Diazepam Accelerates GABAAR Synaptic Exchange and Alters Intracellular Trafficking

Despite 50+ years of clinical use as anxiolytics, anti-convulsants, and sedative/hypnotic agents, the mechanisms underlying benzodiazepine (BZD) tolerance are poorly understood. BZDs potentiate the actions of gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the adult brain, through positive allosteric modulation of γ2 subunit containing GABA type A receptors (GABAARs). Here we define key molecular events impacting γ2 GABAAR and the inhibitory synapse gephyrin scaffold following initial sustained BZD exposure in vitro and in vivo. Using immunofluorescence and biochemical experiments, we found that cultured cortical neurons treated with the classical BZD, diazepam (DZP), presented no substantial change in surface or synaptic levels of γ2-GABAARs. In contrast, both γ2 and the postsynaptic scaffolding protein gephyrin showed diminished total protein levels following a single DZP treatment in vitro and in mouse cortical tissue. We further identified DZP treatment enhanced phosphorylation of gephyrin Ser270 and increased generation of gephyrin cleavage products. Selective immunoprecipitation of γ2 from cultured neurons revealed enhanced ubiquitination of this subunit following DZP exposure. To assess novel trafficking responses induced by DZP, we employed a γ2 subunit containing an N terminal fluorogen-activating peptide (FAP) and pH-sensitive green fluorescent protein (γ2pHFAP). Live-imaging experiments using γ2pHFAP GABAAR expressing neurons identified enhanced lysosomal targeting of surface GABAARs and increased overall accumulation in vesicular compartments in response to DZP. Using fluorescence resonance energy transfer (FRET) measurements between α2 and γ2 subunits within a GABAAR in neurons, we identified reductions in synaptic clusters of this subpopulation of surface BZD sensitive receptor. Additional time-series experiments revealed the gephyrin regulating kinase ERK was inactivated by DZP at multiple time points. Moreover, we found DZP simultaneously enhanced synaptic exchange of both γ2-GABAARs and gephyrin using fluorescence recovery after photobleaching (FRAP) techniques. Finally we provide the first proteomic analysis of the BZD sensitive GABAAR interactome in DZP vs. vehicle treated mice. Collectively, our results indicate DZP exposure elicits down-regulation of gephyrin scaffolding and BZD sensitive GABAAR synaptic availability via multiple dynamic trafficking processes

Technological resources and personnel costs required to implement an automated alert system for ambulatory physicians when patients are discharged from hospitals to home

Background With the adoption of electronic medical records by medical group practices, there are opportunities to improve the quality of care for patients discharged from hospitals. However, there is little guidance for medical groups outside integrated hospital systems to automate the flow of patient information during transitions in care.Objective To describe the technological resources, expertise and time needed to develop an automated system providing information to ambulatory physicians when their patients are discharged from hospitals to home.Development Within a medical group practice, we developed an automated alert system that provides notification of discharges, reminders of the need for follow-up visits, drugs added during inpatient stays, and recommendations for laboratory monitoring of high-risk drugs. We tracked components of the information system required and the time spent by team members. We used USA national averages of hourly wages to estimate personnel costs.Application Critical components of the information system are notifications of hospital discharges through an admission, discharge and transfer registration (ADT) interface, linkage to the group’s scheduling system, access to information on pharmacy dispensing and lab tests, and an interface engine. Total personnel cost was $76,314. Nearly half (47%) was for 614 hours by physicians who developed content, provided overall project management, and reviewed alerts to ensure that only ‘actionable’ alerts would be sent.Conclusion Implementing a system to provide information about hospital discharges requires strong internal informatics expertise, cooperation between facilities and ambulatory providers, development of electronic linkages, and extensive commitment of physician time

Report on WRF model sensitivity studies and specifications for the mesoscale wind atlas production runs:Deliverable D4.3

