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BackgroundCytomegalovirus (CMV) infection, either de novo or as reactivation after allotransplantation and chronic immunosuppression, is recognized to cause detrimental alloimmune effects, inclusive of higher susceptibility to graft rejection and substantive impact on chronic graft injury and reduced transplant survival. To obtain further insights into the evolution and pathogenesis of CMV infection in an immunocompromised host we evaluated changes in the circulating host proteome serially, before and after transplantation, and during and after CMV DNA replication (DNAemia), as measured by quantitative polymerase chain reaction (QPCR).MethodsLC-MS-based proteomics was conducted on 168 serially banked plasma samples, from 62 propensity score-matched kidney transplant recipients. Patients were stratified by CMV replication status into 31 with CMV DNAemia and 31 without CMV DNAemia. Patients had blood samples drawn at protocol times of 3- and 12-months post-transplant. Additionally, blood samples were also drawn before and 1 week and 1 month after detection of CMV DNAemia. Plasma proteins were analyzed using an LCMS 8060 triple quadrupole mass spectrometer. Further, public transcriptomic data on time matched PBMCs samples from the same patients was utilized to evaluate integrative pathways. Data analysis was conducted using R and Limma.ResultsSamples were segregated based on their proteomic profiles with respect to their CMV Dnaemia status. A subset of 17 plasma proteins was observed to predict the onset of CMV at 3 months post-transplant enriching platelet degranulation (FDR, 4.83E-06), acute inflammatory response (FDR, 0.0018), blood coagulation (FDR, 0.0018) pathways. An increase in many immune complex proteins were observed at CMV infection. Prior to DNAemia the plasma proteome showed changes in the anti-inflammatory adipokine vaspin (SERPINA12), copper binding protein ceruloplasmin (CP), complement activation (FDR = 0.03), and proteins enriched in the humoral (FDR = 0.01) and innate immune responses (FDR = 0.01).ConclusionPlasma proteomic and transcriptional perturbations impacting humoral and innate immune pathways are observed during CMV infection and provide biomarkers for CMV disease prediction and resolution. Further studies to understand the clinical impact of these pathways can help in the formulation of different types and duration of anti-viral therapies for the management of CMV infection in the immunocompromised host.

Next generation sequencing has demonstrated that alpha diversity of the fecal microbiota is significantly altered in horses with typhlocolitis. The objective of this study was to evaluate the bacterial composition of the cecum content of horses with and without typhlocolitis through direct and culture-enriched 16S gene sequencing of six healthy horses and six horses with acute typhlocolitis; a case-control study design. Cecal content was collected after euthanasia. An aliquot was used for direct 16S gene sequencing. Another was serially diluted with brain heart infusion (BHI) and plated onto five different agar media. All culture medias, except for MacConkey, were incubated anaerobically. Bacterial colonies were harvested in bulk and used for DNA extraction, 16S PCR amplification, and sequenced using the Illumina MiSeq platform. Predominant phyla in healthy and diseased horses were Firmicutes, followed by Bacteroidetes in all cultured medias, except for MacConkey agar, in which Proteobacteria was the dominant phylum. Greater bacterial richness was identified in sequenced cecal contents as compared to cultured plates (P < 0.05). Culture-enriched molecular profiling combined with 16S rRNA gene sequencing offer an alternative method for the study of the gut microbiota of horses. For direct cecum content 16S gene amplification, the alpha diversity indices were lower in diarrheic horses compared to healthy horses (P < 0.05). A higher relative abundance of Fusobacteriota was found in 2/6 samples from diarrheic horses. The role of Fusobacteriota in equine colitis deserves investigation.

Objectives Compare lay expectations of medical development to those of experts in the context of SARS-CoV-2 vaccine development. Methods A short online survey of experts and lay people measuring when participants believe important vaccine milestones would occur and how likely potential setbacks were. Samples of US and Canadian lay people recruited through Qualtrics. The expert sample was created through a contact network in vaccine development and supplemented with corresponding authors of recent scholarly review articles on vaccine development. Results In aggregate, lay people gave responses that were within 3 months of experts, tending to be later than experts for early milestones and earlier for later milestones. Median lay best estimates for when a vaccine would be available to the public were 08/2021 and 09/2021 for the US and Canadian samples, compared with 09-10/2021 for the experts. However, many individual lay responses showed more substantial disagreement with expert opinions, with 54% of lay best estimates of when a vaccine would be available to the public being before the median expert soonest estimate or after the median expert latest estimate. Lay people were much more pessimistic about vaccine development encountering setbacks than experts (median probability 59% of boxed warning compared with only 30% for experts). Misalignment between layperson and expert expectations was not explained by any demographic variables collected in our survey. Conclusion Median lay expectations were generally similar to experts. At the individual level, however, lay people showed substantial variation with many believing milestones would occur much sooner than experts. Lay people were in general much more pessimistic about the prospect of setbacks than were experts.

Conserved IncI1 and IncHI1 plasmids carrying blaCTX-M-1 have been found circulating in chickens and horses from continental Europe, respectively. In Canada, blaCTX-M-1 is overwhelmingly the most common blaCTX-M variant found in Escherichia coli from chicken and horses and can be recovered at lower frequencies in swine, cattle, and dogs. Whole-genome sequencing has identified a large genetic diversity of isolates carrying this variant, warranting further investigations into the plasmids carrying this gene. Therefore, the objective of this study was to describe the genetic profiles of blaCTX-M-1 plasmids circulating in E. coli from Canadian domestic animals and compare them to those recovered in animals in Europe. Fifty-one blaCTX-M-1 positive E. coli isolates from chicken (n = 14), horses (racetrack horses n = 11; community horses n = 3), swine (n = 7), turkey (n = 6), dogs (n = 5), beef cattle (n = 3), and dairy cattle (n = 2) were selected for plasmid characterization. Sequences were obtained through both Illumina and Oxford Nanopore technologies. Genomes were assembled using either Unicycler hybrid assembly or Flye with polishing performed using Pilon. blaCTX-M-1 was found residing on a plasmid in 45 isolates and chromosomally located in six isolates. A conserved IncI1/ST3 plasmid was identified among chicken (n = 12), turkey (n = 4), swine (n = 6), dog (n = 2), and beef cattle (n = 2) isolates. When compared against publicly available data, these plasmids showed a high degree of similarity to those identified in isolates from poultry and swine in Europe. These results suggest that an epidemic IncI1/ST3 plasmid similar to the one found in Europe is contributing to the spread of blaCTX-M-1 in Canada. A conserved IncHI1/FIA(HI1)/ST2 plasmid was also recovered from nearly all racetrack horse isolates (n = 10). Although IncHI1/ST2 plasmids have been reported among European horse isolates, IncHI1/ST9 plasmids appear to be more widespread. Further studies are necessary to understand the factors contributing to these plasmids’ success in their respective populations.

