Relationship between Expression of the Family of M Proteins and Lipoteichoic Acid to Hydrophobicity and Biofilm Formation in Streptococcus pyogenes

Background: Hydrophobicity is an important attribute of bacteria that contributes to adhesion and biofilm formation. Hydrophobicity of Streptococcus pyogenes is primarily due to lipoteichoic acid (LTA) on the streptococcal surface but the mechanism(s) whereby LTA is retained on the surface is poorly understood. In this study, we sought to determine whether members of the M protein family consisting of Emm (M protein), Mrp (M-related protein), Enn (an M-like protein), and the streptococcal protective antigen (Spa) are involved in anchoring LTA in a manner that contributes to hydrophobicity of the streptococci and its ability to form biofilms. Methodology/Principal Findings: Isogenic mutants defective in expression of emm, mrp, enn, and/or spa genes of eight different serotypes and their parental strains were tested for differences in LTA bound to surface proteins, LTA released into the culture media, and membrane-bound LTA. The effect of these mutations on the ability of streptococci to form a hydrophobic surface and to generate biofilms was also investigated. A recombinant strain overexpressing Emm1 was also engineered and similarly tested. The serotypes tested ranged from those that express only a single M protein gene to those that express two or three members of the M protein family. Overexpression of Emm1 led to enhanced hydrophobicity an

Calpains Are Downstream Effectors of bax-Dependent Excitotoxic Apoptosis

Excitotoxicity resulting from excessive Ca2+ influx through glutamate receptors contributes to neuronal injury after stroke, trauma, and seizures. Increased cytosolic Ca2+ levels activate a family of calcium-dependent proteases with papain-like activity, the calpains. Here we investigated the role of calpain activation during NMDA-induced excitotoxic injury in embryonic (E16-E18) murine cortical neurons that (1) underwent excitotoxic necrosis, characterized by immediate deregulation of Ca2+ homeostasis, a persistent depolarization of mitochondrial membrane potential (Delta psi(m)), and insensitivity to bax-gene deletion, (2) underwent excitotoxic apoptosis, characterized by recovery of NMDA-induced cytosolic Ca2+ increases, sensitivity to bax gene deletion, and delayed Delta psi(m) depolarization and Ca2+ deregulation, or (3) that were tolerant to excitotoxic injury. Interestingly, treatment with the calpain inhibitor calpeptin, overexpression of the endogenous calpain inhibitor calpastatin, or gene silencing of calpain protected neurons against excitotoxic apoptosis but did not influence excitotoxic necrosis. Calpeptin failed to exert a protective effect in bax-deficient neurons but protected bid-deficient neurons similarly to wild-type cells. To identify when calpains became activated during excitotoxic apoptosis, we monitored calpain activation dynamics by time-lapse fluorescence microscopy using a calpain-sensitive Forster resonance energy transfer probe. We observed a delayed calpain activation that occurred downstream of mitochondrial engagement and directly preceded neuronal death. In contrast, we could not detect significant calpain activity during excitotoxic necrosis or in neurons that were tolerant to excitotoxic injury. Oxygen/glucose deprivation-induced injury in organotypic hippocampal slice cultures confirmed that calpains were specifically activated during bax-dependent apoptosis and in this setting function as downstream cell-death executioners

CRISPR/Cas9 DNA cleavage at SNP-derived PAM enables both in vitro and in vivo KRT12 mutation-specific targeting

CRISPR/Cas9-based therapeutics hold the possibility for permanent treatment of genetic disease. The potency and specificity of this system has been used to target dominantly inherited conditions caused by heterozygous missense mutations through inclusion of the mutated base in the short-guide RNA (sgRNA) sequence. This research evaluates a novel approach for targeting heterozygous single-nucleotide polymorphisms (SNPs) using CRISPR/Cas9. We determined that a mutation within KRT12, which causes Meesmann's epithelial corneal dystrophy (MECD), leads to the occurrence of a novel protospacer adjacent motif (PAM). We designed an sgRNA complementary to the sequence adjacent to this SNP-derived PAM and evaluated its potency and allele specificity both in vitro and in vivo. This sgRNA was found to be highly effective at reducing the expression of mutant KRT12 mRNA and protein in vitro. To assess its activity in vivo we injected a combined Cas9/sgRNA expression construct into the corneal stroma of a humanized MECD mouse model. Sequence analysis of corneal genomic DNA revealed non-homologous end-joining repair resulting in frame-shifting deletions within the mutant KRT12 allele. This study is the first to demonstrate in vivo gene editing of a heterozygous disease-causing SNP that results in a novel PAM, further highlighting the potential for CRISPR/Cas9-based therapeutics

