Genetic Organisation, Mobility and Predicted Functions of Genes on Integrated, Mobile Genetic Elements in Sequenced Strains of Clostridium difficile

Background: Clostridium difficile is the leading cause of hospital-associated diarrhoea in the US and Europe. Recently the incidence of C. difficile-associated disease has risen dramatically and concomitantly with the emergence of 'hypervirulent' strains associated with more severe disease and increased mortality. C. difficile contains numerous mobile genetic elements, resulting in the potential for a highly plastic genome. In the first sequenced strain, 630, there is one proven conjugative transposon (CTn), Tn5397, and six putative CTns (CTn1, CTn2 and CTn4-7), of which, CTn4 and CTn5 were capable of excision. In the second sequenced strain, R20291, two further CTns were described.Results: CTn1, CTn2 CTn4, CTn5 and CTn7 were shown to excise from the genome of strain 630 and transfer to strain CD37. A putative CTn from R20291, misleadingly termed a phage island previously, was shown to excise and to contain three putative mobilisable transposons, one of which was capable of excision. In silico probing of C. difficile genome sequences with recombinase gene fragments identified new putative conjugative and mobilisable transposons related to the elements in strains 630 and R20291. CTn5-like elements were described occupying different insertion sites in different strains, CTn1-like elements that have lost the ability to excise in some ribotype 027 strains were described and one strain was shown to contain CTn5-like and CTn7-like elements arranged in tandem. Additionally, using bioinformatics, we updated previous gene annotations and predicted novel functions for the accessory gene products on these new elements.Conclusions: The genomes of the C. difficile strains examined contain highly related CTns suggesting recent horizontal gene transfer. Several elements were capable of excision and conjugative transfer. The presence of antibiotic resistance genes and genes predicted to promote adaptation to the intestinal environment suggests that CTns play a role in the interaction of C. difficile with its human host

Inactivation of the dnaK gene in Clostridium difficile 630 Δerm yields a temperature-sensitive phenotype and increases biofilm-forming ability

Abstract Clostridium difficile infection is a growing problem in healthcare settings worldwide and results in a considerable socioeconomic impact. New hypervirulent strains and acquisition of antibiotic resistance exacerbates pathogenesis; however, the survival strategy of C. difficile in the challenging gut environment still remains incompletely understood. We previously reported that clinically relevant heat-stress (37–41 °C) resulted in a classical heat-stress response with up-regulation of cellular chaperones. We used ClosTron to construct an insertional mutation in the dnaK gene of C. difficile 630 Δerm. The dnaK mutant exhibited temperature sensitivity, grew more slowly than C. difficile 630 Δerm and was less thermotolerant. Furthermore, the mutant was non-motile, had 4-fold lower expression of the fliC gene and lacked flagella on the cell surface. Mutant cells were some 50% longer than parental strain cells, and at optimal growth temperatures, they exhibited a 4-fold increase in the expression of class I chaperone genes including GroEL and GroES. Increased chaperone expression, in addition to the non-flagellated phenotype of the mutant, may account for the increased biofilm formation observed. Overall, the phenotype resulting from dnaK disruption is more akin to that observed in Escherichia coli dnaK mutants, rather than those in the Gram-positive model organism Bacillus subtilis

MR fluoroscopy in vascular and cardiac interventions (review)

Vascular and cardiac disease remains a leading cause of morbidity and mortality in developed and emerging countries. Vascular and cardiac interventions require extensive fluoroscopic guidance to navigate endovascular catheters. X-ray fluoroscopy is considered the current modality for real time imaging. It provides excellent spatial and temporal resolution, but is limited by exposure of patients and staff to ionizing radiation, poor soft tissue characterization and lack of quantitative physiologic information. MR fluoroscopy has been introduced with substantial progress during the last decade. Clinical and experimental studies performed under MR fluoroscopy have indicated the suitability of this modality for: delivery of ASD closure, aortic valves, and endovascular stents (aortic, carotid, iliac, renal arteries, inferior vena cava). It aids in performing ablation, creation of hepatic shunts and local delivery of therapies. Development of more MR compatible equipment and devices will widen the applications of MR-guided procedures. At post-intervention, MR imaging aids in assessing the efficacy of therapies, success of interventions. It also provides information on vascular flow and cardiac morphology, function, perfusion and viability. MR fluoroscopy has the potential to form the basis for minimally invasive image–guided surgeries that offer improved patient management and cost effectiveness