INTRODUCTION:In recent years, numerous methods have been proposed to predict tissue outcome in acute stroke patients using machine learning methods incorporating multiparametric imaging data. Most methods include diffusion and perfusion parameters as image-based parameters but do not include any spatial information although these parameters are spatially dependent, e.g. different perfusion properties in white and gray brain matter. This study aims to investigate if including spatial features improves the accuracy of multi-parametric tissue outcome prediction. MATERIALS AND METHODS:Acute and follow-up multi-center MRI datasets of 99 patients were available for this study. Logistic regression, random forest, and XGBoost machine learning models were trained and tested using acute MR diffusion and perfusion features and known follow-up lesions. Different combinations of atlas coordinates and lesion probability maps were included as spatial information. The stroke lesion predictions were compared to the true tissue outcomes using the area under the receiver operating characteristic curve (ROC AUC) and the Dice metric. RESULTS:The statistical analysis revealed that including spatial features significantly improves the tissue outcome prediction. Overall, the XGBoost and random forest models performed best in every setting and achieved state-of-the-art results regarding both metrics with similar improvements achieved including Montreal Neurological Institute (MNI) reference space coordinates or voxel-wise lesion probabilities. CONCLUSION:Spatial features should be integrated to improve lesion outcome prediction using machine learning models.

[This corrects the article DOI: 10.1371/journal.pone.0228113.].

The goal of this study was to determine the frequency of resistance to extended-spectrum cephalosporins (ESCs) in Escherichia coli and other Enterobacterales from turkeys in Canada and characterize the associated resistance determinants. Pooled fecal samples were collected in 77 turkey farms across British Columbia, Québec, and Ontario. Isolates were obtained with and without selective enrichment cultures and compared to isolates from diagnostic submissions of suspected colibacillosis cases in Ontario. Isolates were identified using MALDI-TOF and susceptibility to ESCs was assessed by disk diffusion. The presence of blaCMY, blaCTX-M, blaTEM, and blaSHV was tested by PCR. Transformation experiments were used to characterize blaCMY plasmids. Genome sequencing with short and long reads was performed on a representative sample of blaCTX-M-positive isolates to assess isolates relatedness and characterize blaCTX-M plasmids. For the positive enrichment cultures (67% of total samples), 93% (587/610) were identified as E. coli, with only a few other Enterobacterales species identified. The frequency of ESC resistance was low in E. coli isolates from diagnostic submission (4%) and fecal samples without selective enrichment (5%). Of the ESC-resistant Enterobacterales isolates from selective enrichments, 71%, 18%, 14%, and 8% were positive for blaCMY, blaTEM, blaCTX-M, and blaSHV, respectively. IncI1 followed by IncK were the main incompatibility groups identified for blaCMY plasmids. The blaCTX-M-1 gene was found repeatedly on IncI1 plasmids of the pMLST type 3, while blaCTX-M-15, blaCTX-M-55, and blaCTX-M-65 were associated with a variety of IncF plasmids. Clonal spread of strains carrying blaCTX-M genes between turkey farms was observed, as well as the presence of an epidemic blaCTX-M-1 plasmid in unrelated E. coli strains. In conclusion, Enterobacterales resistant to ESCs were still widespread at low concentration in turkey feces two years after the cessation of ceftiofur use. Although blaCMY-2 is the main ESC resistance determinant in E. coli from Canadian turkeys, blaCTX-M genes also occur which are often carried by multidrug resistance plasmids. Both clonal spread and horizontal gene transfer are involved in parallel in the spread of blaCTX-M genes in Enterobacterales from Canadian turkeys.

BackgroundAn accurate prediction of tissue outcome in acute ischemic stroke patients is of high interest for treatment decision making. To date, various machine learning models have been proposed that combine multi-parametric imaging data for this purpose. However, most of these machine learning models were trained using voxel information extracted from the whole brain, without taking differences in susceptibility to ischemia into account that exist between brain regions. The aim of this study was to develop and evaluate a local tissue outcome prediction approach, which makes predictions using locally trained machine learning models and thus accounts for regional differences.Material and methodsMulti-parametric MRI data from 99 acute ischemic stroke patients were used for the development and evaluation of the local tissue outcome prediction approach. Diffusion (ADC) and perfusion parameter maps (CBF, CBV, MTT, Tmax) and corresponding follow-up lesion masks for each patient were registered to the MNI brain atlas. Logistic regression (LR) and random forest (RF) models were trained employing a local approach, which makes predictions using models individually trained for each specific voxel position using the corresponding local data. A global approach, which uses a single model trained using all voxels of the brain, was used for comparison. Tissue outcome predictions resulting from the global and local RF and LR models, as well as a combined (hybrid) approach were quantitatively evaluated and compared using the area under the receiver operating characteristic curve (ROC AUC), the Dice coefficient, and the sensitivity and specificity metrics.ResultsStatistical analysis revealed the highest ROC AUC and Dice values for the hybrid approach. With 0.872 (ROC AUC; LR) and 0.353 (Dice; RF), these values were significantly higher (p < 0.01) than the values of the two other approaches. In addition, the local approach achieved the highest sensitivity of 0.448 (LR). Overall, the hybrid approach was only outperformed in sensitivity (LR) by the local approach and in specificity by both other approaches. However, in these cases the effect sizes were comparatively small.ConclusionThe results of this study suggest that using locally trained machine learning models can lead to better lesion outcome prediction results compared to a single global machine learning model trained using all voxel information independent of the location in the brain.

Worldwide, testing capacity for SARS-CoV-2 is limited and bottlenecks in the scale up of polymerase chain reaction (PCR-based testing exist. Our aim was to develop and evaluate a machine learning algorithm to diagnose COVID-19 in the inpatient setting. The algorithm was based on basic demographic and laboratory features to serve as a screening tool at hospitals where testing is scarce or unavailable. We used retrospectively collected data from the UCLA Health System in Los Angeles, California. We included all emergency room or inpatient cases receiving SARS-CoV-2 PCR testing who also had a set of ancillary laboratory features (n = 1,455) between 1 March 2020 and 24 May 2020. We tested seven machine learning models and used a combination of those models for the final diagnostic classification. In the test set (n = 392), our combined model had an area under the receiver operator curve of 0.91 (95% confidence interval 0.87-0.96). The model achieved a sensitivity of 0.93 (95% CI 0.85-0.98), specificity of 0.64 (95% CI 0.58-0.69). We found that our machine learning algorithm had excellent diagnostic metrics compared to SARS-CoV-2 PCR. This ensemble machine learning algorithm to diagnose COVID-19 has the potential to be used as a screening tool in hospital settings where PCR testing is scarce or unavailable.

