cGMP-dependent protein kinase Iα associates with the antidepressant-sensitive serotonin transporter and dictates rapid modulation of serotonin uptake

<p>Abstract</p> <p>Background</p> <p>The Na⁺/Cl^--dependent serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) is a critical element in neuronal 5-HT signaling, being responsible for the efficient elimination of 5-HT after release. SERTs are not only targets for exogenous addictive and therapeutic agents but also can be modulated by endogenous, receptor-linked signaling pathways. We have shown that neuronal A3 adenosine receptor activation leads to enhanced presynaptic 5-HT transport <it>in vitro </it>and an increased rate of SERT-mediated 5-HT clearance <it>in vivo</it>. SERT stimulation by A3 adenosine receptors derives from an elevation of cGMP and subsequent activation of both cGMP-dependent protein kinase (PKG) and p38 mitogen-activated protein kinase. PKG activators such as 8-Br-cGMP are known to lead to transporter phosphorylation, though how this modification supports SERT regulation is unclear.</p> <p>Results</p> <p>In this report, we explore the kinase isoform specificity underlying the rapid stimulation of SERT activity by PKG activators. Using immortalized, rat serotonergic raphe neurons (RN46A) previously shown to support 8-Br-cGMP stimulation of SERT surface trafficking, we document expression of PKGI, and to a lower extent, PKGII. Quantitative analysis of staining profiles using permeabilized or nonpermeabilized conditions reveals that SERT colocalizes with PKGI in both intracellular and cell surface domains of RN46A cell bodies, and exhibits a more restricted, intracellular pattern of colocalization in neuritic processes. In the same cells, SERT demonstrates a lack of colocalization with PKGII in either intracellular or surface membranes. In keeping with the ability of the membrane permeant kinase inhibitor DT-2 to block 8-Br-cGMP stimulation of SERT, we found that DT-2 treatment eliminated cGMP-dependent kinase activity in PKGI-immunoreactive extracts resolved by liquid chromatography. Similarly, treatment of SERT-transfected HeLa cells with small interfering RNAs targeting endogenous PKGI eliminated 8-Br-cGMP-induced regulation of SERT activity. Co-immunoprecipitation studies show that, in transporter/kinase co-transfected cells, PKGIα specifically associates with hSERT.</p> <p>Conclusion</p> <p>Our findings provide evidence of a physical and compartmentalized association between SERT and PKGIα that supports rapid, 8-Br-cGMP-induced regulation of SERT. We discuss a model wherein SERT-associated PKGIα supports sequentially the mobilization of intracellular transporter-containing vesicles, leading to enhanced surface expression, and the production of catalytic-modulatory SERT phosphorylation, leading to a maximal enhancement of 5-HT clearance capacity.</p

BioHackathon series in 2011 and 2012: penetration of ontology and linked data in life science domains

The application of semantic technologies to the integration of biological data and the interoperability of bioinformatics analysis and visualization tools has been the common theme of a series of annual BioHackathons hosted in Japan for the past five years. Here we provide a review of the activities and outcomes from the BioHackathons held in 2011 in Kyoto and 2012 in Toyama. In order to efficiently implement semantic technologies in the life sciences, participants formed various sub-groups and worked on the following topics: Resource Description Framework (RDF) models for specific domains, text mining of the literature, ontology development, essential metadata for biological databases, platforms to enable efficient Semantic Web technology development and interoperability, and the development of applications for Semantic Web data. In this review, we briefly introduce the themes covered by these sub-groups. The observations made, conclusions drawn, and software development projects that emerged from these activities are discussed

The Changing Landscape for Stroke\ua0Prevention in AF: Findings From the GLORIA-AF Registry Phase 2

