Low-density lipoprotein modified by myeloperoxidase oxidants induces endothelial dysfunction

Low-density lipoprotein (LDL) modified by hypochlorous acid (HOCl) produced by myeloperoxidase (MPO) is present in atherosclerotic lesions, where it is implicated in the propagation of inflammation and acceleration of lesion development by multiple pathways, including the induction of endothelial dysfunction. Thiocyanate (SCN-) ions are utilised by MPO to produce the oxidant hypothiocyanous acid (HOSCN), which reacts with LDL in a different manner to HOCl. Whilst the reactivity of HOCl-modified LDL has been previously studied, the role of HOSCN in the modification of LDL in vivo is poorly defined, although emerging evidence suggests that these particles have distinct biological properties. This is important because elevated plasma SCN- is linked with both the propagation and prevention of atherosclerosis. In this study, we demonstrate that both HOSCN- and HOCl-modified LDL inhibit endothelium-mediated vasorelaxation ex vivo in rat aortic ring segments. In vitro experiments with human coronary artery endothelial cells show that HOSCN-modified LDL decreases in the production of nitric oxide (NO•) and induces the loss of endothelial nitric oxide synthase (eNOS) activity. This occurs to a similar extent to that seen with HOCl-modified LDL. In each case, these effects are related to eNOS uncoupling, rather than altered expression, phosphorylation or cellular localisation. Together, these data provide new insights into role of MPO and LDL modification in the induction of endothelial dysfunction, which has implications for both the therapeutic use of SCN- within the setting of atherosclerosis and for smokers, who have elevated plasma levels of SCN-, and are more at risk of developing cardiovascular disease

Assessment of the stress relaxation characteristics of critical gels formed under unidirectional shear flow by controlled stress parallel superposition rheometry

Processes involving a unidirectional shear flow component are widespread in industrial manufacturing techniques such as printing and coating, or in physiological events such as blood coagulation. Standard rheometric techniques are usually employed under quiescent conditions and as such are inappropriate for the study of microstructural modification induced by the presence of a unidirectional shear flow. We demonstrate how controlled stress parallel superposition (CSPS) may be exploited to enable accurate detection of the Gel Point and analysis of Gel Point parameters for systems undergoing a viscoelastic liquid (VEL) to viscoelastic solid (VES) transition in the presence of a unidirectional flow field. Specifically, we note that certain features of the CSPS experiment, when performed near the Gel Point, may obviate previously reported concerns regarding the experiment. A biopolymer system (gelatin) which forms gels by thermoreversible gelation is employed as a model gelling material to confirm the ability of CSPS to characterise the stress relaxation characteristics of critical-gels in the presence of a) progressively decreasing and b) progressively increasing unidirectional strain rate and oscillatory strain amplitude. Additional validation of CSPS results is reported for a silicone dielectric gel used in the industrial production of printed electronic products. Finally, CSPS is used to investigate microstructural modification of fibrin-thrombin gels as a consequence of clot formation under a unidirectional shear stress. The results confirm the validity of the CSPS technique in gelation studies and the technique is used, for the first time, to directly record the thermally induced VES to VEL transition in aqueous gelatin systems

The treatment effect of rivaroxaban on clot characteristics in patients who present acutely with first time deep vein thrombosis

