isotypes were not restricted to specific clones or clonal complexes but were more widely distributed. It is highly likely that certain <it>fnbA </it>genes have been transferred horizontally. Residues lining the putative ligand-binding trench were conserved, which is consistent with the ability of each A domain isotype to bind immobilized fibrinogen and elastin by the dock-latch-lock mechanism. Variant amino acid residues were mapped on a three-dimensional model of the FnBPA A domain and were predicted to be surface-exposed. Polyclonal antibodies raised against the recombinant isotype I A domain bound that protein with a 4 – 7 fold higher apparent affinity compared to the A domains of isotypes II – VII, while some monoclonal antibodies generated against the isotype I A domain showed reduced or no binding to the other isotypes.</p> <p>Conclusion</p> <p>The FnBPA A domain occurs in at least 7 different isotypes which differ antigenically and exhibit limited immuno-crossreactivity, yet retain their ligand-binding functions. Antigenic variation of the FnBPA A domain may aid <it>S. aureus </it>to evade the host's immune responses. These findings have implications for the development of vaccines or immunotherapeutics that target FnBPA.</p

Restoring mucosal barrier function and modifying macrophage phenotype with an extracellular matrix hydrogel: potential therapy for ulcerative colitis

Background & Aims: Despite advances in therapeutic options, more than half of all patients with ulcerative colitis (UC) do not achieve long-term remission, many require colectomy, and the disease still has a marked negative impact on quality of life. Extracellular matrix (ECM) bioscaffolds facilitate the functional repair of many soft tissues by mechanisms that include mitigation of pro-inflammatory macrophage phenotype and mobilization of endogenous stem/progenitor cells. The aim of the present study was to determine if an ECM hydrogel therapy could influence outcomes in an inducible rodent model of UC. Methods: The dextran sodium sulfate (DSS)-colitis model was used in male Sprague Dawley rats. Animals were treated via enema with an ECM hydrogel and the severity of colitis was determined by clinical and histologic criteria. Lamina propria cells were isolated and the production of inflammatory mediators was quantified. Mucosal permeability was assessed in-vivo by administering TRITC-dextran and in-vitro using transepithelial electrical resistance (TEER). Results: ECM hydrogel therapy accelerated healing and improved outcome. The hydrogel was adhesive to colonic tissue, which allowed for targeted delivery of the therapy, and resulted in a reduction in clinical and histologic signs of disease. ECM hydrogel facilitated functional improvement of colonic epithelial barrier function and the resolution of the pro-inflammatory state of tissue macrophages. Conclusions: The present study shows that a nonsurgical and nonpharmacologic ECM-based therapy can abate DSS-colitis not by immunosuppression but by promoting phenotypic change in local macrophage phenotype and rapid replacement of the colonic mucosal barrie

The impact of detergents on the tissue decellularization process: a ToF-SIMS study

Biologic scaffolds are derived from mammalian tissues, which must be decellularized to remove cellular antigens that would otherwise incite an adverse immune response. Although widely used clinically, the optimum balance between cell removal and the disruption of matrix architecture and surface ligand landscape remains a considerable challenge. Here we describe the use of time of flight secondary ion mass spectroscopy (ToF-SIMS) to provide sensitive, molecular specific, localized analysis of detergent decellularized biologic scaffolds. We detected residual detergent fragments, specifically from Triton X-100, sodium deoxycholate and sodium dodecyl sulphate (SDS) in decellularized scaffolds; increased SDS concentrations from 0.1% to 1.0% increased both the intensity of SDS fragments and adverse cell outcomes. We also identified cellular remnants, by detecting phosphate and phosphocholine ions in PAA and CHAPS decellularized scaffolds. The present study demonstrates ToF-SIMS is not only a powerful tool for characterization of biologic scaffold surface molecular functionality, but also enables sensitive assessment of decellularization efficacy