ImportanceCertain individuals, when infected by SARS-CoV-2, tend to develop the more severe forms of Covid-19 illness for reasons that remain unclear.ObjectiveTo determine the demographic and clinical characteristics associated with increased severity of Covid-19 infection.DesignRetrospective observational study. We curated data from the electronic health record, and used multivariable logistic regression to examine the association of pre-existing traits with a Covid-19 illness severity defined by level of required care: need for hospital admission, need for intensive care, and need for intubation.SettingA large, multihospital healthcare system in Southern California.ParticipantsAll patients with confirmed Covid-19 infection (N = 442).ResultsOf all patients studied, 48% required hospitalization, 17% required intensive care, and 12% required intubation. In multivariable-adjusted analyses, patients requiring a higher levels of care were more likely to be older (OR 1.5 per 10 years, PConclusions and relevanceIn our healthcare system, greater Covid-19 illness severity is seen in patients who are older, male, African American, obese, with diabetes, and with greater overall comorbidity burden. Certain comorbidities paradoxically augment risk to a greater extent in younger patients. In hospitalized patients, male sex is the main determinant of needing more intensive care. Further investigation is needed to understand the mechanisms underlying these findings.

Antimicrobial resistance in Campylobacter, common in poultry, is a global public health issue. The emergence and spread of antimicrobial resistant Campylobacter has been linked to the use of antimicrobials in food animals. Small poultry flocks are becoming increasingly popular not only as a source of food but also as pets, yet not all small flock owners are aware of proper antimicrobial use practices and safe food handling protocols. This trend could contribute to antimicrobial resistance. In order to determine the prevalence of antimicrobial resistance in Campylobacter in small poultry flocks, we analyzed data from birds that had been submitted to a diagnostic laboratory in Ontario between October 2015 and September 2017. A pooled cecal sample was obtained from each submission and cultured for Campylobacter jejuni and Campylobacter coli. Three isolates were recovered from each positive sample and tested for susceptibility to nine antimicrobials using a broth microdilution method. Overall, 176 isolates were recovered (141 chicken, 21 turkey, 6 duck, and 8 game bird). A high frequency of resistance to tetracycline was observed in the C. jejuni isolates from chickens (77%) and turkeys (100%), and in the C. coli isolates from turkeys (50%) and game birds (40%). Campylobacter jejuni isolates had higher odds of resistance to tetracycline (OR = 3.54, P ≤ 0.01) compared to C. coli isolates. Overall, there was a low frequency of resistance to quinolones and a very low frequency of resistance to macrolides. Multidrug resistance was uncommon. The high prevalence of tetracycline resistance emphasizes the importance of prudent antimicrobial use in small flocks. Although low, the presence of resistance to macrolides and quinolones, which are used to treat campylobacteriosis in humans, highlights the need for proper food safety and infection control practices by small flock owners to prevent exposure to antimicrobial resistant Campylobacter.

Background and purposeFew data are available concerning the safety of bevacizumab (B) in combination with locoregional radiation therapy (RT). The objective of this study was to evaluate the 5-year late toxicity of concurrent B and RT in non-metastatic breast cancer.Materials and methodsThis multicentre prospective study included non-metastatic breast cancer patients enrolled in phase 3 clinical trials evaluating B with concurrent RT versus RT alone. All patients received neoadjuvant or adjuvant chemotherapy and normofractionated breast or chest wall RT, with or without regional lymph node RT. B was administered at an equivalent dose of 5 mg/kg once a week for 1 year. The safety profile was evaluated 1, 3 and 5 years after completion of radiotherapy.ResultsA total of 64 patients were included between November 2007 and April 2010. Median follow-up was 60 months (12-73) and 5-year late toxicity data were available for 46 patients. The majority of tumours were triple-negative (68.8%), tumour size ConclusionConcurrent B and locoregional RT are associated with acceptable 5-year toxicity in patients with non-metastatic breast cancer. No grade ≥3 toxicity was observed.

BACKGROUND:In most emergency departments (EDs), few patients account for a relatively high number of ED visits. To improve the management of these patients, the university hospital of Lausanne, Switzerland, implemented an interdisciplinary case management (CM) intervention. This study examined whether the CM intervention-compared with standard care (SC) in the ED-reduced costs generated by frequent ED users, not only from the hospital perspective, but also from the third-party payer perspective, that is, from a broader perspective that takes into account the costs of health care services used outside the hospital offering the intervention. METHODS:In this randomized controlled trial, 250 frequent ED users (>5 visits during the previous 12 months) were allocated to either the CM or the SC group and followed up for 12 months. Cost data were obtained from the hospital's analytical accounting system for the entire sample and from health insurance companies for a subgroup (n = 140). Descriptive statistics and multivariate regressions were used to make comparisons between groups and assess the contribution of patient characteristics to the main cost components. RESULTS:At the end of the 12-month follow-up, 115 patients were in the CM group and 115 in the SC group (20 had died). Despite differences in economic costs between patients in the CM intervention and the SC groups, our results do not show any statistically significant reduction in costs associated with the intervention, either for the hospital that housed the intervention or for the third-party payer. Frequent ED users were big users of health services provided by both the hospital and community-based services, with 40% of costs generated outside the hospital that housed the intervention. Higher age, Swiss citizenship, and having social difficulty increased costs significantly. CONCLUSIONS:As the role of the CM team is to guide patients through the entire care process, the intervention location is not limited to the hospital but often extends into the community.

NetF-producing type A Clostridium perfringens is an important cause of canine and foal necrotizing enteritis. NetF, related to the β-sheet pore-forming Leukocidin/Hemolysin superfamily, is considered a major virulence factor for this disease. The main purpose of this work is to demonstrate the pore-forming activity of NetF and characterize the chemical nature of its binding site. Electron microscopy using recombinant NetF (rNetF) confirmed that NetF is able to oligomerize and form large pores in equine ovarian (EO) cell membranes and sheep red blood cells. These oligomeric pores appear to be about 4-6 nm in diameter, and the number of oligomer subunits to vary from 6 to 9. Sodium periodate treatment rendered EO cells non-susceptible to NetF, suggesting that NetF binding requires cell surface carbohydrates. NetF cytotoxicity was also inhibited by a lectin that binds sialic acid, by sialidase, and by free sialic acid in excess, all of which clearly implicate sialic acid-containing membrane carbohydrates in NetF binding and/or toxicity for EO cells. Binding of NetF to sheep red blood cells was not inhibited by the gangliosides GM1, GM2 and GM3, nor did the latter promote membrane permeabilization in liposomes, suggesting that they do not constitute the cellular receptors. In contrast, treatment of EO cells with different proteases reduced their susceptibility to NetF, suggesting that the NetF receptor is a sialic acid-containing glycoprotein.

