Phosphorylation of ezrin on Thr567 is required for the synergistic activation of cell spreading by EPAC1 and protein kinase A in HEK293T cells

Recent studies have demonstrated that the actin binding protein, ezrin, and the cAMP-sensor, EPAC1, cooperate to induce cell spreading in response to elevations in intracellular cAMP. To investigate the mechanisms underlying these effects we generated a model of EPAC1-dependent cell spreading based on the stable transfection of EPAC1 into HEK293T (HEK293T–EPAC1) cells. We found that direct activation of EPAC1 with the EPAC-selective analogue, 8-pCPT-2′-O-Me-cAMP (007), promoted cell spreading in these cells. In addition, co-activation of EPAC1 and PKA, with a combination of the adenylate cyclase activator, forskolin, and the cAMP phosphodiesterase inhibitor, rolipram, was found to synergistically enhance cell spreading, in association with cortical actin bundling and mobilisation of ezrin to the plasma membrane. PKA activation was also associated with phosphorylation of ezrin on Thr567, as detected by an electrophoretic band mobility shift during SDS-PAGE. Inhibition of PKA activity blocked ezrin phosphorylation and reduced the cell spreading response to cAMP elevation to levels induced by EPAC1-activation alone. Transfection of HEK293T–EPAC1 cells with inhibitory ezrin mutants lacking the key PKA phosphorylation site, ezrin-Thr567Ala, or the ability to associate with actin, ezrin-Arg579Ala, promoted cell arborisation and blocked the ability of EPAC1 and PKA to further promote cell spreading. The PKA phospho-mimetic mutants of ezrin, ezrin-Thr567Asp had no effect on EPAC1-driven cell spreading. Our results indicate that association of ezrin with the actin cytoskeleton and phosphorylation on Thr567 are required, but not sufficient, for PKA and EPAC1 to synergistically promote cell spreading following elevations in intracellular cAMP

Moving epidemic method (MEM) applied to virology data as a novel real time tool to predict peak in seasonal influenza healthcare utilisation. The Scottish experience of the 2017/18 season to date

Scotland observed an unusual influenza A(H3N2)- dominated 2017/18 influenza season with healthcare services under significant pressure. We report the application of the moving epidemic method (MEM) to virology data as a tool to predict the influenza peak activity period and peak week of swab positivity in the current season. This novel MEM application has been successful locally and is believed to be of potential use to other countries for healthcare planning and building wider community resilience

Association of Blood Biomarkers With Acute Sport-Related Concussion in Collegiate Athletes: Findings From the NCAA and Department of Defense CARE Consortium

