Improved multiple-locus variable-number tandem-repeat assay for Staphylococcus aureus genotyping, providing a highly informative technique together with strong phylogenetic value.

We describe an improved multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) scheme for genotyping Staphylococcus aureus. We compare its performance to those of multilocus sequence typing (MLST) and spa typing in a survey of 309 strains. This collection includes 87 epidemic methicillin-resistant S. aureus (MRSA) strains of the Harmony collection, 75 clinical strains representing the major MLST clonal complexes (CCs) (50 methicillin-sensitive S. aureus [MSSA] and 25 MRSA), 135 nasal carriage strains (133 MSSA and 2 MRSA), and 13 published S. aureus genome sequences. The results show excellent concordance between the techniques' results and demonstrate that the discriminatory power of MLVA is higher than those of both MLST and spa typing. Two hundred forty-two genotypes are discriminated with 14 VNTR loci (diversity index, 0.9965; 95% confidence interval, 0.9947 to 0.9984). Using a cutoff value of 45%, 21 clusters are observed, corresponding to the CCs previously defined by MLST. The variability of the different tandem repeats allows epidemiological studies, as well as follow-up of the evolution of CCs and the identification of potential ancestors. The 14 loci can conveniently be analyzed in two steps, based upon a first-line simplified assay comprising a subset of 10 loci (panel 1) and a second subset of 4 loci (panel 2) that provides higher resolution when needed. In conclusion, the MLVA scheme proposed here, in combination with available on-line genotyping databases (including http://mlva.u-psud.fr/), multiplexing, and automatic sizing, can provide a basis for almost-real-time large-scale population monitoring of S. aureus.

Rapid Biodiversity Assessment and Monitoring Method for Highly Diverse Benthic Communities: A Case Study of Mediterranean Coralligenous Outcrops

Increasing anthropogenic pressures urge enhanced knowledge and understanding of the current state of marine biodiversity. This baseline information is pivotal to explore present trends, detect future modifications and propose adequate management actions for marine ecosystems. Coralligenous outcrops are a highly diverse and structurally complex deep-water habitat faced with major threats in the Mediterranean Sea. Despite its ecological, aesthetic and economic value, coralligenous biodiversity patterns are still poorly understood. There is currently no single sampling method that has been demonstrated to be sufficiently representative to ensure adequate community assessment and monitoring in this habitat. Therefore, we propose a rapid non-destructive protocol for biodiversity assessment and monitoring of coralligenous outcrops providing good estimates of its structure and species composition, based on photographic sampling and the determination of presence/absence of macrobenthic species. We used an extensive photographic survey, covering several spatial scales (100s of m to 100s of km) within the NW Mediterranean and including 2 different coralligenous assemblages: Paramuricea clavata (PCA) and Corallium rubrum assemblage (CRA). This approach allowed us to determine the minimal sampling area for each assemblage (5000 cm² for PCA and 2500 cm²for CRA). In addition, we conclude that 3 replicates provide an optimal sampling effort in order to maximize the species number and to assess the main biodiversity patterns of studied assemblages in variability studies requiring replicates. We contend that the proposed sampling approach provides a valuable tool for management and conservation planning, monitoring and research programs focused on coralligenous outcrops, potentially also applicable in other benthic ecosystems

Streptococcus tigurinus is highly virulent in a rat model of experimental endocarditis.

Streptococcus tigurinus is responsible for systemic infections in humans including infective endocarditis. We investigated whether the invasive trait of S. tigurinus in humans correlated with an increased ability to induce IE in rats. Rats with catheter-induced aortic vegetations were inoculated with 10⁴ CFU/ml of either of four S. tigurinus strains AZ_3a(T), AZ_4a, AZ_8 and AZ_14, isolated from patients with infective endocarditis or with the well known IE pathogen Streptococcus gordonii (Challis). Aortic infection was assessed after 24 h. S. tigurinus AZ_3a(T), AZ_4a and AZ_14 produced endocarditis in ≥80% of rats whereas S. gordonii produced endocarditis in only 33% of animals (P<0.05). S. tigurinus AZ_8 caused vegetation infection in 56% of the animals. The capacity of S. tigurinus to induce aortic infection was not related to their ability to bind extracellular matrix proteins (fibrinogen, fibronectin or collagen) or to trigger platelet aggregation. However, all S. tigurinus isolates showed an enhanced resistance to phagocytosis by macrophages and two of them had an increased ability to enter endothelial cells, key attributes of invasive streptococcal species.

