Synthesis and structural characterization of a mimetic membrane-anchored prion protein

During pathogenesis of transmissible spongiform encephalopathies (TSEs) an abnormal form (PrPSc) of the host encoded prion protein (PrPC) accumulates in insoluble fibrils and plaques. The two forms of PrP appear to have identical covalent structures, but differ in secondary and tertiary structure. Both PrPC and PrPSc have glycosylphospatidylinositol (GPI) anchors through which the protein is tethered to cell membranes. Membrane attachment has been suggested to play a role in the conversion of PrPC to PrPSc, but the majority of in vitro studies of the function, structure, folding and stability of PrP use recombinant protein lacking the GPI anchor. In order to study the effects of membranes on the structure of PrP, we synthesized a GPI anchor mimetic (GPIm), which we have covalently coupled to a genetically engineered cysteine residue at the C-terminus of recombinant PrP. The lipid anchor places the protein at the same distance from the membrane as does the naturally occurring GPI anchor. We demonstrate that PrP coupled to GPIm (PrP-GPIm) inserts into model lipid membranes and that structural information can be obtained from this membrane-anchored PrP. We show that the structure of PrP-GPIm reconstituted in phosphatidylcholine and raft membranes resembles that of PrP, without a GPI anchor, in solution. The results provide experimental evidence in support of previous suggestions that NMR structures of soluble, anchor-free forms of PrP represent the structure of cellular, membrane-anchored PrP. The availability of a lipid-anchored construct of PrP provides a unique model to investigate the effects of different lipid environments on the structure and conversion mechanisms of PrP

Mechanism of adherence of Candida albicans to epithelial cells

The research in this thesis was aimed at determining the chemical nature of the surface structures on Candida albicans and on host epithelial cells that are involved in yeast-epithelial adhesion. From previous studies it was known that C. albicans from active infections (I strains) were more adherent to buccal and vaginal epithelial cells after growth in medium containing 500 mM galactose than after growth on 50 mM glucose. Extracellular polymeric material (EPM) was isolated from culture supernates of C. albicans after growth in medium containing 500 mM galactose. EPM was composed of carbohydrate (70%), protein (10%) and phosphorus (0.05%) and was therefore considered to be glycoprotein. When used to pretreat buccal epithelial cells, EPM inhibited adherence, which suggested that it contains an adhesin that binds to, and blocks, epithelial-cell receptors. Fractionation of EPM by gel filtration and ion-exchange chromatography gave five components, all of which were capable of inhibiting adherence but to different extentso An index, the adherence inhibition index (All) was devised to compare activities of the various components. Fractionation of EPM by affinity chromatography on Con A-Sepharose and application of Con A-bound material to a DEAE-cellulose column yielded material which was 30 times more active as an inhibitor than crude EPM. Attempts to resolve EPM by SDS-polyacrylamide gel electrophoresis proved unsuccessful even though a variety of buffer and staining procedures was investigated. Chemical and enzymic treatment of EPM indicated that the protein portion of the (postulated) glycoprotein was more important than the carbohydrate at inhibiting adherence. Proteolytic enzymes, mild acid and heat treatment of EPM abolished its ability to inhibit adherence. Periodate and a-mannosidase had no effect, whereas papain and mild alkaline treatment of EPM enhanced its ability to inhibit adherence. The nature of the epithelial cell receptor for C. albicans was investigated with potential receptor analogues such as sugars and lectins and inhibitors. The adhesion of C. albicans GDH 2346 to buccal, cells was inhibited by L-fucose and pretreatment of the buccal cells with winged-pea lectin (which is specific for L-fucose), However, the adhesion of another C. albicans strain (GDH 2023) to buccal cells was inhibited by N-acetyl-D-glucosamine and by pretreatment of the buccal cells with wheat-germ agglutinin (which is specific for N-acetyl-D-glucosamine). These experiments indicated that glycosides function as epithelial cell receptors for C. albicans, but with the sugar residue varying between strains. Samples of EPM from various strains of C. albicans bound to affinity columns of Sepharose containing coupled sugars. The bound fractions were capable of inhibiting adherence, suggesting that they contained an adhesin component. These results indicate that 'lectin-like' interactions may be involved in yeast adherence to host epithelial cells