Importance: There is potential scientific and clinical value in validation of objective biomarkers for sport-related concussion (SRC). Objective: To investigate the association of acute-phase blood biomarker levels with SRC in collegiate athletes. Design, Setting, and Participants: This multicenter, prospective, case-control study was conducted by the National Collegiate Athletic Association (NCAA) and the US Department of Defense Concussion Assessment, Research, and Education (CARE) Consortium from February 20, 2015, to May 31, 2018, at 6 CARE Advanced Research Core sites. A total of 504 collegiate athletes with concussion, contact sport control athletes, and non-contact sport control athletes completed clinical testing and blood collection at preseason baseline, the acute postinjury period, 24 to 48 hours after injury, the point of reporting being asymptomatic, and 7 days after return to play. Data analysis was conducted from March 1 to November 30, 2019. Main Outcomes and Measures: Glial fibrillary acidic protein (GFAP), ubiquitin C-terminal hydrolase-L1 (UCH-L1), neurofilament light chain, and tau were quantified using the Quanterix Simoa multiplex assay. Clinical outcome measures included the Sport Concussion Assessment Tool-Third Edition (SCAT-3) symptom evaluation, Standardized Assessment of Concussion, Balance Error Scoring System, and Brief Symptom Inventory 18. Results: A total of 264 athletes with concussion (mean [SD] age, 19.08 [1.24] years; 211 [79.9%] male), 138 contact sport controls (mean [SD] age, 19.03 [1.27] years; 107 [77.5%] male), and 102 non-contact sport controls (mean [SD] age, 19.39 [1.25] years; 82 [80.4%] male) were included in the study. Athletes with concussion had significant elevation in GFAP (mean difference, 0.430 pg/mL; 95% CI, 0.339-0.521 pg/mL; P < .001), UCH-L1 (mean difference, 0.449 pg/mL; 95% CI, 0.167-0.732 pg/mL; P < .001), and tau levels (mean difference, 0.221 pg/mL; 95% CI, 0.046-0.396 pg/mL; P = .004) at the acute postinjury time point compared with preseason baseline. Longitudinally, a significant interaction (group × visit) was found for GFAP (F7,1507.36 = 16.18, P < .001), UCH-L1 (F7,1153.09 = 5.71, P < .001), and tau (F7,1480.55 = 6.81, P < .001); the interaction for neurofilament light chain was not significant (F7,1506.90 = 1.33, P = .23). The area under the curve for the combination of GFAP and UCH-L1 in differentiating athletes with concussion from contact sport controls at the acute postinjury period was 0.71 (95% CI, 0.64-0.78; P < .001); the acute postinjury area under the curve for all 4 biomarkers combined was 0.72 (95% CI, 0.65-0.79; P < .001). Beyond SCAT-3 symptom score, GFAP at the acute postinjury time point was associated with the classification of athletes with concussion from contact controls (β = 12.298; 95% CI, 2.776-54.481; P = .001) and non-contact sport controls (β = 5.438; 95% CI, 1.676-17.645; P = .005). Athletes with concussion with loss of consciousness or posttraumatic amnesia had significantly higher levels of GFAP than athletes with concussion with neither loss of consciousness nor posttraumatic amnesia at the acute postinjury time point (mean difference, 0.583 pg/mL; 95% CI, 0.369-0.797 pg/mL; P < .001). Conclusions and Relevance: The results suggest that blood biomarkers can be used as research tools to inform the underlying pathophysiological mechanism of concussion and provide additional support for future studies to optimize and validate biomarkers for potential clinical use in SRC

Rotation and activity in the solar-metallicity open cluster NGC2516

We report new measures of radial velocities and rotation rates (v sin i) for 51 F and early-G stars in the open cluster NGC2516, and combine these with previously published data. From high signal-to-noise spectra of two stars, we show that NGC2516 has a relative iron abundance with respect to the Pleiades of delta([Fe/H])= +0.04 +/- 0.07 at the canonical reddening of E(B - V) = 0.12, in contrast to previous photometric studies that placed the cluster 0.2 to 0.4 dex below solar. We construct a color-magnitude diagram based on radial velocity members, and explore the sensitivity of photometric determinations of the metallicity and distance to assumed values of the reddening. For a metal abundance near solar, the Hipparcos distance to NGC2516 is probably underestimated. Finally, we show that the distribution of rotation rates and X-ray emission does not differ greatly from that of the Pleiades, when allowance is made for the somewhat older age of NGC2516.Comment: Accepted for publication in the Astrophysical Journal. 35 pages including 7 figure

Affinity enrichment of extracellular vesicles from plasma reveals mRNA changes associated with acute ischemic stroke

Currently there is no in vitro diagnostic test for acute ischemic stroke (AIS), yet rapid diagnosis is crucial for effective thrombolytic treatment. We previously demonstrated the utility of CD8(+) T-cells’ mRNA expression for AIS detection; however extracellular vesicles (EVs) were not evaluated as a source of mRNA for AIS testing. We now report a microfluidic device for the rapid and efficient affinity-enrichment of CD8(+) EVs and subsequent EV’s mRNA analysis using droplet digital PCR (ddPCR). The microfluidic device contains a dense array of micropillars modified with anti-CD8α monoclonal antibodies that enriched 158 ± 10 nm sized EVs at 4.3 ± 2.1 × 109 particles/100 µL of plasma. Analysis of mRNA from CD8(+) EVs and their parental T-cells revealed correlation in the expression for AIS-specific genes in both cell lines and healthy donors. In a blinded study, 80% test positivity for AIS patients and controls was revealed with a total analysis time of 3.7 h