OBJECTIVES:In countries with universal health coverage (UHC), national public health insurances cover 70% of health expenditures on average, but health care user fees and out-of-pocket expenditures have been neglected in empirical patient-centered health inequality research. This study is the first to investigate how health care-related factors are associated with health status among middle-aged and elderly people-vulnerable groups for the burden of illness-in countries with UHC. DESIGN:Longitudinal observational cohort study. SETTING:Population-based cohort Survey of Health, Ageing and Retirement in Europe (SHARE) in twelve countries with UHC. PARTICIPANTS:Non-institutionalized people aged 50 and older (n = 29,260). Two subsamples were also used: participants without global activity limitation at baseline (n = 16,879) and participants without depression at baseline (n = 21,178). MAIN OUTCOME MEASURES:Risk of death, risk of global activity limitations, and risk of depression. We used mixed-effects Cox proportional hazards regressions to estimate hazard ratios (HR) for all-cause mortality, physical limitations, and depression. RESULTS:Having a voluntary private insurance to cover health expenses not included in the public health care system (44.1% of the total sample) was a protective factor for all outcomes (HR≤0.91), controlling for a large range of socio-economic variables. On the contrary, having out-of-pocket expenditures (62.4%) was a risk factor (HR≥1.12). CONCLUSIONS:UHC systems are not free from health inequalities: there is a potential effect of lack of voluntary private insurance and out-of-pocket expenditures on mortality and health. Health care-related factors should be at focus in future researches designed to understand and address health inequalities. Reducing out-of-pocket expenditures and developing voluntary private insurance may protect against premature illness and death.

BACKGROUND:Deprivation usually encompasses material, social, and health components. It has been shown to be associated with greater risks of developing chronic health conditions and of worse outcome in multimorbidity. The DipCare questionnaire, an instrument developed and validated in Switzerland for use in primary care, identifies patients subject to potentially higher levels of deprivation. OBJECTIVES:To identifying determinants of the material, social, and health profiles associated with deprivation in a sample of multimorbid, primary care patients, and thus set priorities in screening for deprivation in this population. DESIGN:Secondary analysis from a nationwide cross-sectional study in Switzerland. PARTICIPANTS:A random sample of 886 adult patients suffering from at least three chronic health conditions. MAIN MEASURES:The outcomes of interest were the patients' levels of deprivation as measured using the DipCare questionnaire. Classification And Regression Tree analysis identified the independent variables that separated the examined population into groups with increasing deprivation scores. Finally, a sensitivity analysis (multivariate regression) confirmed the robustness of our results. KEY RESULTS:Being aged under 64 years old was associated with higher overall, material, and health deprivation; being aged over 77 years old was associated with higher social deprivation. Other variables associated with deprivation were the level of education, marital status, and the presence of depression or chronic pain. CONCLUSION:Specific profiles, such as being younger, were associated with higher levels of overall, material, and health deprivation in multimorbid patients. In contrast, patients over 77 years old reported higher levels of social deprivation. Furthermore, chronic pain and depression added to the score for health deprivation. It is important that GPs consider the possibility of deprivation in these multimorbid patients and are able to identify it, both in order to encourage treatment adherence and limit any forgoing of care for financial reasons.

Salmonella has been detected in the feces of many wildlife species, including raccoons (Procyon lotor), but little is known about the epidemiology of Salmonella in wildlife living in different habitat types. Our objective was to investigate demographic, temporal, and climatic factors associated with the carriage of Salmonella in raccoons and their environment on swine farms and conservation areas. Using a repeated cross-sectional study design, we collected fecal samples from raccoons and environmental samples (soil, manure pits, dumpsters) on 5 swine farms and 5 conservation areas in Ontario, Canada once every five weeks from May to November, 2011-2013. Salmonella was detected in 26% (279/1093; 95% CI 22.9-28.2) of raccoon fecal samples, 6% (88/1609; 95% CI 4.5-6.8) of soil samples, 30% (21/69; 95% CI 20.0-42.7) of manure pit samples, and 23% (7/31; 95% CI 9.6-41.0) of dumpster samples. Of samples testing positive for Salmonella, antimicrobial resistance was detected in 5% (14/279; 95% CI 2.8-8.3) of raccoon fecal, 8% (7/89; 95% CI 3.2-15.5) of soil, 10% (2/21; 95% CI 1.2-30.4) of manure pit, and 0/7 dumpster samples. Using multi-level multivariable logistic regression analyses, we found location type (swine farm or conservation area) was not a significant explanatory variable for Salmonella occurrence in raccoon feces or soil (p > 0.05). However, detection of Salmonella in raccoon feces was associated with rainfall, season, and sex with various interaction effects among these variables. We detected a variety of Salmonella serovars that infect humans and livestock in the feces of raccoons indicating that raccoons living near humans, regardless of location type, may play a role in the epidemiology of salmonellosis in livestock and humans in southwestern Ontario.

The recent discovery of a novel beta-pore-forming toxin, NetF, which is strongly associated with canine and foal necrotizing enteritis should improve our understanding of the role of type A Clostridium perfringens associated disease in these animals. The current study presents the complete genome sequence of two netF-positive strains, JFP55 and JFP838, which were recovered from cases of foal necrotizing enteritis and canine hemorrhagic gastroenteritis, respectively. Genome sequencing was done using Single Molecule, Real-Time (SMRT) technology-PacBio and Illumina Hiseq2000. The JFP55 and JFP838 genomes include a single 3.34 Mb and 3.53 Mb chromosome, respectively, and both genomes include five circular plasmids. Plasmid annotation revealed that three plasmids were shared by the two newly sequenced genomes, including a NetF/NetE toxins-encoding tcp-conjugative plasmid, a CPE/CPB2 toxins-encoding tcp-conjugative plasmid and a putative bacteriocin-encoding plasmid. The putative beta-pore-forming toxin genes, netF, netE and netG, were located in unique pathogenicity loci on tcp-conjugative plasmids. The C. perfringens JFP55 chromosome carries 2,825 protein-coding genes whereas the chromosome of JFP838 contains 3,014 protein-encoding genes. Comparison of these two chromosomes with three available reference C. perfringens chromosome sequences identified 48 (~247 kb) and 81 (~430 kb) regions unique to JFP55 and JFP838, respectively. Some of these divergent genomic regions in both chromosomes are phage- and plasmid-related segments. Sixteen of these unique chromosomal regions (~69 kb) were shared between the two isolates. Five of these shared regions formed a mosaic of plasmid-integrated segments, suggesting that these elements were acquired early in a clonal lineage of netF-positive C. perfringens strains. These results provide significant insight into the basis of canine and foal necrotizing enteritis and are the first to demonstrate that netF resides on a large and unique plasmid-encoded locus.