Experimental and numerical study of auxetic sandwich panels on 160 grams of PE4 blast loading

Mines, specifically as Anti-Tank (AT) mines are a significant threat for defence vehicles. While approaches such as v-shaped hulls are currently used to deflect the blast products from such threats, such a solution is not always usable when hull standoff is limited. As such the development of a low profile, energy absorbing solution is desirable. One approach that has potential to achieve these requirements are sandwich panels. While sandwich panel cores can be constructed from various materials, one material of particular interest are auxetics. Auxetic are materials that exhibit a negative Poisson’s ratio. This material has potential to be an efficient an impact energy absorber by increasing stiffness at local deformation by gathering mass at the impact location. This study investigates the effectiveness of novel auxetic core infills alongside three other panel types (monolithic, air gap, polymer foam sandwich) against buried charges. 160 grams of PE4 were buried in 100 mm depth and 500 mm stand off the target. Laser and High Speed Video (HSV) system were used to capture the deflection-time profile and load cell sensors were used to record the loading profile received by the panels. Experimental works were compared with numerical model. Explicit model were generated in LSDYNA software as ‘initial impulse mine’ keyword. The result found that the auxetic and foam core panels were effective in reducing peak structural loading and impulse by up to 33% and 34% respectively. Air-filled panels were the most effective to reduce the deflection of the rear of the plate, however variation between capture methods (HSV and Laser system) were reported, while numerical modelling provided comparable plate deflections responses. When normalised against panel weight, the air filled panels were experimentally the most efficient per unit mass system with the auxetics being the least effective

The E2 ubiquitin-conjugating enzymes UBE2D1 and UBE2D2 regulate VEGFR2 dynamics and endothelial function

Vascular endothelial growth factor receptor 2 (VEGFR2, encoded by KDR) regulates endothelial function and angiogenesis. VEGFR2 undergoes ubiquitination that programs this receptor for trafficking and proteolysis, but the ubiquitin-modifying enzymes involved are ill-defined. Herein, we used a reverse genetics screen for the human E2 family of ubiquitin-conjugating enzymes to identify gene products that regulate VEGFR2 ubiquitination and proteolysis. We found that depletion of either UBE2D1 or UBE2D2 in endothelial cells caused a rise in steady-state VEGFR2 levels. This rise in plasma membrane VEGFR2 levels impacted on VEGF-A-stimulated signalling, with increased activation of canonical MAPK, phospholipase Cγ1 and Akt pathways. Analysis of biosynthetic VEGFR2 is consistent with a role for UBE2D enzymes in influencing plasma membrane VEGFR2 levels. Cell-surface-specific biotinylation and recycling studies showed an increase in VEGFR2 recycling to the plasma membrane upon reduction in UBE2D levels. Depletion of either UBE2D1 or UBE2D2 stimulated endothelial tubulogenesis, which is consistent with increased VEGFR2 plasma membrane levels promoting the cellular response to exogenous VEGF-A. Our studies identify a key role for UBE2D1 and UBE2D2 in regulating VEGFR2 function in angiogenesis

E2 ubiquitin-conjugating enzymes, UBE2D1 and UBE2D2, regulate VEGFR2 dynamics and endothelial function

