Development and validation of high-resolution melting assays for the detection of potentially virulent strains of \u3ci\u3eEscherichia coli\u3c/i\u3e O103 and O121

Virulent strains of Shiga toxin-producing Escherichia coli (STEC) serogroups O103 and O121 are considered adulterants in beef. Two high-resolution melting (HRM) real-time PCR assays were standardized for the specific detection and discrimination of potentially virulent and avirulent strains of E. coli O103 and O121. The O103 HRM assay offered the possibility to distinguish clearly STEC O103:H2 from STEC O103:H25. The two standardized assays were extensively validated using 215 pure culture strains, laboratory inoculated food samples, and naturally contaminated beef (n = 84) and pork (n = 84) enrichments collected from the red meat surveillance program. Both HRM assays showed 100% inclusivity and exclusivity using pure culture strains and enriched spiked food samples. Data from this study shows the ability of the standardized assays to specifically detect the strains of each target serogroup and, most importantly, to differentiate the strains present into potentially virulent or avirulent groups. The assays standardized in this study can be helpful for food surveillance programs and help mitigate product loss due to the presence of avirulent strains lacking crucial virulence genes (stx and eae)

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ABSTRACT Introduction: Exposure to second-hand smoke (SHS) is a serious public health concern. The Indian smoke-free legislation 'Prohibition of Smoking in Public Places Rules, 2008' prohibits smoking in public places, including workplaces. Objective: To measure the status of compliance to legal provisions that protects the public against harms of SHS exposure, identifies the potential areas of violations and informs policy makers for strengthening enforcement measures. Design: A cross-sectional survey in 1401 public places across 11 district headquarters in Himachal Pradesh, India, using a compliance guide developed by partners of the Bloomberg initiatives to reduce tobacco use. Results: In 1401 public places across 11 district headquarters, 42.8% public places had signage; in 84.2% public places, no smoking was observed and in 83.7%, there was absence of smoking accessories such as ashtray, matchbox and lighter. Tobacco litter like cigarette butts was absent in 64.7% of the public places. Overall, at the state level, there was more than 80% compliance on at least three of the five indicators. Among all categories of public places, educational institutions and offices demonstrated highest compliance, whereas most frequently visited public places, eateries and accommodation facilities had least compliance. Conclusions: The compliance to 'Prohibition of Smoking in Public Places Rules, 2008' was variable in various district headquarters of Himachal Pradesh. This study identified the potential areas of violations that need attention from enforcement agencies and policymakers

Measurement of gut permeability using fluorescent tracer agent technology

Abstract The healthy gut restricts macromolecular and bacterial movement across tight junctions, while increased intestinal permeability accompanies many intestinal disorders. Dual sugar absorption tests, which measure intestinal permeability in humans, present challenges. Therefore, we asked if enterally administered fluorescent tracers could ascertain mucosal integrity, because transcutaneous measurement of differentially absorbed molecules could enable specimen-free evaluation of permeability. We induced small bowel injury in rats using high- (15 mg/kg), intermediate- (10 mg/kg), and low- (5 mg/kg) dose indomethacin. Then, we compared urinary ratios of enterally administered fluorescent tracers MB-402 and MB-301 to urinary ratios of sugar tracers lactulose and rhamnose. We also tested the ability of transcutaneous sensors to measure the ratios of absorbed fluorophores. Urinary fluorophore and sugar ratios reflect gut injury in an indomethacin dose dependent manner. The fluorophores generated smooth curvilinear ratio trajectories with wide dynamic ranges. The more chaotic sugar ratios had narrower dynamic ranges. Fluorophore ratios measured through the skin distinguished indomethacin-challenged from same day control rats. Enterally administered fluorophores can identify intestinal injury in a rat model. Fluorophore ratios are measureable through the skin, obviating drawbacks of dual sugar absorption tests. Pending validation, this technology should be considered for human use

Rhamnose is superior to mannitol as a monosaccharide in the dual sugar absorption test: A prospective randomized study in children with treatment-naïve celiac disease

BACKGROUND AND AIM: We sought to correlate two different measures of gut permeability [lactulose:mannitol (L:M) and lactulose:rhamnose (L:R)] to the severity of duodenal histopathology in children with and without elevated antibodies to tissue transglutaminase (tTG). A secondary objective was to correlate gut permeability with celiac disease (CD) serology and indices of inflammation and bacterial product translocation. METHODS: We prospectively randomized children undergoing endoscopy with abnormal ( RESULTS: Of the 54 cases with positive celiac serology, 31 and 69% had modified Marsh 0/1 scores or ≥3a, respectively. Circulating tTG IgA correlated with the modified Marsh score ( CONCLUSIONS: L:R, but not L:M, is associated with modified Marsh scores in children undergoing small bowel biopsy for suspected CD. Despite increased intestinal permeability, we see scant evidence of systemic exposure to gut microbes in these children. Gut permeability testing with L:R may predict which patients with abnormal celiac serology will have biopsy evidence for celiac disease and reduce the proportion of such patients undergoing endoscopy whose Marsh scores are ≤1. M should not be used as a monosaccharide for permeability testing in children