Quality of anticoagulation and use of warfarin-interacting medications in long-term care: A chart review

<p>Abstract</p> <p>Background</p> <p>Maintenance of therapeutic International Normalized Ratio (INR) in the community is generally poor. The supervised environment in long-term care facilities may represent a more ideal setting for warfarin therapy since laboratory monitoring, compliance, dose adjustment, and interacting medications can all be monitored and controlled. The objectives of this study were to determine how effectively warfarin was administered to a cohort of residents in long-term care facilities, to identify the proportion of residents prescribed warfarin-interacting drugs and to ascertain factors associated with poor INR control.</p> <p>Methods</p> <p>A chart review of 105 residents receiving warfarin therapy in five long-term care facilities in Hamilton, Ontario was performed. Data were collected on INR levels, warfarin prescribing and monitoring practices, and use of interacting medications.</p> <p>Results</p> <p>Over a 12 month period (28,555 resident-days, 78.2 resident years) 3065 INR values were available. Residents were within, below and above the therapeutic range 54%, 35% and 11% of the time, respectively. Seventy-nine percent of residents were prescribed at least one warfarin-interacting medication during the period in review. Residents receiving interacting medications spent less time in the therapeutic range (53.0% vs. 58.2%, OR = 0.93, 95% confidence interval 0.88 to 0.97, P = 0.002). Adequacy of anticoagulation varied significantly between physicians (time in therapeutic range 45.9 to 63.9%).</p> <p>Conclusion</p> <p>In this group of long-term care residents, warfarin control was suboptimal. Both prescriber and co-prescription of interacting medications were associated with poorer INR control. Future studies should seek strategies to improve prescriber skill and decrease use of interacting medications.</p

Equine Cyathostominae can develop to infective third-stage larvae on straw bedding

Background Domesticated grazing animals including horses and donkeys are frequently housed using deep litter bedding systems, where it is commonly presumed that there is no risk of infection from the nematodes that are associated with grazing at pasture. We use two different approaches to test whether equids could become infected with cyathostomines from the ingestion of deep litter straw bedding. Methods Two herbage plot studies were performed in horticultural incubators set up to simulate three straw bedding scenarios and one grass turf positive control. Faeces were placed on 16 plots, and larval recoveries performed on samples of straw/grass substrate over 2- to 3-week periods. Within each incubator, a thermostat was set to maintain an environmental temperature of approximately 10 °C to 20 °C. To provide further validation, 24 samples of straw bedding were collected over an 8-week period from six barns in which a large number of donkeys were housed in a deep litter straw bedding system. These samples were collected from the superficial bedding at 16 sites along a “W” route through each barn. Results No infective larvae were recovered from any of the plots containing dry straw. However, infective cyathostomine larvae were first detected on day 8 from plots containing moist straw. In the straw bedding study, cyathostomine larvae were detected in 18 of the 24 samples. Additionally, in the two barns which were sampled serially, the level of larval infectivity generally increased from week to week, except when the straw bedding was removed and replaced. Conclusions We have demonstrated that equine cyathostomines can develop to infective larvae on moist straw bedding. It is therefore possible for a horse or donkey bedded in deep litter straw to become infected by ingesting the contaminated straw. This has implications for parasite control in stabled equids and potentially in housed ruminants, and further investigation is required in order to establish the relative infective pressure from pasture versus straw bedding

Association of Plasma Vitamin D Metabolites With Incident Type 2 Diabetes: EPIC-InterAct Case-Cohort Study.