BACKGROUND: The acute vascular disease deep vein thrombosis (DVT) requires oral anticoagulants to prevent progression. Monitoring therapeutic efficacy of direct oral anticoagulants (DOAC), including rivaroxaban, is problematic as no reliable test is available. Advances in rheometry have led to the development of a functional coagulation biomarker using Gel Point (GP) analysis which assesses clot structure formation. The biomarker measures incipient clot formation time (TGP) and quantifies fibrin clot structure in terms of fractal dimension (df).OBJECTIVE: This study aimed to investigate clot structure formation in first time DVT and the effect of rivaroxaban treatment.METHODS: This prospective observational cohort study measured the GP and standard laboratory markers at three sample points: pre-treatment and at 20 and 60 days following 15mg BD and 20mg OD rivaroxaban respectively. RESULTS: Forty DVT patients (mean age 64 years [SD±14.8]; 23 males, 17 female) were recruited. The results show that DVT vs non-DVT patients did not have a significantly different GP profile (df: 1.72±0.06 vs 1.70±0.06 and TGP: 267±68sec vs 262±73sec) with both within the defined healthy index. In addition, rivaroxaban therapy increased TGP to 392s (±135s) after 20 days, and subsequently increased to 395s (±194s) at 60 days but did not significantly increase df (from 1.69±0.05 to 1.71±0.06). CONCLUSIONS: The results indicate in this cohort of DVT patients there was no underlying hypercoagulable effect as determined by gel point analysis. Furthermore, the anticoagulant effect of rivaroxaban prolonged clotting, suggesting a protective effect against clot formation, without significantly reducing clot microstructural properties

Approaches used by parents to keep their children safe at home: a qualitative study to explore the perspectives of parents with children aged under five years

BACKGROUND: Childhood unintentional injury represents an important global health problem. Many unintentional injuries experienced by children aged under 5years occur within the home and are preventable. The aim of this study was to explore the approaches used by parents of children under five in order to help prevent unintentional injuries in the home and the factors which influence their use. Understanding how parents approach risk-management in the home has important implications for injury practitioners. METHODS: A multi-centre qualitative study using semi-structured interviews. A thematic approach was used to analyse the data. Sixty five parents of children aged under 5years, from four study areas were interviewed: Bristol, Newcastle, Norwich and Nottingham. RESULTS: Three main injury prevention strategies used by parents were: a) Environmental such as removal of hazards, and use of safety equipment; b) parental supervision; and c) teaching, for example, teaching children about safety and use of rules and routine. Strategies were often used in combination due to their individual limitations. Parental assessment of injury risk, use of strategy and perceived effectiveness were fluid processes dependent on a child's character, developmental age and the prior experiences of both parent and child. Some parents were more proactive in their approach to home safety while others only reacted if their child demonstrated an interest in a particular object or activity perceived as being an injury risk. CONCLUSION: Parents' injury prevention practices encompass a range of strategies that are fluid in line with the child's age and stage of development; however, parents report that they still find it challenging to decide which strategy to use and when

The influence of nicotinamide on the development of neurons

This document is the Accepted Manuscript version of a published work that appeared in final form in Journal of Neurology, Neurosurgery and Psychiatry. To access the final edited and published work see http://dx.doi.org/10.1136/jnnp-2014-309236.199A major challenge in translating the promise of stem cell therapies to treat a myriad of neurodegenerative disorders is to rapidly and efficiently direct pluripotent stem cells to generate differentiated neurons. The application of active vitamin metabolites known to function in embryonic development and maintenance in the adult brain such as retinoic acid (vitamin A), ascorbic acid (vitamin C) and calcitriol (vitamin D3) have proven effective in current in-vitro differentiation protocols. Therefore, in this study we investigated whether the biologically active vitamin B3 metabolite, nicotinamide could enhance the differentiation of mouse embryonic stem cells, cultured as monolayers, into mature neurons at either early or late stages of development. Interestingly, nicotinamide elicited a dose-responsive increase in the percentage of neurons when added at an early developmental stage to the cells undergoing differentiation (days 0–7). Nicotinamide (10 mM) increased the proportion of β-III tubulin positive neurons by two fold and concomitantly decreased the total number of cells in culture, measured by quantification of 4′, 6-diamidino-2-phenylindole positive cells. This effect could result from induction of cell-cycle exit and/or selective cell death in non-neural populations. Higher levels of nicotinamide (20 mM) induced cytoxicity and cell death. This study supports previous evidence that vitamins and their metabolites can efficiently direct stem cells into neurons. Current work is focusing on the effect of nicotinamide on the process of neural induction and whether nicotinamide influences the generation of particular neuronal subtypes implicated in neurodegenerative diseases, specifically focusing on midbrain dopamine neurons; towards a therapy for Parkinson's disease