The Clostridium difficile Cell Wall Protein CwpV is Antigenically Variable between Strains, but Exhibits Conserved Aggregation-Promoting Function

Clostridium difficile is the main cause of antibiotic-associated diarrhea, leading to significant morbidity and mortality and putting considerable economic pressure on healthcare systems. Current knowledge of the molecular basis of pathogenesis is limited primarily to the activities and regulation of two major toxins. In contrast, little is known of mechanisms used in colonization of the enteric system. C. difficile expresses a proteinaceous array on its cell surface known as the S-layer, consisting primarily of the major S-layer protein SlpA and a family of SlpA homologues, the cell wall protein (CWP) family. CwpV is the largest member of this family and is expressed in a phase variable manner. Here we show CwpV promotes C. difficile aggregation, mediated by the C-terminal repetitive domain. This domain varies markedly between strains; five distinct repeat types were identified and were shown to be antigenically distinct. Other aspects of CwpV are, however, conserved. All CwpV types are expressed in a phase variable manner. Using targeted gene knock-out, we show that a single site-specific recombinase RecV is required for CwpV phase variation. CwpV is post-translationally cleaved at a conserved site leading to formation of a complex of cleavage products. The highly conserved N-terminus anchors the CwpV complex to the cell surface. Therefore CwpV function, regulation and processing are highly conserved across C. difficile strains, whilst the functional domain exists in at least five antigenically distinct forms. This hints at a complex evolutionary history for CwpV

Towards real-time cardiovascular magnetic resonance guided transarterial CoreValve implantation: in vivo evaluation in swine

<p>Abstract</p> <p>Background</p> <p>Real-time cardiovascular magnetic resonance (rtCMR) is considered attractive for guiding TAVI. Owing to an unlimited scan plane orientation and an unsurpassed soft-tissue contrast with simultaneous device visualization, rtCMR is presumed to allow safe device navigation and to offer optimal orientation for precise axial positioning. We sought to evaluate the preclinical feasibility of rtCMR-guided transarterial aortic valve implatation (TAVI) using the nitinol-based Medtronic CoreValve bioprosthesis.</p> <p>Methods</p> <p>rtCMR-guided transfemoral (n = 2) and transsubclavian (n = 6) TAVI was performed in 8 swine using the original CoreValve prosthesis and a modified, CMR-compatible delivery catheter without ferromagnetic components.</p> <p>Results</p> <p>rtCMR using TrueFISP sequences provided reliable imaging guidance during TAVI, which was successful in 6 swine. One transfemoral attempt failed due to unsuccessful aortic arch passage and one pericardial tamponade with subsequent death occurred as a result of ventricular perforation by the device tip due to an operating error, this complication being detected without delay by rtCMR. rtCMR allowed for a detailed, simultaneous visualization of the delivery system with the mounted stent-valve and the surrounding anatomy, resulting in improved visualization during navigation through the vasculature, passage of the aortic valve, and during placement and deployment of the stent-valve. Post-interventional success could be confirmed using ECG-triggered time-resolved cine-TrueFISP and flow-sensitive phase-contrast sequences. Intended valve position was confirmed by ex-vivo histology.</p> <p>Conclusions</p> <p>Our study shows that rtCMR-guided TAVI using the commercial CoreValve prosthesis in conjunction with a modified delivery system is feasible in swine, allowing improved procedural guidance including immediate detection of complications and direct functional assessment with reduction of radiation and omission of contrast media.</p