Targeting proinsulin to local immune cells using an intradermal microneedle delivery system; a potential antigen-specific immunotherapy for type 1 diabetes

Antigen-specific immunotherapy (ASI) has been proposed as an alternative treatment strategy for type 1 diabetes (T1D). ASI aims to induce a regulatory, rather than stimulatory, immune response in order to reduce, or prevent, autoimmune mediated β-cell destruction, thus preserving endogenous insulin production. The abundance of immunocompetent antigen presenting cells (APCs) within the skin makes this organ an attractive target for immunotherapies. Microneedles (MNs) have been proposed as a suitable drug delivery system to facilitate intradermal delivery of autoantigens in a minimally invasive manner. However, studies to date have employed single peptide autoantigens, which would restrict ASI to patients expressing specific Human Leukocyte Antigen (HLA) molecules, thus stratifying the patient population. This study aims to develop, for the first time, an intradermal MN delivery system to target proinsulin, a large multi-epitope protein capable of inducing tolerance in a heterogenous (in terms of HLA status) population of T1D patients, to the immunocompetent cells of the skin. An optimized three component coating formulation containing proinsulin, a diluent and a surfactant, facilitated uniform and reproducible coating of >30 μg of the active pharmaceutical ingredient on a stainless steel MN array consisting of thirty 500 μm projections. When applied to a murine model these proinsulin-coated MNs efficiently punctured the skin and after a limited insertion time (150 s) a significant proportion of the therapeutic payload (86%) was reproducibly delivered into the local tissue. Localized delivery of proinsulin in non-obese diabetic (NOD) mice using the coated MN system stimulated significantly greater proliferation of adoptively transferred antigen-specific CD8+ T cells in the skin draining lymph nodes compared to a conventional intradermal injection. This provides evidence of targeted delivery of the multi-epitope proinsulin antigen to skin-resident APCs, in vivo, in a form that enables antigen presentation to antigen-specific T cells in the local lymph nodes. The development of an innovative coated MN system for highly targeted and reproducible delivery of proinsulin to local immune cells warrants further evaluation to determine translation to a tolerogenic clinical outcome

Genome-Wide Identification of Alternatively Spliced mRNA Targets of Specific RNA-Binding Proteins

BACKGROUND: Alternative splicing plays an important role in generating molecular and functional diversity in multi-cellular organisms. RNA binding proteins play crucial roles in modulating splice site choice. The majority of known binding sites for regulatory proteins are short, degenerate consensus sequences that occur frequently throughout the genome. This poses an important challenge to distinguish between functionally relevant sequences and a vast array of those occurring by chance. METHODOLOGY/PRINCIPAL FINDINGS: Here we have used a computational approach that combines a series of biological constraints to identify uridine-rich sequence motifs that are present within relevant biological contexts and thus are potential targets of the Drosophila master sex-switch protein Sex-lethal (SXL). This strategy led to the identification of one novel target. Moreover, our systematic analysis provides a starting point for the molecular and functional characterization of an additional target, which is dependent on SXL activity, either directly or indirectly, for regulation in a germline-specific manner. CONCLUSIONS/SIGNIFICANCE: This approach has successfully identified previously known, new, and potential SXL targets. Our analysis suggests that only a subset of potential SXL sites are regulated by SXL. Finally, this approach should be directly relevant to the large majority of splicing regulatory proteins for which bonafide targets are unknown

Pigeonpea - A unique jewel in rainfed cropping systems

Pigeonpea is a crop for rainfed environments endowed with several features to thrive harsh climate. It adapts well in sole crop and inter cropped conditions (with cereals, millets, oils seeds and pulses) by enhancing the system productivity and net income to the small and marginal farmers across the globe. The range of maturity duration in the crop allows it to grow in diversified cropping systems and patterns in varied ecoregions of the world. Development of cytoplasmic male sterility based hybrids provided an opportunity for enhancing the yields under marginal environments. With recent interventions in addressing the photo sensitivity and maturity have led to evolving super early varieties with less than 100 days duration, provided a scope for horizontal expansion of the crop in different agro ecological systems

Anastrozole has an association between degree of estrogen suppression and outcomes in early breast cancer and is a ligand for estrogen receptor α