A highly sensitive in vitro infection assay to explore early stages of mouse mammary tumor virus infection.

Mouse mammary tumor virus (MMTV) infection of adult mice induces a strong response to superantigen (Sag) in their draining lymph nodes, which results from the presentation of Sag by MMTV-infected B cells to Sag-reactive T cells. To date, infection with physiologically relevant doses of MMTV can be detected in vivo only after several days of Sag-mediated T-cell-dependent amplification of infected B cells. Furthermore, no efficient in vitro system of detecting MMTV infection is available. Such a model would allow the dissection of the early phase of infection, the assessment of the contributions of different cell types, and the screening of large panels of molecules for their potential roles in infection and Sag response. For these reasons, we have established an in vitro model for detecting infection which is as sensitive and reproducible as the in vivo model. We found that the viral envelope (Env) protein is crucial for target cell infection but not for presentation of Sag. Furthermore, we show that infection of purified B cells with MMTV induces entry of Sag-responsive T cells into the cell cycle, while other professional antigen-presenting cells, such as dendritic cells, are much less efficient in inducing a response.

Characteristics of highly frequent users of a Swiss academic emergency department: a retrospective consecutive case series.

OBJECTIVES: The aim of this study was to describe the demographic, social and medical characteristics, and healthcare use of highly frequent users of a university hospital emergency department (ED) in Switzerland. METHODS: A retrospective consecutive case series was performed. We included all highly frequent users, defined as patients attending the ED 12 times or more within a calendar year (1 January 2009 to 31 December 2009). We collected their characteristics and calculated a score of accumulation of risk factors of vulnerability. RESULTS: Highly frequent users comprised 0.1% of ED patients, and they accounted for 0.8% of all ED attendances (23 patients, 425 attendances). Of all highly frequent users, 87% had a primary care practitioner, 82.6% were unemployed, 73.9% were socially isolated, and 60.9% had a mental health or substance use primary diagnosis. One-third had attempted suicide during study period, all of them being women. They were often admitted (24.0% of attendances), and only 8.7% were uninsured. On average, they cumulated 3.3 different risk factors of vulnerability (SD 1.4). CONCLUSION: Highly frequent users of a Swiss academic ED are a highly vulnerable population. They are in poor health and accumulate several risk factors of being even in poorer health. The small number of patients and their high level of insurance coverage make it particularly feasible to design a specific intervention to approach their needs, in close collaboration with their primary care practitioner. Elaboration of the intervention should focus on social reinsertion and risk-reduction strategies with regard to substance use, hospital admissions and suicide.

Transcription factor PROX1 induces colon cancer progression by promoting the transition from benign to highly dysplastic phenotype.

The Drosophila transcription factor Prospero functions as a tumor suppressor, and it has been suggested that the human counterpart of Prospero, PROX1, acts similarly in human cancers. However, we show here that PROX1 promotes dysplasia in colonic adenomas and colorectal cancer progression. PROX1 expression marks the transition from benign colon adenoma to carcinoma in situ, and its loss inhibits growth of human colorectal tumor xenografts and intestinal adenomas in Apc(min/+) mice, while its transgenic overexpression promotes colorectal tumorigenesis. Furthermore, in intestinal tumors PROX1 is a direct and dose-dependent target of the beta-catenin/TCF signaling pathway, responsible for the neoplastic transformation. Our data underscore the complexity of cancer pathogenesis and implicate PROX1 in malignant tumor progression through the regulation of cell polarity and adhesion.