Whole shaft visibility and mechanical performance for active MR catheters using copper-nitinol braided polymer tubes

<p>Abstract</p> <p>Background</p> <p>Catheter visualization and tracking remains a challenge in interventional MR.</p> <p>Active guidewires can be made conspicuous in "profile" along their whole shaft exploiting metallic core wire and hypotube components that are intrinsic to their mechanical performance. Polymer-based catheters, on the other hand, offer no conductive medium to carry radio frequency waves. We developed a new "active" catheter design for interventional MR with mechanical performance resembling braided X-ray devices. Our 75 cm long hybrid catheter shaft incorporates a wire lattice in a polymer matrix, and contains three distal loop coils in a flexible and torquable 7Fr device. We explored the impact of braid material designs on radiofrequency and mechanical performance.</p> <p>Results</p> <p>The incorporation of copper wire into in a superelastic nitinol braided loopless antenna allowed good visualization of the whole shaft (70 cm) <it>in vitro </it>and <it>in vivo </it>in swine during real-time MR with 1.5 T scanner. Additional distal tip coils enhanced tip visibility. Increasing the copper:nitinol ratio in braiding configurations improved flexibility at the expense of torquability. We found a 16-wire braid of 1:1 copper:nitinol to have the optimum balance of mechanical (trackability, flexibility, torquability) and antenna (signal attenuation) properties. With this configuration, the temperature increase remained less than 2°C during real-time MR within 10 cm horizontal from the isocenter. The design was conspicuous <it>in vitro </it>and <it>in vivo</it>.</p> <p>Conclusion</p> <p>We have engineered a new loopless antenna configuration that imparts interventional MR catheters with satisfactory mechanical and imaging characteristics. This compact loopless antenna design can be generalized to visualize the whole shaft of any general-purpose polymer catheter to perform safe interventional procedures.</p

Mutator dynamics in sexual and asexual experimental populations of yeast

<p>Abstract</p> <p>Background</p> <p>In asexual populations, mutators may be expected to hitchhike with associated beneficial mutations. In sexual populations, recombination is predicted to erode such associations, inhibiting mutator hitchhiking. To investigate the effect of recombination on mutators experimentally, we compared the frequency dynamics of a mutator allele (<it>msh2</it>Δ) in sexual and asexual populations of <it>Saccharomyces cerevisiae</it>.</p> <p>Results</p> <p>Mutator strains increased in frequency at the expense of wild-type strains in all asexual diploid populations, with some approaching fixation in 150 generations of propagation. Over the same period of time, mutators declined toward loss in all corresponding sexual diploid populations as well as in haploid populations propagated asexually.</p> <p>Conclusions</p> <p>We report the first experimental investigation of mutator dynamics in sexual populations. We show that a strong mutator quickly declines in sexual populations while hitchhiking to high frequency in asexual diploid populations, as predicted by theory. We also show that the <it>msh2Δ </it>mutator has a high and immediate realized cost that is alone sufficient to explain its decline in sexual populations. We postulate that this cost is indirect; namely, that it is due to a very high rate of recessive lethal or strongly deleterious mutation. However, we cannot rule out the possibility that <it>msh2</it>Δ also has unknown directly deleterious effects on fitness, and that these effects may differ between haploid asexual and sexual populations. Despite these reservations, our results prompt us to speculate that the short-term cost of highly deleterious recessive mutations can be as important as recombination in preventing mutator hitchhiking in sexual populations.</p

Clinical impact of a targeted next-generation sequencing gene panel for autoinflammation and vasculitis.