Antimicrobial resistance is a global threat to livestock, human and environmental health. Although resistant bacteria have been detected in wildlife, their role in the epidemiology of antimicrobial resistance is not clear. Our objective was to investigate demographic, temporal and climatic factors associated with carriage of antimicrobial resistant Escherichia coli in raccoons and the environment. We collected samples from raccoon paws and feces and from soil, manure pit and dumpsters on five swine farms and five conservation areas in Ontario, Canada once every five weeks from May to November, 2011-2013 and tested them for E. coli and susceptibility to 15 antimicrobials. Of samples testing positive for E. coli, resistance to ≥ 1 antimicrobials was detected in 7.4% (77/1044; 95% CI, 5.9-9.1) of raccoon fecal samples, 6.3% (23/365; 95% CI, 4.0-9.3) of paw samples, 9.6% (121/1260; 8.0-11.4) of soil samples, 57.4% (31/54; 95% CI, 43.2-70.8) of manure pit samples, and 13.8% (4/29; 95% CI, 3.9-31.7) of dumpster samples. Using univariable logistic regression, there was no significant difference in the occurrence of resistant E. coli in raccoon feces on conservation areas versus farms; however, E. coli isolates resistant to ≥ 1 antimicrobials were significantly less likely to be detected from raccoon paw samples on swine farms than conservation areas and significantly more likely to be detected in soil samples from swine farms than conservation areas. Resistant phenotypes and genotypes that were absent from the swine farm environment were detected in raccoons from conservation areas, suggesting that conservation areas and swine farms may have different exposures to resistant bacteria. However, the similar resistance patterns and genes in E. coli from raccoon fecal and environmental samples from the same location types suggest that resistant bacteria may be exchanged between raccoons and their environment.

BackgroundTheileriosis, caused by a number of species within the genus Theileria, is a common disease of livestock in Oman. It is a major constraint to the development of the livestock industry due to a high rate of morbidity and mortality in both cattle and sheep. Since little is currently known about the genetic diversity of the parasites causing theileriosis in Oman, the present study was designed to address this issue with specific regard to T. annulata in cattle.MethodsBlood samples were collected from cattle from four geographically distinct regions in Oman for genetic analysis of the Theileria annulata population. Ten genetic markers (micro- and mini-satellites) representing all four chromosomes of T. annulata were applied to these samples using a combination of PCR amplification and fragment analysis. The resultant genetic data was analysed to provide a first insight into the structure of the T. annulata population in Oman.ResultsWe applied ten micro- and mini-satellite markers to a total of 310 samples obtained from different regions (174 [56%] from Dhofar, 68 [22%] from Dhira, 44 [14.5%] from Batinah and 24 [8%] from Sharqia). A high degree of allelic diversity was observed among the four parasite populations. Expected heterozygosity for each site ranged from 0.816 to 0.854. A high multiplicity of infection was observed in individual hosts, with an average of 3.3 to 3.4 alleles per locus, in samples derived from Batinah, Dhofar and Sharqia regions. In samples from Dhira region, an average of 2.9 alleles per locus was observed. Mild but statistically significant linkage disequilibrium between pairs of markers was observed in populations from three of the four regions. In contrast, when the analysis was performed at farm level, no significant linkage disequilibrium was observed. Finally, no significant genetic differentiation was seen between the four populations, with most pair-wise FST values being less than 0.03. Slightly higher FST values (GST' = 0.075, θ = 0.07) were detected when the data for T. annulata parasites in Oman was compared with that previously generated for Turkey and Tunisia.ConclusionGenetic analyses of T. annulata samples representing four geographical regions in Oman revealed a high level of genetic diversity in the parasite population. There was little evidence of genetic differentiation between parasites from different regions, and a high level of genetic diversity was maintained within each sub-population. These findings are consistent with a high parasite transmission rate and frequent movement of animals between different regions in Oman.

Although speech and language deficits are common in children and strongly associated with poor educational and social outcomes, little attention has been paid to the antecedents. In this study we used the information from the Avon Longitudinal Study of Parents and Children to examine preconception and prenatal environmental risk factors that were related to communication difficulties in children using the Children's Communication Checklist (CCC). We used an exposome-wide approach to identify environmental factors univariably associated with the CCC. Taking account of the False Discovery rate, we used a P value of 0.000157 to identify 621 of 3855 items tested. These were then subjected to a series of stepwise linear regression analyses, firstly within 10 domains: personal characteristics, health, development, education, socio-economic variables, lifestyle, home and social environments, life events and chemical and other exposures; and then with the predictive variables from each domain. The final model consisted of 19 variables independently associated with the communication scale. These variables suggested 6 possible mechanisms: stressors primarily associated with socio-economic disadvantage although other lifestyle choices such as a social network of family or friends can ameliorate these effects; indicators of future parenting skills primarily associated with aspects of parental personality; aspects of the home environment; poor maternal health with a novel finding concerning maternal hearing loss; and maternal education which was partially mediated by the child's IQ. Finally, there may be a mechanism via the maternal diet in pregnancy in particular the consumption of fatty or processed foods. This is the subject of ongoing investigation.

Interest in 3D inertial motion tracking devices (AHRS) has been growing rapidly among the biomechanical community. Although the convenience of such tracking devices seems to open a whole new world of possibilities for evaluation in clinical biomechanics, its limitations haven't been extensively documented. The objectives of this study are: 1) to assess the change in absolute and relative accuracy of multiple units of 3 commercially available AHRS over time; and 2) to identify different sources of errors affecting AHRS accuracy and to document how they may affect the measurements over time.This study used an instrumented Gimbal table on which AHRS modules were carefully attached and put through a series of velocity-controlled sustained motions including 2 minutes motion trials (2MT) and 12 minutes multiple dynamic phases motion trials (12MDP). Absolute accuracy was assessed by comparison of the AHRS orientation measurements to those of an optical gold standard. Relative accuracy was evaluated using the variation in relative orientation between modules during the trials.Both absolute and relative accuracy decreased over time during 2MT. 12MDP trials showed a significant decrease in accuracy over multiple phases, but accuracy could be enhanced significantly by resetting the reference point and/or compensating for initial Inertial frame estimation reference for each phase.The variation in AHRS accuracy observed between the different systems and with time can be attributed in part to the dynamic estimation error, but also and foremost, to the ability of AHRS units to locate the same Inertial frame.Mean accuracies obtained under the Gimbal table sustained conditions of motion suggest that AHRS are promising tools for clinical mobility assessment under constrained conditions of use. However, improvement in magnetic compensation and alignment between AHRS modules are desirable in order for AHRS to reach their full potential in capturing clinical outcomes.