BACKGROUND: Existing evidence for the prospective association of vitamin D status with type 2 diabetes (T2D) is focused almost exclusively on circulating total 25-hydroxyvitamin D [25(OH)D] without distinction between its subtypes: nonepimeric and epimeric 25(OH)D3 stereoisomers, and 25(OH)D2, the minor component of 25(OH)D. We aimed to investigate the prospective associations of circulating levels of the sum and each of these three metabolites with incident T2D. METHODS: This analysis in the European Prospective Investigation into Cancer and Nutrition (EPIC)-InterAct case-cohort study for T2D included 9671 incident T2D cases and 13,562 subcohort members. Plasma vitamin D metabolites were quantified by liquid chromatography-mass spectrometry. We used a multivariable Prentice-weighted Cox regression to estimate hazard ratios (HRs) of T2D for each metabolite. Analyses were performed separately within country, and estimates were combined across countries using random-effects meta-analysis. RESULTS: The mean concentrations (SD) of total 25(OH)D, nonepimeric 25(OH)D3, epimeric 25(OH)D3, and 25(OH)D2 were 41.1 (17.2), 40.7 (17.3), 2.13 (1.31), and 8.16 (6.52) nmol/L, respectively. Plasma total 25(OH)D and nonepimeric 25(OH)D3 were inversely associated with incident T2D [multivariable-adjusted HR per 1 SD = 0.81 (95% CI, 0.77, 0.86) for both variables], whereas epimeric 25(OH)D3 was positively associated [per 1 SD HR = 1.16 (1.09, 1.25)]. There was no statistically significant association with T2D for 25(OH)D2 [per 1 SD HR = 0.94 (0.76, 1.18)]. CONCLUSIONS: Plasma nonepimeric 25(OH)D3 was inversely associated with incident T2D, consistent with it being the major metabolite contributing to total 25(OH)D. The positive association of the epimeric form of 25(OH)D3 with incident T2D provides novel information to assess the biological relevance of vitamin D epimerization and vitamin D subtypes in diabetes etiology.The InterAct project was funded by the European Union Framework 6 (LSHM_CT_2006_037197). Biomarker measurements for vitamin D metabolites were funded jointly by the InterAct project, the MRC Cambridge Initiative (RG71466, SJAH/004) and the EPIC-CVD project. EPIC-CVD has been supported by the European Union Framework 7 (HEALTH-F2-2012-279233), the European Research Council (268834), the UK Medical Research Council (G0800270 and MR/L003120/1), the British Heart Foundation (SP/09/002 and RG/08/014 and RG13/13/30194) and the UK National Institute of Health Research. In addition, InterAct investigators acknowledge funding from the following agencies: Medical Research Council Epidemiology Unit MC_UU_12015/1 and MC_UU_12015/5, NIHR Biomedical Research Centre Cambridge: Nutrition, Diet, and Lifestyle Research Theme (IS-BRC-1215-20014), Regional Government of Asturias and Regional Governments of Basque Country. Dr. Ivonne Sluijs is supported by the Junior Dr. Dekker grant (2015T019) from the Dutch Heart Foundation. Dr. Guy Fagherazzi is supported by the French Research Agency (Agence Nationale de la Recherche) via an “Investissement d’Avenir” grant (investment for the future grant, ANR-10-COHO-0006) and by the IDEX Paris Saclay Nutriperso Project. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 701708

Interplay between genetic predisposition, macronutrient intake and type 2 diabetes incidence: analysis within EPIC-InterAct across eight European countries.

AIMS/HYPOTHESIS: Gene-macronutrient interactions may contribute to the development of type 2 diabetes but research evidence to date is inconclusive. We aimed to increase our understanding of the aetiology of type 2 diabetes by investigating potential interactions between genes and macronutrient intake and their association with the incidence of type 2 diabetes. METHODS: We investigated the influence of interactions between genetic risk scores (GRSs) for type 2 diabetes, insulin resistance and BMI and macronutrient intake on the development of type 2 diabetes in the European Prospective Investigation into Cancer and Nutrition (EPIC)-InterAct, a prospective case-cohort study across eight European countries (N = 21,900 with 9742 incident type 2 diabetes cases). Macronutrient intake was estimated from diets reported in questionnaires, including proportion of energy derived from total carbohydrate, protein, fat, plant and animal protein, saturated, monounsaturated and polyunsaturated fat and dietary fibre. Using multivariable-adjusted Cox regression, we estimated country-specific interaction results on the multiplicative scale, using random-effects meta-analysis. Secondary analysis used isocaloric macronutrient substitution. RESULTS: No interactions were identified between any of the three GRSs and any macronutrient intake, with low-to-moderate heterogeneity between countries (I2 range 0-51.6%). Results were similar using isocaloric macronutrient substitution analyses and when weighted and unweighted GRSs and individual SNPs were examined. CONCLUSIONS/INTERPRETATION: Genetic susceptibility to type 2 diabetes, insulin resistance and BMI did not modify the association between macronutrient intake and incident type 2 diabetes. This suggests that macronutrient intake recommendations to prevent type 2 diabetes do not need to account for differences in genetic predisposition to these three metabolic conditions

