Predictive validity of the UK clinical aptitude test in the final years of medical school:a prospective cohort study

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Using zebrafish larval models to study brain injury, locomotor and neuroinflammatory outcomes following intracerebral haemorrhage.

Intracerebral haemorrhage (ICH) is a devastating condition with limited treatment options, and current understanding of pathophysiology is incomplete. Spontaneous cerebral bleeding is a characteristic of the human condition that has proven difficult to recapitulate in existing pre-clinical rodent models. Zebrafish larvae are frequently used as vertebrate disease models and are associated with several advantages, including high fecundity, optical translucency and non-protected status prior to 5 days post-fertilisation. Furthermore, other groups have shown that zebrafish larvae can exhibit spontaneous ICH. The aim of this study was to investigate whether such models can be utilised to study the pathological consequences of bleeding in the brain, in the context of pre-clinical ICH research. Here, we compared existing genetic (bubblehead) and chemically inducible (atorvastatin) zebrafish larval models of spontaneous ICH and studied the subsequent disease processes. Through live, non-invasive imaging of transgenic fluorescent reporter lines and behavioural assessment we quantified brain injury, locomotor function and neuroinflammation following ICH. We show that ICH in both zebrafish larval models is comparable in timing, frequency and location. ICH results in increased brain cell death and a persistent locomotor deficit. Additionally, in haemorrhaged larvae we observed a significant increase in macrophage recruitment to the site of injury. Live in vivo imaging allowed us to track active macrophage-based phagocytosis of dying brain cells 24 hours after haemorrhage. Morphological analyses and quantification indicated that an increase in overall macrophage activation occurs in the haemorrhaged brain. Our study shows that in zebrafish larvae, bleeding in the brain induces quantifiable phenotypic outcomes that mimic key features of human ICH. We hope that this methodology will enable the pre-clinical ICH community to adopt the zebrafish larval model as an alternative to rodents, supporting future high throughput drug screening and as a complementary approach to elucidating crucial mechanisms associated with ICH pathophysiology

An Open Randomized Comparison of Gatifloxacin versus Cefixime for the Treatment of Uncomplicated Enteric Fever

OBJECTIVE: To assess the efficacy of gatifloxacin versus cefixime in the treatment of uncomplicated culture positive enteric fever. DESIGN: A randomized, open-label, active control trial with two parallel arms. SETTING: Emergency Room and Outpatient Clinics in Patan Hospital, Lagankhel, Lalitpur, Nepal. PARTICIPANTS: Patients with clinically diagnosed uncomplicated enteric fever meeting the inclusion criteria. INTERVENTIONS: Patients were allocated to receive one of two drugs, Gatifloxacin or Cefixime. The dosages used were Gatifloxacin 10 mg/kg, given once daily for 7 days, or Cefixime 20 mg/kg/day given in two divided doses for 7 days. OUTCOME MEASURES: The primary outcome measure was fever clearance time. The secondary outcome measure was overall treatment failure (acute treatment failure and relapse). RESULTS: Randomization was carried out in 390 patients before enrollment was suspended on the advice of the independent data safety monitoring board due to significant differences in both primary and secondary outcome measures in the two arms and the attainment of a priori defined endpoints. Median (95% confidence interval) fever clearance times were 92 hours (84-114 hours) for gatifloxacin recipients and 138 hours (105-164 hours) for cefixime-treated patients (Hazard Ratio[95%CI] = 2.171 [1.545-3.051], p<0.0001). 19 out of 70 (27%) patients who completed the 7 day trial had acute clinical failure in the cefixime group as compared to 1 out of 88 patients (1%) in gatifloxacin group(Odds Ratio [95%CI] = 0.031 [0.004 - 0.237], p<0.001). Overall treatment failure patients (relapsed patients plus acute treatment failure patients plus death) numbered 29. They were determined to be (95% confidence interval) 37.6 % (27.14%-50.2%) in the cefixime group and 3.5% (2.2%-11.5%) in the gatifloxacin group (HR[95%CI] = 0.084 [0.025-0.280], p<0.0001). There was one death in the cefixime group. CONCLUSIONS: Based on this study, gatifloxacin is a better treatment for uncomplicated enteric fever as compared to cefixime. TRIAL REGISTRATION: Current Controlled Trials ISRCTN75784880

The sensitivity of real-time PCR amplification targeting invasive Salmonella serovars in biological specimens