To develop guidance on what information to include and how to present it in tables summarizing the evidence from systematic reviews of test accuracy following the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach.To design and refine the evidence tables, we used an iterative process based on the analysis of data from four rounds of discussions, feedback and user testing. During the final round, we conducted one-on-one user testing with target end users. We presented a number of alternative formats of evidence tables to participants and obtained information about users' understanding and preferences.More than 150 users participated in initial discussions and provided their formal and informal feedback. 20 users completed one-on-one user testing interviews. Almost all participants preferred summarizing the results of systematic reviews of test accuracy in tabular format rather than plain text. Users generally preferred less complex tables but found presenting sensitivity and specificity estimates only as too simplistic. Users found the presentation of test accuracy for several values of prevalence initially confusing but modifying table layout and adding sample clinical scenarios for each prevalence reduced this confusion. Providing information about clinical consequences of testing result was viewed as not feasible for authors of systematic reviews.We present the current formats for tables presenting test accuracy following the GRADE approach. These tables can be developed using GRADEpro guidelines development tool (www.guidelinedevelopment.org or www.gradepro.org) and are being further developed into electronic interactive tables that will suit the needs of different end users. The formatting of these tables, and how they influence result interpretation and decision-making will be further evaluated in a randomized trial.

Biotic indices, which reflect the quality of the environment, are widely used in the marine realm. Sometimes, key species or ecosystem engineers are selected for this purpose. This is the case of the Mediterranean seagrass Posidonia oceanica, widely used as a biological quality element in the context of the European Union Water Framework Directive (WFD). The good quality of a water body and the apparent health of a species, whether or not an ecosystem engineer such as P. oceanica, is not always indicative of the good structure and functioning of the whole ecosystem. A key point of the recent Marine Strategy Framework Directive (MSFD) is the ecosystem-based approach. Here, on the basis of a simplified conceptual model of the P. oceanica ecosystem, we have proposed an ecosystem-based index of the quality of its functioning, compliant with the MSFD requirements. This index (EBQI) is based upon a set of representative functional compartments, the weighting of these compartments and the assessment of the quality of each compartment by comparison of a supposed baseline. The index well discriminated 17 sites in the north-western Mediterranean (French Riviera, Provence, Corsica, Catalonia and Balearic Islands) covering a wide range of human pressure levels. The strong points of the EBQI are that it is easy to implement, non-destructive, relatively robust, according to the selection of the compartments and to their weighting, and associated with confidence indices that indicate possible weakness and biases and therefore the need for further field data acquisition.

BackgroundGrowing social inequities have made it important for general practitioners to verify if patients can afford treatment and procedures. Incorporating social conditions into clinical decision-making allows general practitioners to address mismatches between patients' health-care needs and financial resources.ObjectivesIdentify a screening question to, indirectly, rule out patients' social risk of forgoing health care for economic reasons, and estimate prevalence of forgoing health care and the influence of physicians' attitudes toward deprivation.DesignMulticenter cross-sectional survey.ParticipantsForty-seven general practitioners working in the French-speaking part of Switzerland enrolled a random sample of patients attending their private practices.Main measuresPatients who had forgone health care were defined as those reporting a household member (including themselves) having forgone treatment for economic reasons during the previous 12 months, through a self-administered questionnaire. Patients were also asked about education and income levels, self-perceived social position, and deprivation levels.Key resultsOverall, 2,026 patients were included in the analysis; 10.7% (CI95% 9.4-12.1) reported a member of their household to have forgone health care during the 12 previous months. The question "Did you have difficulties paying your household bills during the last 12 months" performed better in identifying patients at risk of forgoing health care than a combination of four objective measures of socio-economic status (gender, age, education level, and income) (R(2) = 0.184 vs. 0.083). This question effectively ruled out that patients had forgone health care, with a negative predictive value of 96%. Furthermore, for physicians who felt powerless in the face of deprivation, we observed an increase in the odds of patients forgoing health care of 1.5 times.ConclusionGeneral practitioners should systematically evaluate the socio-economic status of their patients. Asking patients whether they experience any difficulties in paying their bills is an effective means of identifying patients who might forgo health care.

OBJECTIVE: To identify which physician and patient characteristics are associated with physicians' estimation of their patient social status. DESIGN: Cross-sectional multicentric survey. SETTING: Fourty-seven primary care private offices in Western Switzerland. PARTICIPANTS: Random sample of 2030 patients ≥ 16, who encountered a general practitioner (GP) between September 2010 and February 2011. MAIN MEASURES: PRIMARY OUTCOME: patient social status perceived by GPs, using the MacArthur Scale of Subjective Social Status, ranging from the bottom (0) to the top (10) of the social scale.Secondary outcome: Difference between GP's evaluation and patient's own evaluation of their social status. Potential patient correlates: material and social deprivation using the DiPCare-Q, health status using the EQ-5D, sources of income, and level of education. GP characteristics: opinion regarding patients' deprivation and its influence on health and care. RESULTS: To evaluate patient social status, GPs considered the material, social, and health aspects of deprivation, along with education level, and amount and type of income. GPs declaring a frequent reflexive consideration of their own prejudice towards deprived patients, gave a higher estimation of patients' social status (+1.0, p = 0.002). Choosing a less costly treatment for deprived patients was associated with a lower estimation (-0.7, p = 0.002). GP's evaluation of patient social status was 0.5 point higher than the patient's own estimate (p

Using a prospective, longitudinal study design, this paper addresses the impact of genetic counseling and testing for deafness on deaf adults and the Deaf community. This study specifically evaluated the effect of genetic counseling and Connexin-26 and Connexin-30 genetic test results on participants' deaf identity and understanding of their genetic test results. Connexin-26 and Connexin-30 genetic testing was offered to participants in the context of linguistically and culturally appropriate genetic counseling. Questionnaire data collected from 209 deaf adults at four time points (baseline, immediately following pre-test genetic counseling, 1-month following genetic test result disclosure, and 6-months after result disclosure) were analyzed. Four deaf identity orientations (hearing, marginal, immersion, bicultural) were evaluated using subscales of the Deaf Identity Development Scale-Revised. We found evidence that participants understood their specific genetic test results following genetic counseling, but found no evidence of change in deaf identity based on genetic counseling or their genetic test results. This study demonstrated that culturally and linguistically appropriate genetic counseling can improve deaf clients' understanding of genetic test results, and the formation of deaf identity was not directly related to genetic counseling or Connexin-26 and Connexin-30 genetic test results.

To evaluate the safety, tolerability and immunogenicity of three different doses (5, 25 and 50 µg) of the novel antituberculous vaccine RUTI compared to placebo in subjects with latent tuberculosis infection.Double-blind, randomized, placebo-controlled Phase II Clinical Trial (95 patients randomized). Three different RUTI doses and placebo were tested, randomized both in HIV-positive (n = 47) and HIV-negative subjects (n = 48), after completion of one month isoniazid (INH) pre-vaccination. Each subject received two vaccine administrations, 28 Days apart. Five patients withdrew and 90 patients completed the study. Assessment of safety showed no deaths during study. Two subjects had serious adverse events one had a retinal detachment while taking INH and was not randomized and the other had a severe local injection site abscess on each arm and was hospitalized; causality was assessed as very likely and by the end of the study the outcome had resolved. All the patients except 5 (21%) patients of the placebo group (3 HIV+ and 2 HIV-) reported at least one adverse event (AE) during the study. The most frequently occurring AEs among RUTI recipients were (% in HIV+/-): injection site reactions [erythema (91/92), induration (94/92), local nodules (46/25), local pain (66/75), sterile abscess (6/6), swelling (74/83), ulcer (20/11), headache (17/22) and nasopharyngitis (20/5)]. These events were mostly mild and well tolerated. Overall, a polyantigenic response was observed, which differed by HIV- status. The best polyantigenic response was obtained when administrating 25 µg RUTI, especially in HIV-positive subjects which was not increased after the second inoculation.This Phase II clinical trial demonstrates reasonable tolerability of RUTI. The immunogenicity profile of RUTI vaccine in LTBI subjects, even being variable among groups, allows us considering one single injection of one of the highest doses in future trials, preceded by an extended safety clinical phase.ClinicalTrials.gov NCT01136161.