Purpose: To determine if the degree of estrogen suppression with aromatase inhibitors (AI: anastrozole, exemestane, letrozole) is associated with efficacy in early-stage breast cancer, and to examine for differences in the mechanism of action between the three AIs. Experimental design: Matched case-control studies [247 matched sets from MA.27 (anastrozole vs. exemestane) and PreFace (letrozole) trials] were undertaken to assess whether estrone (E1) or estradiol (E2) concentrations after 6 months of adjuvant therapy were associated with risk of an early breast cancer event (EBCE). Preclinical laboratory studies included luciferase activity, cell proliferation, radio-labeled ligand estrogen receptor binding, surface plasmon resonance ligand receptor binding, and nuclear magnetic resonance assays. Results: Women with E1 ≥1.3 pg/mL and E2 ≥0.5 pg/mL after 6 months of AI treatment had a 2.2-fold increase in risk (P = 0.0005) of an EBCE, and in the anastrozole subgroup, the increase in risk of an EBCE was 3.0-fold (P = 0.001). Preclinical laboratory studies examined mechanisms of action in addition to aromatase inhibition and showed that only anastrozole could directly bind to estrogen receptor α (ERα), activate estrogen response element-dependent transcription, and stimulate growth of an aromatase-deficient CYP19A1-/- T47D breast cancer cell line. Conclusions: This matched case-control clinical study revealed that levels of estrone and estradiol above identified thresholds after 6 months of adjuvant anastrozole treatment were associated with increased risk of an EBCE. Preclinical laboratory studies revealed that anastrozole, but not exemestane or letrozole, is a ligand for ERα. These findings represent potential steps towards individualized anastrozole therapy

The development and validation of a scoring tool to predict the operative duration of elective laparoscopic cholecystectomy

Background: The ability to accurately predict operative duration has the potential to optimise theatre efficiency and utilisation, thus reducing costs and increasing staff and patient satisfaction. With laparoscopic cholecystectomy being one of the most commonly performed procedures worldwide, a tool to predict operative duration could be extremely beneficial to healthcare organisations. Methods: Data collected from the CholeS study on patients undergoing cholecystectomy in UK and Irish hospitals between 04/2014 and 05/2014 were used to study operative duration. A multivariable binary logistic regression model was produced in order to identify significant independent predictors of long (> 90 min) operations. The resulting model was converted to a risk score, which was subsequently validated on second cohort of patients using ROC curves. Results: After exclusions, data were available for 7227 patients in the derivation (CholeS) cohort. The median operative duration was 60 min (interquartile range 45–85), with 17.7% of operations lasting longer than 90 min. Ten factors were found to be significant independent predictors of operative durations > 90 min, including ASA, age, previous surgical admissions, BMI, gallbladder wall thickness and CBD diameter. A risk score was then produced from these factors, and applied to a cohort of 2405 patients from a tertiary centre for external validation. This returned an area under the ROC curve of 0.708 (SE = 0.013, p  90 min increasing more than eightfold from 5.1 to 41.8% in the extremes of the score. Conclusion: The scoring tool produced in this study was found to be significantly predictive of long operative durations on validation in an external cohort. As such, the tool may have the potential to enable organisations to better organise theatre lists and deliver greater efficiencies in care

Microneedle delivery of autoantigen for immunotherapy in type 1 diabetes

Antigen specific immunotherapy mediated via the sustained generation of regulatory T cells arguably represents the ideal therapeutic approach to preventing beta cell destruction in type 1 diabetes. However, there is a need to enhance the efficacy of this approach to achieve disease modification in man. Previous studies suggest that prolonged expression of self-antigen in skin in a non-inflammatory context is beneficial for tolerance induction. We therefore sought to develop a dry-coated microneedle (MN) delivery system and combine it with topical steroid to minimise local inflammation and promote prolonged antigen presentation in the skin. Here we show that a combination of surface-modified MNs coated with appropriate solvent systems can deliver therapeutically relevant quantities of peptide to mouse and human skin even with hydrophobic peptides. Compared to conventional “wet” intradermal (ID) administration, “dry” peptide delivered via MNs was retained for longer in the skin and whilst topical hydration of the skin with vehicle or steroid accelerated loss of ID-delivered peptide from the skin, MN delivery of peptide was unaffected. Furthermore, MN delivery resulted in enhanced presentation of antigen to T cells in skin draining lymph nodes (LNs) both 3 and 10 days after administration. Repeated administration of islet antigen peptide via MN was effective at reducing antigen-specific T cell proliferation in the pancreatic LN, although topical steroid therapy did not enhance this. Taken together, these data show auto-antigenic peptide delivery into skin using coated MNs results in prolonged retention and enhanced antigen presentation compared to conventional ID delivery and this approach may have potential in individuals identified as being at a high risk of developing type 1 diabetes and other autoimmune diseases