Probing the extragalactic fast transient sky at minute timescales with DECam

Searches for optical transients are usually performed with a cadence of days to weeks, optimised for supernova discovery. The optical fast transient sky is still largely unexplored, with only a few surveys to date having placed meaningful constraints on the detection of extragalactic transients evolving at sub-hour timescales. Here, we present the results of deep searches for dim, minute-timescale extragalactic fast transients using the Dark Energy Camera, a core facility of our all-wavelength and all-messenger Deeper, Wider, Faster programme. We used continuous 20s exposures to systematically probe timescales down to 1.17 minutes at magnitude limits $g > 23$ (AB), detecting hundreds of transient and variable sources. Nine candidates passed our strict criteria on duration and non-stellarity, all of which could be classified as flare stars based on deep multi-band imaging. Searches for fast radio burst and gamma-ray counterparts during simultaneous multi-facility observations yielded no counterparts to the optical transients. Also, no long-term variability was detected with pre-imaging and follow-up observations using the SkyMapper optical telescope. We place upper limits for minute-timescale fast optical transient rates for a range of depths and timescales. Finally, we demonstrate that optical $g$-band light curve behaviour alone cannot discriminate between confirmed extragalactic fast transients such as prompt GRB flashes and Galactic stellar flares.Comment: Published in MNRA

Detection of a glitch in the pulsar J1709-4429

We report the detection of a glitch event in the pulsar J1709$-$4429 (also known as B1706$-$44) during regular monitoring observations with the Molonglo Observatory Synthesis Telescope (UTMOST). The glitch was found during timing operations, in which we regularly observe over 400 pulsars with up to daily cadence, while commensally searching for Rotating Radio Transients, pulsars, and FRBs. With a fractional size of $\Delta\nu/\nu \approx 52.4 \times10^{-9}$, the glitch reported here is by far the smallest known for this pulsar, attesting to the efficacy of glitch searches with high cadence using UTMOST.Comment: 3 pages, 1 figur

A simple method for directional transcriptome sequencing using Illumina technology.

High-throughput sequencing of cDNA has been used to study eukaryotic transcription on a genome-wide scale to single base pair resolution. In order to compensate for the high ribonuclease activity in bacterial cells, we have devised an equivalent technique optimized for studying complete prokaryotic transcriptomes that minimizes the manipulation of the RNA sample. This new approach uses Illumina technology to sequence single-stranded (ss) cDNA, generating information on both the direction and level of transcription throughout the genome. The protocol, and associated data analysis programs, are freely available from http://www.sanger.ac.uk/Projects/Pathogens/Transcriptome/. We have successfully applied this method to the bacterial pathogens Salmonella bongori and Streptococcus pneumoniae and the yeast Schizosaccharomyces pombe. This method enables experimental validation of genetic features predicted in silico and allows the easy identification of novel transcripts throughout the genome. We also show that there is a high correlation between the level of gene expression calculated from ss-cDNA and double-stranded-cDNA sequencing, indicting that ss-cDNA sequencing is both robust and appropriate for use in quantitative studies of transcription. Hence, this simple method should prove a useful tool in aiding genome annotation and gene expression studies in both prokaryotes and eukaryotes

A cardinal role for cathepsin D in co-ordinating the host-mediated apoptosis of macrophages and killing of pneumococci

The bactericidal function of macrophages against pneumococci is enhanced by their apoptotic demise, which is controlled by the anti-apoptotic protein Mcl-1. Here, we show that lysosomal membrane permeabilization (LMP) and cytosolic translocation of activated cathepsin D occur prior to activation of a mitochondrial pathway of macrophage apoptosis. Pharmacological inhibition or knockout of cathepsin D during pneumococcal infection blocked macrophage apoptosis. As a result of cathepsin D activation, Mcl-1 interacted with its ubiquitin ligase Mule and expression declined. Inhibition of cathepsin D had no effect on early bacterial killing but inhibited the late phase of apoptosis-associated killing of pneumococci in vitro. Mice bearing a cathepsin D-/- hematopoietic system demonstrated reduced macrophage apoptosis in vivo, with decreased clearance of pneumococci and enhanced recruitment of neutrophils to control pulmonary infection. These findings establish an unexpected role for a cathepsin D-mediated lysosomal pathway of apoptosis in pulmonary host defense and underscore the importance of apoptosis-associated microbial killing to macrophage function