Recruitment and retention strategies and the examination of attrition bias in a randomised controlled trial in children’s centres serving families in disadvantaged areas of England

Background Failure to retain participants in randomised controlled trials and longitudinal studies can cause significant methodological problems. We report the recruitment and retention strategies of a randomised controlled trial to promote fire-related injury prevention in families with pre-school children attending children’s centres in disadvantaged areas in England. Methods Thirty-six children’s centres were cluster randomised into one of three arms of a 12-month fire-related injury prevention trial. Two arms delivered safety interventions and there was one control arm. Retention rates compared the numbers of participants responding to the 12-month questionnaire to the number recruited to the trial. Multivariable random effects logistic regression was used to explore factors independently associated with participant retention. Results The trial exceeded its required sample size through the use of multiple recruitment strategies. All children’s centres remained in the study, despite increased reorganisation. Parent retention was 68% at 12 months, ranging from 65% to 70% across trial arms and from 62% to 74% across trial sites. There was no significant difference in the rates of retention between trial arms (p = 0.58) or between trial sites (p = 0.16). Retention was significantly lower amongst mothers aged 16–25 years than older mothers [adjusted odds ratio (AOR) 0.57, 95% CI 0.41, 0.78], those living in non-owner occupied accommodation than in owner occupied accommodation (AOR 0.53, 95% CI 0.38, 0.73) and those living in more disadvantaged areas (most versus least disadvantaged quintiles AOR 0.50, 95% CI 0.30, 0.82). Conclusions Studies recruiting disadvantaged populations should measure and report attrition by socioeconomic factors to enable determination of the extent of attrition bias and estimation of its potential impact on findings. Where differential attrition is anticipated, consideration should be given to over-sampling during recruitment and targeted and more intensive strategies of participant retention in these sub-groups. In transient populations collection of multiple sources of contact information at recruitment and throughout the study may aid retention

Estradiol effects on the dopamine transporter – protein levels, subcellular location, and function

BACKGROUND: The effects of estrogens on dopamine (DA) transport may have important implications for the increased incidence of neurological disorders in women during life stages when hormonal fluctuations are prevalent, e.g. during menarche, reproductive cycling, pregnancy, and peri-menopause. RESULTS: The activity of the DA transporter (DAT) was measured by the specific uptake of (3)H-DA. We found that low concentrations (10(-14 )to 10(-8 )M) of 17β-estradiol (E(2)) inhibit uptake via the DAT in PC12 cells over 30 minutes, with significant inhibition taking place due to E(2 )exposure during only the last five minutes of the uptake period. Such rapid action suggests a non-genomic, membrane-initiated estrogenic response mechanism. DAT and estrogen receptor-α (ERα) were elevated in cell extracts by a 20 ng/ml 2 day NGFβ treatment, while ERβ was not. DAT, ERα and ERβ were also detectable on the plasma membrane of unpermeabilized cells by immunocytochemical staining and by a fixed cell, quantitative antibody (Ab)-based plate assay. In addition, PC12 cells contained RNA coding for the alternative membrane ER GPR30; therefore, all 3 ER subtypes are candidates for mediating the rapid nongenomic actions of E(2). At cell densities above 15,000 cells per well, the E(2)-induced inhibition of transport was reversed. Uptake activity oscillated with time after a 10 nM E(2 )treatment; in a slower room temperature assay, inhibition peaked at 9 min, while uptake activity increased at 3 and 20–30 min. Using an Ab recognizing the second extracellular loop of DAT (accessible only on the outside of unpermeabilized cells), our immunoassay measured membrane vs. intracellular/nonvesicular DAT; both were found to decline over a 5–60 min E(2 )treatment, though immunoblot analyses demonstrated no total cellular loss of protein. CONCLUSION: Our results suggest that physiological levels of E(2 )may act to sequester DAT in intracellular compartments where the transporter's second extramembrane loop is inaccessible (inside vesicles) and that rapid estrogenic actions on this differentiated neuronal cell type may be regulated via membrane ERs of several types