Amplification of 16S rRNA gene sequences to differentiate two highly related bradyrhizobia species

A 16S rRNA gene PCR-based assay was developed aiming at a fast molecular diagnostic method to differentiate the two phylogenetically closely related species Bradyrhizobium japonicum and B. elkanii, isolated from soybean nodules, in order to identify those more competitive and comprising greater nitrogen fixation ability for use in the formulation of commercial inoculants. The assay used was able to discriminate ten reference strains belonging to these two Bradyrhizobium species, as well as to efficiently identify 37 strains isolated from fields cultivated with soybean.

Selecting highly affine and well-expressed TCRs for gene therapy of melanoma.

A recent phase 1 trial has demonstrated that the generation of tumor-reactive T lymphocytes by transfer of specific T-cell receptor (TCR) genes into autologous lymphocytes is feasible. However, compared with results obtained by infusion of tumor-infiltrating lymphocytes, the response rate observed in this first TCR gene therapy trial is low. One strategy that is likely to enhance the success rate of TCR gene therapy is the use of tumor-reactive TCRs with a higher capacity for tumor cell recognition. We therefore sought to develop standardized procedures for the selection of well-expressed, high-affinity, and safe human TCRs. Here we show that TCR surface expression can be improved by modification of TCR alpha and beta sequences and that such improvement has a marked effect on the in vivo function of TCR gene-modified T cells. From a panel of human, melanoma-reactive TCRs we subsequently selected the TCR with the highest affinity. Furthermore, a generally applicable assay was used to assess the lack of alloreactivity of this TCR against a large series of common human leukocyte antigen alleles. The procedures described in this study should be of general value for the selection of well- and stably expressed, high-affinity, and safe human TCRs for subsequent clinical testing.

Zinc adsorption in highly weathered soils

The objective of this work was to assess the effects of pH and ionic strength upon zinc adsorption, in three highly weathered variable charge soils. Adsorption isotherms were elaborated from batch adsorption experiments, with increasing Zn concentrations (0-80 mg L-1), and adsorption envelopes were constructed through soil samples reactions with 0.01, 0.1 and 1 mol L-1 Ca(NO3)2 solutions containing 5 mg L-1 of Zn, with an increasing pH value from 3 to 8. Driving force of reaction was quantified by Gibbs free energy and separation factor. Isotherms were C-, H- and L-type and experimental results were fitted to nonlinear Langmuir model. Maximum adsorption ranged from 59-810 mg kg-1, and Zn affinity was greater in subsoil (0.13-0.81 L kg-1) than in the topsoil samples (0.01-0.34 L kg-1). Zinc adsorption was favorable and spontaneous, and showed sharply increase (20-90%) in the 4-6 pH range. No effect of ionic strength was observed at pH values below 5, because specific adsorption mechanisms predominated in the 3-5 pH range. Above pH 5, and in subsoil samples, Zn was adsorbed by electrostatic mechanisms, since ionic strength effect was observed. Despite depth and ionic strength effects, Zn adsorption depends mainly on the pH.

Anti-HLA antibody repertoire after IVIg infusion in highly sensitized patients waiting for kidney transplantation.

Polyclonal intravenous immunoglobulin (IVIg) treatment reduces crossmatch positivity and increases rates of transplantation in highly sensitised patients (HS). We quantified the panel reactive antibody (PRA) by microlymphocytotoxicity (MLCC), and we analysed anti-HLA class I and class II IgG specific antibody repertoire by Luminex before and after IVIg infusion alone in HS patients awaiting kidney transplantation. Five patients received three monthly infusions of 1 g/kg of IVIg. Serum samples collected pre and post IVIg treatment were submitted for PRA analysis by MLCC. Anti-class I and anti-class II antibody specificities were then tested by Luminex. We focused on the anti-HLA class I and class II antibodies directed against HLA expressed by a previous graft. We also analysed the anti-HLA antibody repertoire in three patients who had not received IVIg infusion. The PRA level determined by MLCC decreased significantly in one of the five patients, dropping from 40% to 17%. The Luminex assay showed fluctuations of the anti-HLA antibody levels over time, but no significant longterm modifications of the anti-HLA antibody repertoire were observed, even in the patient with a strong and prolonged reduction of the PRA determined by MLCC. Our results show that IVIg at 1 g/kg is not sufficient to reduce PRA and does not modify the repertoire of specific anti-HLA antibody determined by Luminex.