332 research outputs found
Expression of SCCmec cassette chromosome recombinases in methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis
Objectives Methicillin resistance in staphylococci is mediated by the mecA gene, which is carried on the staphylococcal cassette chromosome mec (SCCmec). SCCmec is responsible for vertical and horizontal transfer of methicillin resistance. Horizontal transfer implies first SCCmec excision from the chromosome. Site-specific excision is catalysed by the Ccr recombinases, which are encoded by ccrAB genes located on the cassette. The aim of this study is to determine the promoter activity of ccrAB genes in individual cells of methicillin-resistant Staphylococcus aureus (N315, COL and MW2) and Staphylococcus epidermidis (RP62A). One mutant cured of its SCCmec (N315EX) was also used. Exposure to various stresses was included in the study. Methods For each strain, translational promoter-green fluorescent protein (gfp) fusions were used to assess the levels of ccr promoter activity in individual cells. Analyses were performed using epifluorescence microscopy and flow cytometry. Results ccr promoter activity was observed in only a small percentage of cell populations. This ‘bistable' phenotype was strain dependent (GFP was expressed in N315 and RP62A, but not in COL and MW2) and growth dependent (GFP-expressing cells decreased from approximately 3% to 1% between logarithmic and stationary growth phases). The ccr promoter of strain N315 displayed normal promoter activity when expressed in SCCmec-negative N315EX. Likewise, the ccr promoter of strain COL (which was inactive in COL) showed normal N315-like activity when transformed into N315 and N315EX. Conclusions SCCmec excision operates through bistability, favouring a small fraction of cells to ‘sacrifice' their genomic islands for transfer, while the rest of the population remains intact. Determinants responsible for the activity of the ccr promoter were not located on SCCmec, but were elsewhere on the genome. Thus, the staphylococcal chromosome plays a key role in determining SCCmec stability and transferabilit
Excision of staphylococcal cassette chromosome mec in methicillin-resistant Staphylococcus aureus assessed by quantitative PCR.
BACKGROUND: Methicillin-resistance in staphylococci is conferred by the mecA gene, located on the genomic island Staphylococcal Cassette Chromosome mec (SCCmec). SCCmec mobility relies on the Ccr recombinases, which catalyze insertion and excision form the host's chromosome. Although being a crucial step in its horizontal transfer, little is known about the dynamics of SCCmec excision.
RESULTS: A quantitative PCR-based method was used to measure the rate of SCCmec excision by amplifying the chromosome-chromosome junction and the circularized SCCmec resulting from excision. SCCmec excision rate was measured in methicillin-resistant Staphylococcus aureus (MRSA) strain N315 at various growth times in broth cultures. In the present experimental settings, excision of SCCmec occurred at a rate of approximately 2 × 10(-6) in MRSA N315.
CONCLUSION: This work brings new insights in the poorly understood SCCmec excision process. The results presented herein suggest a model in which excision occurs during a limited period of time at the early stages of growth
Hausdorff dimension of three-period orbits in Birkhoff billiards
We prove that the Hausdorff dimension of the set of three-period orbits in
classical billiards is at most one. Moreover, if the set of three-period orbits
has Hausdorff dimension one, then it has a tangent line at almost every point.Comment: 10 pages, 1 figur
Clinical and functional characterisation of a novel TNFRSF1A c.605T > A/V173D cleavage site mutation associated with tumour necrosis factor receptor-associated periodic fever syndrome (TRAPS), cardiovascular complications and excellent response to etanercept treatment.
Objectives: To study the clinical outcome, treatment
response, T-cell subsets and functional consequences of a
novel tumour necrosis factor (TNF) receptor type 1
(TNFRSF1A) mutation affecting the receptor
cleavage site.
Methods: Patients with symptoms suggestive of tumour
necrosis factor receptor-associated periodic syndrome
(TRAPS) and 22 healthy controls (HC) were screened for
mutations in the TNFRSF1A gene. Soluble TNFRSF1A and
inflammatory cytokines were measured by ELISAs.
TNFRSF1A shedding was examined by stimulation of
peripheral blood mononuclear cells (PBMCs) with phorbol
12-myristate 13-acetate followed by flow cytometric
analysis (FACS). Apoptosis of PBMCs was studied by
stimulation with TNFa in the presence of cycloheximide
and annexin V staining. T cell phenotypes were monitored
by FACS.
Results: TNFRSF1A sequencing disclosed a novel V173D/
p.Val202Asp substitution encoded by exon 6 in one
family, the c.194–14G.A splice variant in another and
the R92Q/p.Arg121Gln substitution in two families.