Contour integration is a fundamental visual process. The constraints on integrating discrete contour elements and the associated neural mechanisms have typically been investigated using static contour paths. However, in our dynamic natural environment objects and scenes vary over space and time. With the aim of investigating the parameters affecting spatiotemporal contour path integration, we measured human contrast detection performance of a briefly presented foveal target embedded in dynamic collinear stimulus sequences (comprising five short 'predictor' bars appearing consecutively towards the fovea, followed by the 'target' bar) in four experiments. The data showed that participants' target detection performance was relatively unchanged when individual contour elements were separated by up to 2° spatial gap or 200 ms temporal gap. Randomising the luminance contrast or colour of the predictors, on the other hand, had similar detrimental effect on grouping dynamic contour path and subsequent target detection performance. Randomising the orientation of the predictors reduced target detection performance greater than introducing misalignment relative to the contour path. The results suggest that the visual system integrates dynamic path elements to bias target detection even when the continuity of path is disrupted in terms of spatial (2°), temporal (200 ms), colour (over 10 colours) and luminance (-25% to 25%) information. We discuss how the findings can be largely reconciled within the functioning of V1 horizontal connections.

African forest elephants- taxonomically and functionally unique-are being poached at accelerating rates, but we lack range-wide information on the repercussions. Analysis of the largest survey dataset ever assembled for forest elephants (80 foot-surveys; covering 13,000 km; 91,600 person-days of fieldwork) revealed that population size declined by ca. 62% between 2002-2011, and the taxon lost 30% of its geographical range. The population is now less than 10% of its potential size, occupying less than 25% of its potential range. High human population density, hunting intensity, absence of law enforcement, poor governance, and proximity to expanding infrastructure are the strongest predictors of decline. To save the remaining African forest elephants, illegal poaching for ivory and encroachment into core elephant habitat must be stopped. In addition, the international demand for ivory, which fuels illegal trade, must be dramatically reduced.

Inertial measurement of motion with Attitude and Heading Reference Systems (AHRS) is emerging as an alternative to 3D motion capture systems in biomechanics. The objectives of this study are: 1) to describe the absolute and relative accuracy of multiple units of commercially available AHRS under various types of motion; and 2) to evaluate the effect of motion velocity on the accuracy of these measurements.The criterion validity of accuracy was established under controlled conditions using an instrumented Gimbal table. AHRS modules were carefully attached to the center plate of the Gimbal table and put through experimental static and dynamic conditions. Static and absolute accuracy was assessed by comparing the AHRS orientation measurement to those obtained using an optical gold standard. Relative accuracy was assessed by measuring the variation in relative orientation between modules during trials.Evaluated AHRS systems demonstrated good absolute static accuracy (mean error < 0.5(o)) and clinically acceptable absolute accuracy under condition of slow motions (mean error between 0.5(o) and 3.1(o)). In slow motions, relative accuracy varied from 2(o) to 7(o) depending on the type of AHRS and the type of rotation. Absolute and relative accuracy were significantly affected (p

A randomized controlled field trial was conducted to evaluate the effects of two sets of treatment strategies on ceftiofur and tetracycline resistance in feedlot cattle. The strategies consisted of ceftiofur crystalline-free acid (CCFA) administered to either one or all of the steers within a pen, followed by feeding or not feeding a therapeutic dose of chlortetracycline (CTC). Eighty-eight steers were randomly allocated to eight pens of 11 steers each. Both treatment regimens were randomly assigned to the pens in a two-way full factorial design. Non-type-specific (NTS) E. coli (n = 1,050) were isolated from fecal samples gathered on Days 0, 4, 12, and 26. Antimicrobial susceptibility profiles were determined using a microbroth dilution technique. PCR was used to detect tet(A), tet(B), and bla CMY-2 genes within each isolate. Chlortetracycline administration greatly exacerbated the already increased levels of both phenotypic and genotypic ceftiofur resistance conferred by prior CCFA treatment (P

Triacylglycerol lipases (EC 3.1.1.3) catalyze both hydrolysis and synthesis reactions with a broad spectrum of substrates rendering them especially suitable for many biotechnological applications. Most lipases used today originate from mesophilic organisms and are susceptible to thermal denaturation whereas only few possess high thermotolerance. Here, we report on the identification and characterization of two novel thermostable bacterial lipases identified by functional metagenomic screenings. Metagenomic libraries were constructed from enrichment cultures maintained at 65 to 75 °C and screened resulting in the identification of initially 10 clones with lipolytic activities. Subsequently, two ORFs were identified encoding lipases, LipS and LipT. Comparative sequence analyses suggested that both enzymes are members of novel lipase families. LipS is a 30.2 kDa protein and revealed a half-life of 48 h at 70 °C. The lipT gene encoded for a multimeric enzyme with a half-life of 3 h at 70 °C. LipS had an optimum temperature at 70 °C and LipT at 75 °C. Both enzymes catalyzed hydrolysis of long-chain (C(12) and C(14)) fatty acid esters and additionally hydrolyzed a number of industry-relevant substrates. LipS was highly specific for (R)-ibuprofen-phenyl ester with an enantiomeric excess (ee) of 99%. Furthermore, LipS was able to synthesize 1-propyl laurate and 1-tetradecyl myristate at 70 °C with rates similar to those of the lipase CalB from Candida antarctica. LipS represents the first example of a thermostable metagenome-derived lipase with significant synthesis activities. Its X-ray structure was solved with a resolution of 1.99 Å revealing an unusually compact lid structure.