Evaluation of a brief pilot nutrition and exercise intervention for the prevention of weight gain in general practice patients

Objective To pilot-test a brief written prescription recommending lifestyle changes delivered by general practitioners (GPs) to their patients.Design The Active Nutrition Script (ANS) included five nutrition messages and personalised exercise advice for a healthy lifestyle and/or the prevention of weight gain. GPs were asked to administer 10 scripts over 4 weeks to 10 adult patients with a body mass index (BMI) of between 23 and 30 kg m&minus; 2. Information recorded on the script consisted of patients\u27 weight, height, waist circumference, gender and date of birth, type and frequency of physical activity prescribed, and the selected nutrition messages. GPs also recorded reasons for administering the script. Interviews recorded GPs views on using the script.Setting General practices located across greater Melbourne.Subjects and results Nineteen GPs (63% female) provided a median of nine scripts over 4 weeks. Scripts were administered to 145 patients (mean age: 54 &plusmn; 13.2 years, mean BMI: 31.7 &plusmn; 6.3 kg m&minus; 2; 57% female), 52% of whom were classified as obese (BMI &gt;30 kg m&minus; 2). GPs cited &lsquo;weight reduction&rsquo; as a reason for writing the script for 78% of patients. All interviewed GPs (90%, n = 17) indicated that the messages were clear and simple to deliver.Conclusions GPs found the ANS provided clear nutrition messages that were simple to deliver. However, GPs administered the script to obese patients for weight loss rather than to prevent weight gain among the target group. This has important implications for future health promotion interventions designed for general practice.<br /

Confidence intervals for a spatially generalized, continuous simulation flood frequency model for Great Britain

There is growing interest in the application of "continuous simulation'' conceptual rainfall-runoff models for flood frequency estimation as an adjunct to event-based or statistical design methodology. The approach has advantages that stem from the use of models with continuous water balance accounting. Conceptual rainfall-runoff models usually require calibration, which in turn requires gauged rainfall and flow data. One of the key challenges is therefore to develop ways of generalizing models for use at ungauged sites. Recent work has produced a prototype scheme for achieving this aim in Great Britain for two catchment models by relating model parameters to spatial catchment properties, such as soils, topography, and geology. In this paper we present an analysis of the uncertainty associated with one of the generalized models ( the "probability distributed model'') in terms of confidence intervals for simulations at test sites that are treated as if they were ungauged. This is done by fitting regression relationships between hydrological model parameters and catchment properties so as to estimate the parameters as distribution functions for the ungauged site case. Flood flow outputs are then simulated from the parameter distributions and used to construct approximate confidence intervals. Comparison with gauged data suggests that the generalized model may be tentatively accepted. Uncertainty in the modeled flood flows is often of a similar order to the uncertainty surrounding a more conventional statistical model, in this case a single-site generalized Pareto distribution fitted to the gauged data

TLR7-mediated skin inflammation remotely triggers chemokine expression and leukocyte accumulation in the brain