Cardiovascular complications (lethal heart attack and
peripheral arterial thrombosis) developed in two V173D
patients. Subsequent etanercept treatment of the V173D
carriers was highly effective over an 18-month follow-up
period. Serum TNFRSF1A levels did not differ between
TRAPS patients and HC, while TNFRSF1A cleavage from
monocytes was significantly reduced in V173D and R92Q
patients. TNFa-induced apoptosis of PBMCs and T-cell
senescence were comparable between V173D patients
and HC.
Conclusions: The TNFRSF1A V173D cleavage site
mutation may be associated with an increased risk for
cardiovascular complications and shows a strong
response to etanercept. T-cell senescence does not seem
to have a pathogenetic role in affected patients
Predicting the outcome of heart failure against chronic-ischemic heart disease in elderly population – Machine learning approach based on logistic regression, case to Villa Scassi hospital Genoa, Italy
Totally 167 patients were admitted at cardiology ward in Villa Scassi hospital, Genoa, Italy. We worked with two control groups: heart failure 59 patients (mean age: 71.37 ± 13.27 years) and chronic-ischemic heart disease 108 patients (mean age: 68.85 ± 11.3 years). Nine parameters: Hb, Serum Creatinine, LDL, HDL, Triglycerides, ALT, AST, hs-cTnI, CRP were evaluated onset to hospitalization. We aimed to identify significant independent predictors relative to the outcome of heart failure versus chronic-ischemic heart disease and select combination of biochemical parameters in logistic regression-based model that would provide on average excellent discrimination to the outcome of heart failure versus chronic-ischemic heart
disease in elderly population. Applying 20-fold repeated stratified cross-validation, 4:1 train/test ratio split, we have found that probability of heart failure, provides best discrimination of the outcome of heart failure against chronic-ischemic heart disease
Development and evaluation of double locus sequence typing for molecular epidemiological investigations of Clostridium difficile.
Despite the development of novel typing methods based on whole genome sequencing, most laboratories still rely on classical molecular methods for outbreak investigation or surveillance. Reference methods for Clostridium difficile include ribotyping and pulsed-field gel electrophoresis, which are band-comparing methods often difficult to establish and which require reference strain collections. Here, we present the double locus sequence typing (DLST) scheme as a tool to analyse C. difficile isolates. Using a collection of clinical C. difficile isolates recovered during a 1-year period, we evaluated the performance of DLST and compared the results to multilocus sequence typing (MLST), a sequence-based method that has been used to study the structure of bacterial populations and highlight major clones. DLST had a higher discriminatory power compared to MLST (Simpson's index of diversity of 0.979 versus 0.965) and successfully identified all isolates of the study (100 % typeability). Previous studies showed that the discriminatory power of ribotyping was comparable to that of MLST; thus, DLST might be more discriminatory than ribotyping. DLST is easy to establish and provides several advantages, including absence of DNA extraction [polymerase chain reaction (PCR) is performed on colonies], no specific instrumentation, low cost and unambiguous definition of types. Moreover, the implementation of a DLST typing scheme on an Internet database, such as that previously done for Staphylococcus aureus and Pseudomonas aeruginosa ( http://www.dlst.org ), will allow users to easily obtain the DLST type by submitting directly sequencing files and will avoid problems associated with multiple databases
Nano-Motion Analysis for Rapid and Label Free Assessing of Cancer Cell Sensitivity to Chemotherapeutics.
Background and Objectives: Optimization of chemotherapy is crucial for cancer patients. Timely and costly efficient treatments are emerging due to the increasing incidence of cancer worldwide. Here, we present a methodology of nano-motion analysis that could be developed to serve as a screening tool able to determine the best chemotherapy option for a particular patient within hours. Materials and Methods: Three different human cancer cell lines and their multidrug resistant (MDR) counterparts were analyzed with an atomic force microscope (AFM) using tipless cantilevers to adhere the cells and monitor their nano-motions. Results: The cells exposed to doxorubicin (DOX) differentially responded due to their sensitivity to this chemotherapeutic. The death of sensitive cells corresponding to the drop in signal variance occurred in less than 2 h after DOX application, while MDR cells continued to move, even showing an increase in signal variance. Conclusions: Nano-motion sensing can be developed as a screening tool that will allow simple, inexpensive and quick testing of different chemotherapeutics for each cancer patient. Further investigations on patient-derived tumor cells should confirm the method's applicability
Correction: Absence of Zika virus among pregnant women in Vietnam in 2008.
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