ObjectivesTo determine 1) HIV testing practices in a 1400-bed university hospital where local HIV prevalence is 0.4% and 2) the effect on testing practices of national HIV testing guidelines, revised in March 2010, recommending Physician-Initiated Counselling and Testing (PICT).MethodsUsing 2 hospital databases, we determined the number of HIV tests performed by selected clinical services, and the number of patients tested as a percentage of the number seen per service ('testing rate'). To explore the effect of the revised national guidelines, we examined testing rates for two years pre- and two years post-PICT guideline publication.ResultsCombining the clinical services, 253,178 patients were seen and 9,183 tests were performed (of which 80 tested positive, 0.9%) in the four-year study period. The emergency department (ED) performed the second highest number of tests, but had the lowest testing rates (0.9-1.1%). Of inpatient services, neurology and psychiatry had higher testing rates than internal medicine (19.7% and 9.6% versus 8%, respectively). There was no significant increase in testing rates, either globally or in the majority of the clinical services examined, and no increase in new HIV diagnoses post-PICT recommendations.ConclusionsUsing a simple two-database tool, we observe no global improvement in HIV testing rates in our hospital following new national guidelines but do identify services where testing practices merit improvement. This study may show the limit of PICT strategies based on physician risk assessment, compared to the opt-out approach.

Brugada syndrome (BrS) is an inherited autosomal dominant cardiac channelopathy. Several mutations on the cardiac sodium channel Na(v)1.5 which are responsible for BrS lead to misfolded proteins that do not traffic properly to the plasma membrane. In order to mimic patient heterozygosity, a trafficking defective mutant, R1432G was co-expressed with Wild Type (WT) Na(v)1.5 channels in HEK293T cells. This mutant significantly decreased the membrane Na current density when it was co-transfected with the WT channel. This dominant negative effect did not result in altered biophysical properties of Na(v)1.5 channels. Luminometric experiments revealed that the expression of mutant proteins induced a significant reduction in membrane expression of WT channels. Interestingly, we have found that the auxiliary Na channel β(1)-subunit was essential for this dominant negative effect. Indeed, the absence of the β(1)-subunit prevented the decrease in WT sodium current density and surface proteins associated with the dominant negative effect. Co-immunoprecipitation experiments demonstrated a physical interaction between Na channel α-subunits. This interaction occurred only when the β(1)-subunit was present. Our findings reveal a new role for β(1)-subunits in cardiac voltage-gated sodium channels by promoting α-α subunit interaction which can lead to a dominant negative effect when one of the α-subunits shows a trafficking defective mutation.

In some pathological conditions carnitine concentration is high while in others it is low. In both cases,cardiac arrhythmias can occur and lead to sudden cardiac death. It has been proposed that in ischaemia, acylcarnitine (acyl-CAR), but not carnitine, is involved in arrhythmias through modulation of ionic currents. We studied the effects of acyl-CARs on hERG, K(IR)2.1 and K(v)7.1/minK channels (channels responsible for I(KR), I(K1) and I(KS) respectively).HEK293 cells stably expressing hERG, K(IR)2.1 or Kv7.1/minK were studied using the patch clamp technique. Free carnitine (CAR) and acyl-CAR derivatives from medium- (C8 and C10) and long-chain (C16 and C18:1) fatty acids were applied intra- and extracellularly at different concentrations. For studies on hERG, C16 and C18:1 free fatty acid were also used.Extracellular long-chain (LCAC), but not medium-chain, acyl-CAR,induced an increase of I(hERG) amplitude associated with a dose-dependent speeding of deactivation kinetics. They had no effect on K(IR)2.1 or Kv7.1/minK currents.Computer simulations of these effects were consistent with changes in action potential profile. CONCLUSIONS AND APPLICATIONS: Extracellular LCAC tonically regulates I(hERG) amplitude and kinetics under physiological conditions. This modulation may contribute to the changes in action potential duration that precede cardiac arrhythmias in ischaemia, diabetes and primary systemic carnitine deficiency.

Lactococcus lactis is a biotechnological workhorse for food fermentations and potentially therapeutic products and is therefore widely consumed by humans. It is predominantly used as a starter microbe for fermented dairy products, and specialized strains have adapted from a plant environment through reductive evolution and horizontal gene transfer as evidenced by the association of adventitious traits with mobile elements. Specifically, L. lactis has armed itself with a myriad of plasmid-encoded bacteriophage defensive systems to protect against viral predation. This known arsenal had not included CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins), which forms a remarkable microbial immunity system against invading DNA. Although CRISPR/Cas systems are common in the genomes of closely related lactic acid bacteria (LAB), none was identified within the eight published lactococcal genomes. Furthermore, a PCR-based search of the common LAB CRISPR/Cas systems (Types I and II) in 383 industrial L. lactis strains proved unsuccessful. Here we describe a novel, Type III, self-transmissible, plasmid-encoded, phage-interfering CRISPR/Cas discovered in L. lactis. The native CRISPR spacers confer resistance based on sequence identity to corresponding lactococcal phage. The interference is directed at phages problematic to the dairy industry, indicative of a responsive system. Moreover, targeting could be modified by engineering the spacer content. The 62.8-kb plasmid was shown to be conjugally transferrable to various strains. Its mobility should facilitate dissemination within microbial communities and provide a readily applicable system to naturally introduce CRISPR/Cas to industrially relevant strains for enhanced phage resistance and prevention against acquisition of undesirable genes.

Major genetic factors for age-related macular degeneration (AMD) have recently been identified as susceptibility risk factors, including variants in the CFH gene and the ARMS2 LOC387715/HTRA1locus. Our purpose was to perform a case-control study in two populations among individuals who did not carry risk variants for CFHY402H and LOC387715 A69S (ARMS2), called "study" individuals, in order to identify new genetic risk factors. Based on a candidate gene approach, we analyzed SNP rs5888 of the SCARB1 gene, coding for SRBI, which is involved in the lipid and lutein pathways. This study was conducted in a French series of 1241 AMD patients and 297 controls, and in a North American series of 1257 patients with advanced AMD and 1732 controls. Among these individuals, we identified 61 French patients, 77 French controls, 85 North American patients and 338 North American controls who did not carry the CFH nor ARMS2 polymorphisms. An association between AMD and the SCARB1 gene was seen among the study subjects. The genotypic distribution of the rs5888 polymorphism was significantly different between cases and controls in the French population (p

MicroRNAs (miRNAs) are endogenously expressed single-stranded ~21-23 nucleotide RNAs that inhibit gene expression post-transcriptionally by binding imperfectly to elements usually within the 3'untranslated region (3'UTR) of mRNAs. Small interfering RNAs (siRNAs) mediate site-specific cleavage by binding with perfect complementarity to RNA. Here, a cell-based miRNA reporter system was developed to screen for compounds from marine and plant extracts that inhibit miRNA or siRNA activity. The daphnane diterpenoid genkwanine M (GENK) isolated from the plant Wikstroemia polyantha induces an early inflammatory response and can moderately inhibit miR-122 activity in the liver Huh-7 cell line. GENK does not alter miR-122 levels nor does it directly inhibit siRNA activity in an in vitro cleavage assay. Finally, we demonstrate that GENK can inhibit HCV infection in Huh-7 cells. In summary, the development of the cell-based miRNA sensor system should prove useful in identifying compounds that affect miRNA/siRNA activity.