Background: PCR amplification for the detection of pathogens in biological material is generally considered a rapid and informative diagnostic technique. Invasive Salmonella serovars, which cause enteric fever, can be commonly cultured from the blood of infected patients. Yet, the isolation of invasive Salmonella serovars from blood is protracted and potentially insensitive. Methods: We developed and optimised a novel multiplex three colour real-time PCR assay to detect specific target sequences in the genomes of Salmonella serovars Typhi and Paratyphi A. We performed the assay on DNA extracted from blood and bone marrow samples from culture positive and negative enteric fever patients. Results: The assay was validated and demonstrated a high level of specificity and reproducibility under experimental conditions. All bone marrow samples tested positive for Salmonella, however, the sensitivity on blood samples was limited. The assay demonstrated an overall specificity of 100% (75/75) and sensitivity of 53.9% (69/128) on all biological samples. We then tested the PCR detection limit by performing bacterial counts after inoculation into blood culture bottles. Conclusions: Our findings corroborate previous clinical findings, whereby the bacterial load of S. Typhi in peripheral blood is low, often below detection by culture and, consequently, below detection by PCR. Whilst the assay may be utilised for environmental sampling or on differing biological samples, our data suggest that PCR performed directly on blood samples may be an unsuitable methodology and a potentially unachievable target for the routine diagnosis of enteric fever. </p

Directed -in vitro- evolution of Precambrian and extant Rubiscos

Rubisco is an ancient, catalytically conserved yet slow enzyme, which plays a central role in the biosphere’s carbon cycle. The design of Rubiscos to increase agricultural productivity has hitherto relied on the use of in vivo selection systems, precluding the exploration of biochemical traits that are not wired to cell survival. We present a directed -in vitro- evolution platform that extracts the enzyme from its biological context to provide a new avenue for Rubisco engineering. Precambrian and extant form II Rubiscos were subjected to an ensemble of directed evolution strategies aimed at improving thermostability. The most recent ancestor of proteobacteria -dating back 2.4 billion years- was uniquely tolerant to mutagenic loading. Adaptive evolution, focused evolution and genetic drift revealed a panel of thermostable mutants, some deviating from the characteristic trade-offs in CO2-fixing speed and specificity. Our findings provide a novel approach for identifying Rubisco variants with improved catalytic evolution potential.This work was supported by the REPSOL Research contracts Rubolution (RC020401120018), Rubolution 2.0 (RC 020401140042), the CSIC project PIE-201780E043 and the Australian Research Council grant CE140100015

MUC4 activates HER2 signalling and enhances the motility of human ovarian cancer cells

The mucin MUC4 is a high molecular weight transmembrane glycoprotein. It consists of a mucin-type subunit (MUC4α) and a transmembrane growth factor-like subunit (MUC4β). The mucin MUC4 is overexpressed in many epithelial malignancies including ovarian cancer, suggesting a possible role in the pathogenesis of these cancers. In this study, we investigated the functional role of MUC4 in the human ovarian cancer cell line SKOV3. The mucin MUC4 was ectopically expressed by stable transfection, and its expression was examined by western blot and confocal microscopy analyses. The in vitro studies demonstrated an enhanced motility of MUC4-expressing SKOV3 cells compared with the vector-transfected cells. The mucin MUC4 expression was associated with apparent changes in actin organisation, leading to the formation of microspike, lammelopodia and filopodia-like cellular projections. An enhanced protein expression and activation of HER2, a receptor tyrosine kinase, was also seen, although no significant change was observed in HER-2 transcript levels in the MUC4-transfected SKOV3 cells. Reciprocal co-immunoprecipitation revealed an interaction of MUC4 with HER2. Further, the MUC4-overexpressing SKOV3 cells exhibited an increase in the phosphorylation of focal adhesion kinase (FAK), Akt and ERK, downstream effectors of HER2. Taken together, our findings demonstrate that MUC4 plays a role in ovarian cancer cell motility, in part, by altering actin arrangement and potentiating HER2 downstream signalling in these cells

Immunohistochemical and transcriptional expression of Matrix Metalloproteinases in full-term human umbilical cord and Human Umbilical Vein Endothelial Cells

Matrix metalloproteinases (MMPs) are extracellular zinc-dependent endopeptidases involved in the degradation and remodelling of extracellular matrix in physiological and pathological processes. MMPs also have a role on cell proliferation, migration, differentiation, angiogenesis and apoptosis. Umbilical cord is a special organ subjected to many changes during pre-natal life and whose cells can maintain a certain degree of plasticity also in post-natal period; for example recently they have been used as a source of stem cells. In this work we investigated the expression of MMPs in human umbilical cord and Human Umbilical Vein Endothelial Cells (HUVEC) though immunohistochemistry, RT-PCR and gelatin zymography. MMP-2 protein is expressed in the amniotic epithelium of human umbilical cord and in few sub-epithelial fibroblasts, while MMP-3 and MMP-10 only in the umbilical epithelium. MMP-8, MMP-9 and MMP-13 immunoreactivity is localised in the epithelium and in Wharton\u2019s jelly mesenchymal cells. Immunocytochemistry also revealed protein expression for MMP-2, 3, 8, 9 and 10 in cultured HUVEC. In agreement with immunohistochemical data, RT-PCR analysis performed on samples of whole umbilical cord confirmed the transcriptional expression for the genes encoding all the six matrix metalloproteinases investigated, while in HUVEC only the expression of MMP-2, 3, 9, 10 and 13 mRNAs was detected. Gelatin zymograpgy showed a clear MMP-2 and MMP-9 enzymatic activity in the conditioned medium of HUVEC at different culture passages, suggesting that HUVEC secrete gelatinases, that afterwards undergo extracellular activation, and this ability is not affected by passage number