Vascular endothelial growth factor receptor 2 (VEGFR2) regulates endothelial function and angiogenesis. VEGFR2 undergoes ubiquitination which programs this receptor for trafficking and proteolysis but the ubiquitin-modifying enzymes involved are ill-defined. Herein, we used a reverse genetics screen of the human E2 family of ubiquitin-conjugating enzymes to identify gene products which regulate VEGFR2 ubiquitination and proteolysis. We find that depletion of either UBE2D1 or UBE2D2 in endothelial cells cause a rise in steady-state VEGFR2 levels. This rise in plasma membrane VEGFR2 levels impact on VEGF-A-stimulated signalling, with increased activation of canonical MAPK, phospholipase C1, and Akt pathways. Analysis of biosynthetic VEGFR2 is consistent with a role for UBE2D enzymes in influencing plasma membrane VEGFR2 levels. Cell surface biotinylation and recycling studies show an increase in VEGFR2 recycling to the plasma membrane upon reduction in UBE2D levels. Depletion of either UBE2D1 or UBE2D2 stimulates endothelial tubulogenesis which is consistent with increased VEGFR2 plasma membrane levels promoting the cellular response to exogenous VEGF-A. Our studies identify a key role for UBE2D1 and UBE2D2 in regulating VEGFR2 function in angiogenesis

Are there gender differences in acute management and secondary prevention of acute coronary syndromes in Barbados? A cohort study.

OBJECTIVES: In Barbados, high case fatality rates have been reported after myocardial infarction (MI) with higher rates in women than men. To explore this inequality, we examined documented pharmacological interventions for ST-segment elevated myocardial infarction (STEMI), non-STEMI (NSTEMI) and unstable and chronic angina in women and men. DESIGN: Prospective cohort registry data for STEMI and NSTEMI and retrospective chart review for unstable and chronic angina. SETTING: Tertiary care (acute coronary syndromes) and primary care (chronic angina) centres in Barbados. PARTICIPANTS: For the years 2009-2016, a total of 1018 patients with STEMI or NSTEMI were identified via the prospective study. For unstable and chronic angina, 136 and 272 notes were reviewed respectively for the years 2010-2014. OUTCOME MEASURES: The proportions of patients prescribed recommended medication during the first 24 hours after an acute event, at discharge and for chronic care were calculated. Prescribed proportions were analysed by gender after adjustment for age. RESULTS: Between 2009 and 2016, for the acute management of patients with NSTEMI and STEMI, only two (aspirin and clopidogrel) of six drugs had documented prescription rates of 80% or more. Patients with STEMI (n=552) had higher prescription rates than NSTEMI (n=466), with gender differences being more pronounced in the former. Among patients with STEMI, after adjustment for age, diabetes, hypertension and smoking, men were more likely to receive fibrinolytics acutely, OR 2.28 (95% CI 1.24 to 4.21). Compared with men, a higher proportion of women were discharged on all recommended treatments; this was only statistically significant for beta-blockers: age-adjusted OR 1.87 (95% CI 1.16 to 3.00). There were no statistically significant differences in documented prescription of drugs for chronic angina. CONCLUSION: Following acute MI in Barbados, the proportion of patients with documented recommended treatment is relatively low. Although women were less likely to receive appropriate acute care than men, by discharge gender differences were reversed

Long–term hay meadow management maintains the target community despite local-scale species turnover

Hay meadows, which are managed using a low intensity regime, are characterised by highly diverse vegetation but have declined significantly since the mid twentieth century. Remaining species-rich meadows are often protected by statutory designations and conservation management agreements. However, long-term studies of change in the composition of meadow vegetation, and investigations of the success of conservation over the long-term are rare. Fourteen sites, which had a long history of being managed for field dried hay, were resurveyed after 25 years and redundancy analysis was undertaken to investigate changes in community composition. Investigations of the effect of soil conditions, site size and spatial distribution of the meadow sites were carried out. Although overall community composition had changed significantly, the suite of species representative of the meadow community had been maintained, and species usually associated with more intensively managed grasslands had declined. However, there were losses of particular species of conservation importance such as Alchemilla glabra and Conopodium majus, and losses and gains of species varied from site to site. There was a significant increase in the homogeneity of the meadow vegetation between the two survey years. Comparisons of indicators of soil conditions suggested that there had been no significant change for the community as a whole but analyses of the species showing the most change indicated a decrease in soil fertility. Low intensity management has been successful in maintaining the meadow community but consideration of changes in key species and losses at the site level is needed. More research is needed to establish whether fragmentation is starting to have an impact on diversity