This report describes the sensitivity studies performed with the mesoscale model WRF in preparation of the mesoscale wind atlas production runs. The objective of this work was to find a model setup that is not just a best practice setup but well-founded and based on scientific evaluation. We started with performing some initial sensitivity experiments changing the PBL scheme and the initialisation of the model. The work was distributed among several partners, each conducting the same set of experiments but on a different domain. The objective of this first phase was to ensure that everybody speaks the same language in terms of applying WRF in the context of NEWA. The results were analysed and compared in terms of the mean wind climate. To draw conclusions regarding the quality of the experiments, the results of one domain were compared to tall mast observations. Overall the model showed a good performance with slightly better results for one of the two tested PBL schemes (MYNN) and weekly initialisation of simulations (compared to daily). In the next phase, further sensitivity tests were conducted for one of the previously defined domains, varying a multitude of parameters as e.g. model version, vertical resolution, forcing data and land surface parameterisation. These studies showed that virtually each parameter change is affecting the results in some way, while significant effects on the wind climate are mostly obtained by changes in physical parameterisation e.g. PBL scheme, representation of the land surface and surface roughness. However, also non-physical parameters as the simulation length and the domain size affects the results considerably. The results suggest to use rather small domains and not too long simulations (in the order of 1–2 weeks). One of the objectives of NEWA is to create a probabilistic wind atlas, i.e. to provide uncertainty information to the mesoscale wind atlas (see Deliverables D3.1 and D4.4). This will be achieved by generating an ensemble of WRF simulations with different model configurations. While the final ensemble to be run over the complete NEWA domain will only include a few members, a much larger ensemble was run for a smaller sub-domain to find the ensemble members that generate the largest spread and will be used in the final NEWA ensemble. A second objective of this initial large ensemble was to find an optimal setup for the mesoscale production run. Based on the experience gained in the previous sensitivity experiments, a 47-member ensemble was assembled and run. The individual members were compared against each other, as well as against tall mast observations. Different metrics were explored to assess the performance of the members, i.e. not only the usual statistical measures as RMSE, BIAS and correlation but also metrics that compare the wind speed distributions. In the final part of this report we present the ultimate WRF setup for the NEWA production run that was run between August 2018 and March 2019 on the MareNostrum supercomputer in Barcelona

Improving the physician-patient cardiovascular risk dialogue to improve statin adherence

<p>Abstract</p> <p>Background</p> <p>The purpose of this study was to evaluate the effectiveness of a patient education program developed to facilitate statin adherence.</p> <p>Methods</p> <p>A controlled trial was designed to test the effectiveness of a multifaceted patient education program to facilitate statin adherence. The program included a brief, in-office physician counseling kit followed by patient mailings. The primary end point was adherence to filling statin prescriptions during a 120-day period. Patients new to statins enrolled and completed a survey. Data from a national pharmacy claims database were used to track adherence.</p> <p>Results</p> <p>Patients new to statin therapy exposed to a patient counseling and education program achieved a 12.4 higher average number of statin prescription fill days and were 10% more likely to fill prescriptions for at least 120 days (<it>p </it>= .01).</p> <p>Conclusion</p> <p>Brief in-office counseling on cardiovascular risk followed by patient education mailings can be effective in increasing adherence. Physicians found a one-minute counseling tool and pocket guidelines useful in counseling patients.</p