Global, regional, and national comparative risk assessment of 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks, 1990-2015: a systematic analysis for the Global Burden of Disease Study 2015

SummaryBackground The Global Burden of Diseases, Injuries, and Risk Factors Study 2015 provides an up-to-date synthesis of the evidence for risk factor exposure and the attributable burden of disease. By providing national and subnational assessments spanning the past 25 years, this study can inform debates on the importance of addressing risks in context. Methods We used the comparative risk assessment framework developed for previous iterations of the Global Burden of Disease Study to estimate attributable deaths, disability-adjusted life-years (DALYs), and trends in exposure by age group, sex, year, and geography for 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks from 1990 to 2015. This study included 388 risk-outcome pairs that met World Cancer Research Fund-defined criteria for convincing or probable evidence. We extracted relative risk and exposure estimates from randomised controlled trials, cohorts, pooled cohorts, household surveys, census data, satellite data, and other sources. We used statistical models to pool data, adjust for bias, and incorporate covariates. We developed a metric that allows comparisons of exposure across risk factors—the summary exposure value. Using the counterfactual scenario of theoretical minimum risk level, we estimated the portion of deaths and DALYs that could be attributed to a given risk. We decomposed trends in attributable burden into contributions from population growth, population age structure, risk exposure, and risk-deleted cause-specific DALY rates. We characterised risk exposure in relation to a Socio-demographic Index (SDI). Findings Between 1990 and 2015, global exposure to unsafe sanitation, household air pollution, childhood underweight, childhood stunting, and smoking each decreased by more than 25%. Global exposure for several occupational risks, high body-mass index (BMI), and drug use increased by more than 25% over the same period. All risks jointly evaluated in 2015 accounted for 57·8% (95% CI 56·6–58·8) of global deaths and 41·2% (39·8–42·8) of DALYs. In 2015, the ten largest contributors to global DALYs among Level 3 risks were high systolic blood pressure (211·8 million [192·7 million to 231·1 million] global DALYs), smoking (148·6 million [134·2 million to 163·1 million]), high fasting plasma glucose (143·1 million [125·1 million to 163·5 million]), high BMI (120·1 million [83·8 million to 158·4 million]), childhood undernutrition (113·3 million [103·9 million to 123·4 million]), ambient particulate matter (103·1 million [90·8 million to 115·1 million]), high total cholesterol (88·7 million [74·6 million to 105·7 million]), household air pollution (85·6 million [66·7 million to 106·1 million]), alcohol use (85·0 million [77·2 million to 93·0 million]), and diets high in sodium (83·0 million [49·3 million to 127·5 million]). From 1990 to 2015, attributable DALYs declined for micronutrient deficiencies, childhood undernutrition, unsafe sanitation and water, and household air pollution; reductions in risk-deleted DALY rates rather than reductions in exposure drove these declines. Rising exposure contributed to notable increases in attributable DALYs from high BMI, high fasting plasma glucose, occupational carcinogens, and drug use. Environmental risks and childhood undernutrition declined steadily with SDI; low physical activity, high BMI, and high fasting plasma glucose increased with SDI. In 119 countries, metabolic risks, such as high BMI and fasting plasma glucose, contributed the most attributable DALYs in 2015. Regionally, smoking still ranked among the leading five risk factors for attributable DALYs in 109 countries; childhood underweight and unsafe sex remained primary drivers of early death and disability in much of sub-Saharan Africa. Interpretation Declines in some key environmental risks have contributed to declines in critical infectious diseases. Some risks appear to be invariant to SDI. Increasing risks, including high BMI, high fasting plasma glucose, drug use, and some occupational exposures, contribute to rising burden from some conditions, but also provide opportunities for intervention. Some highly preventable risks, such as smoking, remain major causes of attributable DALYs, even as exposure is declining. Public policy makers need to pay attention to the risks that are increasingly major contributors to global burden. Funding Bill & Melinda Gates Foundation