Adjuvant Chemotherapy Guided by a 21-Gene Expression Assay in Breast Cancer

BACKGROUND The recurrence score based on the 21-gene breast cancer assay predicts chemotherapy benefit if it is high and a low risk of recurrence in the absence of chemotherapy if it is low; however, there is uncertainty about the benefit of chemotherapy for most patients, who have a midrange score. METHODS We performed a prospective trial involving 10,273 women with hormone-receptor–positive, human epidermal growth factor receptor 2 (HER2)–negative, axillary node–negative breast cancer. Of the 9719 eligible patients with follow-up information, 6711 (69%) had a midrange recurrence score of 11 to 25 and were randomly assigned to receive either chemoendocrine therapy or endocrine therapy alone. The trial was designed to show noninferiority of endocrine therapy alone for invasive disease–free survival (defined as freedom from invasive disease recurrence, second primary cancer, or death). RESULTS Endocrine therapy was noninferior to chemoendocrine therapy in the analysis of invasive disease–free survival (hazard ratio for invasive disease recurrence, second primary cancer, or death [endocrine vs. chemoendocrine therapy], 1.08; 95% confidence interval, 0.94 to 1.24; P=0.26). At 9 years, the two treatment groups had similar rates of invasive disease–free survival (83.3% in the endocrine-therapy group and 84.3% in the chemoendocrine-therapy group), freedom from disease recurrence at a distant site (94.5% and 95.0%) or at a distant or local–regional site (92.2% and 92.9%), and overall survival (93.9% and 93.8%). The chemotherapy benefit for invasive disease–free survival varied with the combination of recurrence score and age (P=0.004), with some benefit of chemotherapy found in women 50 years of age or younger with a recurrence score of 16 to 25. CONCLUSIONS Adjuvant endocrine therapy and chemoendocrine therapy had similar efficacy in women with hormone-receptor–positive, HER2-negative, axillary node–negative breast cancer who had a midrange 21-gene recurrence score, although some benefit of chemotherapy was found in some women 50 years of age or younger

Clinical and Genomic Risk to Guide the Use of Adjuvant Therapy for Breast Cancer

BACKGROUND The use of adjuvant chemotherapy in patients with breast cancer may be guided by clinicopathological factors and a score based on a 21-gene assay to determine the risk of recurrence. Whether the level of clinical risk of breast cancer recurrence adds prognostic information to the recurrence score is not known. METHODS We performed a prospective trial involving 9427 women with hormone-receptor–positive, human epidermal growth factor receptor 2–negative, axillary node–negative breast cancer, in whom an assay of 21 genes had been performed, and we classified the clinical risk of recurrence of breast cancer as low or high on the basis of the tumor size and histologic grade. The effect of clinical risk was evaluated by calculating hazard ratios for distant recurrence with the use of Cox proportional-hazards models. The initial endocrine therapy was tamoxifen alone in the majority of the premenopausal women who were 50 years of age or younger. RESULTS The level of clinical risk was prognostic of distant recurrence in women with an intermediate 21-gene recurrence score of 11 to 25 (on a scale of 0 to 100, with higher scores indicating a worse prognosis or a greater potential benefit from chemotherapy) who were randomly assigned to endocrine therapy (hazard ratio for the comparison of high vs. low clinical risk, 2.73; 95% confidence interval [CI], 1.93 to 3.87) or to chemotherapy plus endocrine (chemoendocrine) therapy (hazard ratio, 2.41; 95% CI, 1.66 to 3.48) and in women with a high recurrence score (a score of 26 to 100), all of whom were assigned to chemoendocrine therapy (hazard ratio, 3.17; 95% CI, 1.94 to 5.19). Among women who were 50 years of age or younger who had received endocrine therapy alone, the estimated (±SE) rate of distant recurrence at 9 years was less than 5% (≤1.8±0.9%) with a low recurrence score (a score of 0 to 10), irrespective of clinical risk, and 4.7±1.0% with an intermediate recurrence score and low clinical risk. In this age group, the estimated distant recurrence at 9 years exceeded 10% among women with a high clinical risk and an intermediate recurrence score who received endocrine therapy alone (12.3±2.4%) and among those with a high recurrence score who received chemoendocrine therapy (15.2±3.3%). CONCLUSIONS Clinical-risk stratification provided prognostic information that, when added to the 21-gene recurrence score, could be used to identify premenopausal women who could benefit from more effective therapy