BACKGROUND: Monogenic autoinflammatory diseases (AID) are a rapidly expanding group of genetically diverse but phenotypically overlapping systemic inflammatory disorders associated with dysregulated innate immunity. They cause significant morbidity, mortality and economic burden. Here, we aimed to develop and evaluate the clinical impact of a NGS targeted gene panel, the "Vasculitis and Inflammation Panel" (VIP) for AID and vasculitis. METHODS: The Agilent SureDesign tool was used to design 2 versions of VIP; VIP1 targeting 113 genes, and a later version, VIP2, targeting 166 genes. Captured and indexed libraries (QXT Target Enrichment System) prepared for 72 patients were sequenced as a multiplex of 16 samples on an Illumina MiSeq sequencer in 150bp paired-end mode. The cohort comprised 22 positive control DNA samples from patients with previously validated mutations in a variety of the genes; and 50 prospective samples from patients with suspected AID in whom previous Sanger based genetic screening had been non-diagnostic. RESULTS: VIP was sensitive and specific at detecting all the different types of known mutations in 22 positive controls, including gene deletion, small INDELS, and somatic mosaicism with allele fraction as low as 3%. Six/50 patients (12%) with unclassified AID had at least one class 5 (clearly pathogenic) variant; and 11/50 (22%) had at least one likely pathogenic variant (class 4). Overall, testing with VIP resulted in a firm or strongly suspected molecular diagnosis in 16/50 patients (32%). CONCLUSIONS: The high diagnostic yield and accuracy of this comprehensive targeted gene panel validate the use of broad NGS-based testing for patients with suspected AID

Repaired tetralogy of Fallot: the roles of cardiovascular magnetic resonance in evaluating pathophysiology and for pulmonary valve replacement decision support

Surgical management of tetralogy of Fallot (TOF) results in anatomic and functional abnormalities in the majority of patients. Although right ventricular volume load due to severe pulmonary regurgitation can be tolerated for many years, there is now evidence that the compensatory mechanisms of the right ventricular myocardium ultimately fail and that if the volume load is not eliminated or reduced by pulmonary valve replacement the dysfunction might be irreversible. Cardiovascular magnetic resonance (CMR) has evolved during the last 2 decades as the reference standard imaging modality to assess the anatomic and functional sequelae in patients with repaired TOF. This article reviews the pathophysiology of chronic right ventricular volume load after TOF repair and the risks and benefits of pulmonary valve replacement. The CMR techniques used to comprehensively evaluate the patient with repaired TOF are reviewed and the role of CMR in supporting clinical decisions regarding pulmonary valve replacement is discussed

Semiquantitative Analysis of Clinical Heat Stress in Clostridium difficile Strain 630 Using a GeLC/MS Workflow with emPAI Quantitation.

<div><p><i>Clostridium difficile</i> is considered to be the most frequent cause of infectious bacterial diarrhoea in hospitals worldwide yet its adaptive ability remains relatively uncharacterised. Here, we used GeLC/MS and the exponentially modified protein abundance index (emPAI) calculation to determine proteomic changes in response to a clinically relevant heat stress. Reproducibility between both biological and technical replicates was good, and a 37°C proteome of 224 proteins was complemented by a 41°C proteome of 202 proteins at a 1% false discovery rate. Overall, 236 <i>C. difficile</i> proteins were identified and functionally categorised, of which 178 were available for comparative purposes. A total of 65 proteins (37%) were modulated by 1.5-fold or more at 41°C compared to 37°C and we noted changes in the majority of proteins associated with amino acid metabolism, including upregulation of the reductive branch of the leucine fermentation pathway. Motility was reduced at 41°C as evidenced by a 2.7 fold decrease in the flagellar filament protein, FliC, and a global increase in proteins associated with detoxification and adaptation to atypical conditions was observed, concomitant with decreases in proteins mediating transcriptional elongation and the initiation of protein synthesis. Trigger factor was down regulated by almost 5-fold. We propose that under heat stress, titration of the GroESL and dnaJK/grpE chaperones by misfolded proteins will, in the absence of trigger factor, prevent nascent chains from emerging efficiently from the ribosome causing translational stalling and also an increase in secretion. The current work has thus allowed development of a heat stress model for the key cellular processes of protein folding and export.</p></div