Communication in health practices: integrative literature review

Objectivethis study aims to describe the main thematic axes explored in the communication field in health practices in the scenarios of the Unified Health System (SUS). Methodintegrative literature review conducted by means of search for articles in the databases Latin American Literature on Health Sciences (LILACS), International Literature on Health Sciences (MedLine), and Science Direct, using the descriptors: health communication or communication. A crossing of the descriptors communication and health education was provided. Resultfour themes were constructed: 1) communication to establish relationships between health professionals and users; 2) (in)communication: barriers to the communicative act, 3) communication and health professional education; and 4) communicative health models: search for the dialogic model. Conclusionby understanding dialogic communication, which must be observed in communication, the new requirements posed by the legalization of SUS have shown weaknesses of the single-line and vertical communication model and the need to provide health professionals, since the undergraduate course, with knowledge that enable dialogic communication practices. The challenge of reflective and participatory experiences in the various health care settings still remains, in order to promote a sharing of knowledge that leads to understanding between the interlocutors involved in the communicative act.Objetivoeste artigo tem por objetivo descrever os principais eixos temáticos explorados no campo da comunicação nas práticas em saúde nos cenários do Sistema Único de Saúde (SUS). Métodorevisão integrativa da literatura realizada a partir da busca de artigos nas bases de dados Literatura Latino-Americana em Ciências da Saúde (Lilacs), Literatura Internacional em Ciências da Saúde (MedLine) e Science Direct, utilizando os descritores: comunicação em saúde ou comunicação. Procedeu-se ao cruzamento dos descritores comunicação e educação em saúde. Resultadoforam construídas quatro temáticas: 1) a comunicação no estabelecimento de relações entre profissionais da saúde e usuários; 2) (des)comunicação: barreiras ao ato comunicativo; 3) comunicação e formação do profissional da saúde; e 4) modelos comunicativos em saúde: a busca pelo modelo dialógico. Conclusãoa partir do entendimento da comunicação dialógica, que deve estar presente na comunicação, as novas demandas da legalização do SUS vêm mostrando fragilidades do modelo unilinear e verticalizado de comunicação e a necessidade de instrumentalizar os profissionais da saúde, desde a graduação, com saberes que proporcionem práticas comunicativas dialógicas. Persiste o desafio de vivências reflexivas e participativas nos vários cenários de assistência à saúde, de forma a promover um compartilhamento de saberes que conduza ao entendimento entre os interlocutores envolvidos no ato comunicativo.Universidade Federal de PernambucoUniversidade Federal de Pernambuco Departamento de FonoaudiologiaUniversidade Federal de São Paulo (UNIFESP) Centro de Desenvolvimento do Ensino Superior em SaúdeUniversidade Federal de Pernambuco Departamento de EnfermagemUNIFESP, Centro de Desenvolvimento do Ensino Superior em SaúdeSciEL

γ2 GABAAR Trafficking and the Consequences of Human Genetic Variation

GABA type A receptors (GABAARs) mediate the majority of fast inhibitory neurotransmission in the central nervous system (CNS). Most prevalent as heteropentamers composed of two α, two β, and a γ2 subunit, these ligand-gated ionotropic chloride channels are capable of extensive genetic diversity (α1-6, β1-3, γ1-3, δ, , , π, ρ1-3). Part of this selective GABAAR assembly arises from the critical role for γ2 in maintaining synaptic receptor localization and function. Accordingly, mutations in this subunit account for over half of the known epilepsy-associated genetic anomalies identified in GABAARs. Fundamental structure–function studies and cellular pathology investigations have revealed dynamic GABAAR trafficking and synaptic scaffolding as critical regulators of GABAergic inhibition. Here, we introduce in vitro and in vivo findings regarding the specific role of the γ2 subunit in receptor trafficking. We then examine γ2 subunit human genetic variation and assess disease related phenotypes and the potential role of altered GABAAR trafficking. Finally, we discuss new-age imaging techniques and their potential to provide novel insight into critical regulatory mechanisms of GABAAR function

Benzodiazepine treatment induces subtype-specific changes in GABA(A) receptor trafficking and decreases synaptic inhibition

Benzodiazepines potentiate gamma-aminobutyric acid type A receptor (GABA(A)R) activity and are widely prescribed to treat anxiety, insomnia, and seizure disorders. Unfortunately, clinical use of benzodiazepines (BZs) is severely limited by tolerance. The mechanisms leading to BZ tolerance are unknown. BZs bind at the interface between an a and gamma subunit of GABA(A)Rs, preferentially enhancing synaptic receptors largely composed of alpha(1-3, 5), beta 3, and gamma 2 subunits. Using confocal imaging and patch-clamp approaches, we show that treatment with the BZ flurazepam decreases GABA(A)R surface levels and the efficacy of neuronal inhibition in hippocampal neurons. A dramatic decrease in surface and total levels of alpha 2 subunit-containing GABA(A)Rs occurred within 24 h of flurazepam treatment, whereas GABA(A)Rs incorporating alpha 1 subunits showed little alteration. The GABA(A)R surface depletion could be reversed by treatment with the BZ antagonist Ro 15-1788. Coincident with decreased GABA(A)R surface levels, flurazepamtreatment reduced miniature inhibitory postsynaptic current amplitude, which returned to control levels with acute Ro 15-1788 treatment. GABA(A)R endocytosis and insertion rates were unchanged by flurazepam treatment. Treatment with leupeptin restored flurazepam lowered receptor surface levels, strongly suggesting that flurazepam increases lysosomal degradation of GABA(A)Rs. Together, these data suggest that flurazepam exposure enhances degradation of alpha 2 subunit-containing GABA(A)Rs after their removal from the plasma membrane, leading to a reduction in inhibitory synapse size and number along with a decrease in the efficacy of synaptic inhibition. These reported subtype-specific changes in GABA(A)R trafficking provide significant mechanistic insight into the initial neuroadaptive responses occurring with BZ treatment