Background: The relationship between the brain and the immune system has become increasingly topical as, although it is immune-specialised, the CNS is not free from the influences of the immune system. Recent data indicate that peripheral immune stimulation can significantly affect the CNS. But the mechanisms underpinning this relationship remain unclear. The standard approach to understanding this relationship has relied on systemic immune activation using bacterial components, finding that immune mediators, such as cytokines, can have a significant effect on brain function and behaviour. More rarely have studies used disease models that are representative of human disorders. Methods: Here we use a well-characterised animal model of psoriasis-like skin inflammation—imiquimod—to investigate the effects of tissue-specific peripheral inflammation on the brain. We used full genome array, flow cytometry analysis of immune cell infiltration, doublecortin staining for neural precursor cells and a behavioural read-out exploiting natural burrowing behaviour. Results: We found that a number of genes are upregulated in the brain following treatment, amongst which is a subset of inflammatory chemokines (CCL3, CCL5, CCL9, CXCL10, CXCL13, CXCL16 and CCR5). Strikingly, this model induced the infiltration of a number of immune cell subsets into the brain parenchyma, including T cells, NK cells and myeloid cells, along with a reduction in neurogenesis and a suppression of burrowing activity. Conclusions: These findings demonstrate that cutaneous, peripheral immune stimulation is associated with significant leukocyte infiltration into the brain and suggest that chemokines may be amongst the key mediators driving this response

Intercultural ethics: questions of methods in language and intercultural communication

This paper explores how questions of ethics and questions of method are intertwined and unavoidable in any serious study of language and intercultural communication. It argues that the focus on difference and solution orientations to intercultural conflict has been a fundamental driver for theory, data collection and methods in the field. These approaches, the paper argues, have created a considerable consciousness raising industry, with methods, trainings and ‘critical incidents’, which ultimately focus intellectual energy in areas which may be productive in terms of courses and publications but which have a problematic basis in their ethical terrain. Dieser Artikel untersucht wie ethische und methodische Fragen nicht nur ineinander greifen, sondern in keiner ernstzunehmenden Studie ueber Sprache und interkulturelle Kommunikation ausgelassen werden duerfen. Es wird hier argumentiert, dass der Schwerpunkt auf Verschiedenheit und Problemorientierung im interkulturellen Konflikt einen wesentlichen Einfluss auf theoretische Entwicklungen, Datenerhebung und Methoden in diesem Bereich hatte. Dieser Artikel legt auch dar, wie diese Ansaetze eine betraechtliche ‘Bewusstseinsbildungs – Branche' erzeugt haben, mit Methoden, Trainings, und ‘kritischen Interaktionssituationen’, welche letztendlich allen intellektuellen Arbeitseifer auf Bereiche konzentriert hat, die zwar ertragreich sind in Bezug auf Kurse und Publikationen, jedoch eine problematische Grundlage im ethischen Bereich aufweisen

Australian and New Zealand Pulmonary Rehabilitation Guidelines

Background and objective: The aim of the Pulmonary Rehabilitation Guidelines (Guidelines) is to provide evidence-based recommendations for the practice of pulmonary rehabilitation (PR) specific to Australian and New Zealand healthcare contexts. Methods: The Guideline methodology adhered to the Appraisal of Guidelines for Research and Evaluation (AGREE) II criteria. Nine key questions were constructed in accordance with the PICO (Population, Intervention, Comparator, Outcome) format and reviewed by a COPD consumer group for appropriateness. Systematic reviews were undertaken for each question and recommendations made with the strength of each recommendation based on the GRADE (Gradings of Recommendations, Assessment, Development and Evaluation) criteria. The Guidelines were externally reviewed by a panel of experts. Results: The Guideline panel recommended that patients with mild-to-severe COPD should undergo PR to improve quality of life and exercise capacity and to reduce hospital admissions; that PR could be offered in hospital gyms, community centres or at home and could be provided irrespective of the availability of a structured education programme; that PR should be offered to patients with bronchiectasis, interstitial lung disease and pulmonary hypertension, with the latter in specialized centres. The Guideline panel was unable to make recommendations relating to PR programme length beyond 8 weeks, the optimal model for maintenance after PR, or the use of supplemental oxygen during exercise training. The strength of each recommendation and the quality of the evidence are presented in the summary. Conclusion: The Australian and New Zealand Pulmonary Rehabilitation Guidelines present an evaluation of the evidence for nine PICO questions, with recommendations to provide guidance for clinicians and policymakers