Avibactam Pharmacokinetic/Pharmacodynamic Targets

ABSTRACT Avibactam is a novel non-β-lactam β-lactamase inhibitor that has been approved in the United States and Europe for use in combination with ceftazidime. Combinations of avibactam with aztreonam or ceftaroline fosamil have also been clinically evaluated. Until recently, there has been very little precedence of which pharmacokinetic/pharmacodynamic (PK/PD) indices and magnitudes are appropriate to use for β-lactamase inhibitors in population PK modeling for analyzing potential doses and susceptibility breakpoints. For avibactam, several preclinical studies using different in vitro and in vivo models have been conducted to identify the PK/PD index of avibactam and the magnitude of exposure necessary for effect in combination with ceftazidime, aztreonam, or ceftaroline fosamil. The PD driver of avibactam critical for restoring the activity of all three partner β-lactams was found to be time dependent rather than concentration dependent and was defined as the time that the concentration of avibactam exceeded a critical concentration threshold (% f T&gt;C T ). The magnitude of the C T and the time that this threshold needed to be exceeded to elicit particular PD endpoints varied depending on the model and the partner β-lactam. This review describes the preclinical studies used to determine the avibactam PK/PD target in combination with its β-lactam partners. </jats:p

Randomized Trial of Ceftazidime-Avibactam vs Meropenem for Treatment of Hospital-Acquired and Ventilator-Associated Bacterial Pneumonia (REPROVE): Analyses per US FDA-Specified End Points

Background: Hospital-acquired and ventilator-associated pneumonia (HAP/VAP; nosocomial pneumonia) due to Gram-negative pathogens are associated with significant morbidity and mortality; treatment options for multidrug-resistant infections are limited. The pivotal phase III REPROVE trial evaluated the efficacy of ceftazidime-avibactam (CAZ-AVI) vs meropenem in the treatment of patients with HAP/VAP. Study results for prespecified analyses per US Food and Drug Administration-recommended trial end points are reported here. Methods: Hospitalized adults with HAP/VAP proven or suspected to be caused by a Gram-negative pathogen were randomized 1:1 to receive CAZ-AVI or meropenem for 7 to 14 days. The primary outcome was 28-day all-cause mortality in the intent-to-treat (ITT) population. Secondary outcomes included clinical cure at test of cure (TOC) in the ITT and microbiological ITT (micro-ITT) populations, and safety and tolerability throughout the study. Results: hundred seventy randomized patients received treatment and were included in the ITT population (CAZ-AVI, n = 436; meropenem, n = 434). CAZ-AVI was noninferior to meropenem for the primary end point (28-day all-cause mortality; ITT) based on the prespecified 10% noninferiority margin (CAZ-AVI, 9.6%; meropenem, 8.3%; difference, 1.5%; 95% confidence interval [CI], -2.4% to 5.3%) and for the clinical cure end point in the ITT population based on a prespecified -10% noninferiority margin (CAZ-AVI, 67.2%; meropenem, 69.1%; difference, −1.9%; 95% CI, -8.1% to 4.3%). Clinical cure rates at TOC for patients infected with CAZ-nonsusceptible pathogens were similar (CAZ-AVI, 75.5%; meropenem, 71.2%; micro-ITT). Safety data were consistent with established safety profiles for both agents. Conclusions: CAZ-AVI provides an important new treatment option for HAP/VAP due to Gram-negative pathogens, including CAZ-nonsusceptible strains

The management and outcome for patients with chronic subdural hematoma: a prospective, multicenter, observational cohort study in the United Kingdom

Symptomatic chronic subdural hematoma (CSDH) will become an increasingly common presentation in neurosurgical practice as the population ages, but quality evidence is still lacking to guide the optimal management for these patients. The British Neurosurgical Trainee Research Collaborative (BNTRC) was established by neurosurgical trainees in 2012 to improve research by combining the efforts of trainees in each of the United Kingdom (UK) and Ireland's neurosurgical units (NSUs). The authors present the first study by the BNTRC that describes current management and outcomes for patients with CSDH throughout the UK and Ireland. This provides a resource both for current clinical practice and future clinical research on CSDH