NEWA Report on uncertainty quantification Deliverable D4.4

This report explores the quantification of uncertainty in the NEWA project. Uncertainty is un- derstood here as the result of the contributions of model sensitivity to different model setups, and of model errors in a model-data comparison framework. The first part (Sec 3 of this report ex- plores the uncertainty derived from model sensitivity subjected to the decisions taken regarding the use of different models setups and how these produce variability in model output. The range of this variability has been regarded as spread in model output and has been quantified in various manners. The second part of this report (Sec. 4 addresses how model performance can be char- acterised with the data at hand and whether decisions regarding selection of a given model setup for a production run can be taken on the basis of model performance in a variety of situations, using different variables and datasets as observational targets: wind farm data from Vestas; tall masts and wind profiles; surface wind data; satellite data and reanalysis outputs

The making of the New European Wind Atlas – Part 1: Model sensitivity

This is the first of two papers that document the creation of the New European Wind Atlas (NEWA). It describes the sensitivity analysis and evaluation procedures that formed the basis for choosing the final setup of the mesoscale model simulations of the wind atlas. The suitable combination of model setup and parameterizations, bound by practical constraints, was found for simulating the climatology of the wind field at turbine-relevant heights with the Weather Research and Forecasting (WRF) model. Initial WRF model sensitivity experiments compared the wind climate generated by using two commonly used planetary boundary layer schemes and were carried out over several regions in Europe. They confirmed that the most significant differences in annual mean wind speed at 100 m a.g.l. (above ground level) mostly coincide with areas of high surface roughness length and not with the location of the domains or maximum wind speed. Then an ensemble of more than 50 simulations with different setups for a single year was carried out for one domain covering northern Europe for which tall mast observations were available. We varied many different parameters across the simulations, e.g. model version, forcing data, various physical parameterizations, and the size of the model domain. These simulations showed that although virtually every parameter change affects the results in some way, significant changes in the wind climate in the boundary layer are mostly due to using different physical parameterizations, especially the planetary boundary layer scheme, the representation of the land surface, and the prescribed surface roughness length. Also, the setup of the simulations, such as the integration length and the domain size, can considerably influence the results. We assessed the degree of similarity between winds simulated by the WRF ensemble members and the observations using a suite of metrics, including the Earth Mover¿s Distance (EMD), a statistic that measures the distance between two probability distributions. The EMD was used to diagnose the performance of each ensemble member using the full wind speed and direction distribution, which is essential for wind resource assessment. We identified the most realistic ensemble members to determine the most suitable configuration to be used in the final production run, which is fully described and evaluated in the second part of this study (Dörenkämper et al., 2020).The European Commission (EC) partly funded the NEWA project (New European Wind Atlas) through FP7 (topic FP7-ENERGY.2013.10.1.2) The authors of this paper acknowledge the support the Danish Energy Authority (EUDP 14-II, 64014-0590, Denmark); the German Federal Ministry for the Economic Affairs and Energy, on the basis of the decision by the German Bundestag (ref. no. 0325832A/B); Latvijas Zinatnu Akademija (Latvia); Ministerio de Economía y Competitividad (Spain, ref. nos. PCIN-2014-017-C07-03, PCIN-2016-176, PCIN-2014-017-C07-04, and PCIN-2016-009); the Swedish Energy Agency (Sweden); and the Scientific and Technological Research Council of Turkey (grant no. 215M386). Andrea N. Hahmann additionally acknowledges the support of the Danish Ministry of Foreign Affairs, administered by the Danida Fellowship Centre under the project “Multiscale and Model-Chain Evaluation of Wind Atlases” (MEWA) and the ForskEL/EUDP (Denmark) project OffshoreWake (PSO-12521, EUDP 64017-0017)