Identification, characterisation and expression analysis of natural killer receptor genes in Chlamydia pecorum infected koalas (Phascolarctos cinereus)

BACKGROUND: Koalas (Phascolarctos cinereus), an iconic Australian marsupial, are being heavily impacted by the spread of Chlamydia pecorum, an obligate intracellular bacterial pathogen. Koalas vary in their response to this pathogen, with some showing no symptoms, while others suffer severe symptoms leading to infertility, blindness or death. Little is known about the pathology of this disease and the immune response against it in this host. Studies have demonstrated that natural killer (NK) cells, key components of the innate immune system, are involved in the immune response to chlamydial infections in humans. These cells can directly lyse cells infected by intracellular pathogens and their ability to recognise these infected cells is mediated through NK receptors on their surface. These are encoded in two regions of the genome, the leukocyte receptor complex (LRC) and the natural killer complex (NKC). These two families evolve rapidly and different repertoires of genes, which have evolved by gene duplication, are seen in different species. METHODS: In this study we aimed to characterise genes belonging to the NK receptor clusters in the koala by searching available koala transcriptomes using a combination of search methods. We developed a qPCR assay to quantify relative expression of four genes, two encoded within the NK receptor cluster (CLEC1B, CLEC4E) and two known to play a role in NK response to Chalmydia in humans (NCR3, PRF1). RESULTS: We found that the NK receptor repertoire of the koala closely resembles that of the Tasmanian devil, with minimal genes in the NKC, but with lineage specific expansions in the LRC. Additional genes important for NK cell activity, NCR3 and PRF1, were also identified and characterised. In a preliminary study to investigate whether these genes are involved in the koala immune response to infection by its chlamydial pathogen, C. pecorum, we investigated the expression of four genes in koalas with active chlamydia infection, those with past infection and those without infection using qPCR. This analysis revealed that one of these four, CLEC4E, may be upregulated in response to chlamydia infection. CONCLUSION: We have characterised genes of the NKC and LRC in koalas and have discovered evidence that one of these genes may be upregulated in koalas with chlamydia, suggesting that these receptors may play a role in the immune response of koalas to chlamydia infection

Functional Dissection of Streptococcus pyogenes M5 Protein: the Hypervariable Region is Essential for Virulence

The surface-localized M protein of Streptococcus pyogenes is a major virulence factor that inhibits phagocytosis, as determined ex vivo. Because little is known about the role of M protein in vivo we analyzed the contribution of different M protein regions to virulence, using the fibrinogen (Fg)-binding M5 protein and a mouse model of acute invasive infection. This model was suitable, because M5 is required for mouse virulence and binds mouse and human Fg equally well, as shown here. Mixed infection experiments with wild type bacteria demonstrated that mutants lacking the N-terminal hypervariable region (HVR) or the Fg-binding B-repeat region were strongly attenuated, while a mutant lacking the conserved C-repeats was only slightly attenuated. Because the HVR of M5 is not required for phagocytosis resistance, our data imply that this HVR plays a major but unknown role during acute infection. The B-repeat region is required for phagocytosis resistance and specifically binds Fg, suggesting that it promotes virulence by binding Fg. However, B-repeat mutants were attenuated even in Fg-deficient mice, implying that the B-repeats may have a second function, in addition to Fg-binding. These data demonstrate that two distinct M5 regions, including the HVR, are essential to virulence during the early stages of an infection. In particular, our data provide the first in vivo evidence that the HVR of an M protein plays a major role in virulence, focusing interest on the molecular role of this region