Background GLORIA-AF (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients with Atrial Fibrillation) is a prospective, global registry program describing antithrombotic treatment patterns in patients with newly diagnosed nonvalvular atrial fibrillation at risk of stroke. Phase 2 began when dabigatran, the first non\u2013vitamin K antagonist oral anticoagulant (NOAC), became available. Objectives This study sought to describe phase 2 baseline data and compare these with the pre-NOAC era collected during phase&nbsp;1. Methods During phase 2, 15,641 consenting patients were enrolled (November 2011 to December 2014); 15,092 were eligible. This pre-specified cross-sectional analysis describes eligible patients\u2019 baseline characteristics. Atrial fibrillation&nbsp;disease characteristics, medical outcomes, and concomitant diseases and medications were collected. Data were analyzed using descriptive statistics. Results Of the total patients, 45.5% were female; median age was 71 (interquartile range: 64, 78) years. Patients were from Europe (47.1%), North America (22.5%), Asia (20.3%), Latin America (6.0%), and the Middle East/Africa (4.0%). Most had high stroke risk (CHA2DS2-VASc [Congestive heart failure, Hypertension, Age&nbsp; 6575 years, Diabetes mellitus, previous Stroke, Vascular disease, Age 65 to 74 years, Sex category] score&nbsp; 652; 86.1%); 13.9% had moderate risk (CHA2DS2-VASc&nbsp;= 1). Overall, 79.9% received oral anticoagulants, of whom 47.6% received NOAC and 32.3% vitamin K antagonists (VKA); 12.1% received antiplatelet agents; 7.8% received no antithrombotic treatment. For comparison, the proportion of phase 1 patients (of N&nbsp;= 1,063 all eligible) prescribed VKA was 32.8%, acetylsalicylic acid 41.7%, and no therapy 20.2%. In Europe in phase 2, treatment with NOAC was more common than VKA (52.3% and 37.8%, respectively); 6.0% of patients received antiplatelet treatment; and 3.8% received no antithrombotic treatment. In North America, 52.1%, 26.2%, and 14.0% of patients received NOAC, VKA, and antiplatelet drugs, respectively; 7.5% received no antithrombotic treatment. NOAC use was less common in Asia (27.7%), where 27.5% of patients received VKA, 25.0% antiplatelet drugs, and 19.8% no antithrombotic treatment. Conclusions The baseline data from GLORIA-AF phase 2 demonstrate that in newly diagnosed nonvalvular atrial fibrillation patients, NOAC have been highly adopted into practice, becoming more frequently prescribed than VKA in&nbsp;Europe and North America. Worldwide, however, a large proportion of patients remain undertreated, particularly in&nbsp;Asia&nbsp;and North America. (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients With Atrial Fibrillation [GLORIA-AF]; NCT01468701

Effect of remote ischaemic conditioning on clinical outcomes in patients with acute myocardial infarction (CONDI-2/ERIC-PPCI): a single-blind randomised controlled trial.

BACKGROUND: Remote ischaemic conditioning with transient ischaemia and reperfusion applied to the arm has been shown to reduce myocardial infarct size in patients with ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PPCI). We investigated whether remote ischaemic conditioning could reduce the incidence of cardiac death and hospitalisation for heart failure at 12 months. METHODS: We did an international investigator-initiated, prospective, single-blind, randomised controlled trial (CONDI-2/ERIC-PPCI) at 33 centres across the UK, Denmark, Spain, and Serbia. Patients (age >18 years) with suspected STEMI and who were eligible for PPCI were randomly allocated (1:1, stratified by centre with a permuted block method) to receive standard treatment (including a sham simulated remote ischaemic conditioning intervention at UK sites only) or remote ischaemic conditioning treatment (intermittent ischaemia and reperfusion applied to the arm through four cycles of 5-min inflation and 5-min deflation of an automated cuff device) before PPCI. Investigators responsible for data collection and outcome assessment were masked to treatment allocation. The primary combined endpoint was cardiac death or hospitalisation for heart failure at 12 months in the intention-to-treat population. This trial is registered with ClinicalTrials.gov (NCT02342522) and is completed. FINDINGS: Between Nov 6, 2013, and March 31, 2018, 5401 patients were randomly allocated to either the control group (n=2701) or the remote ischaemic conditioning group (n=2700). After exclusion of patients upon hospital arrival or loss to follow-up, 2569 patients in the control group and 2546 in the intervention group were included in the intention-to-treat analysis. At 12 months post-PPCI, the Kaplan-Meier-estimated frequencies of cardiac death or hospitalisation for heart failure (the primary endpoint) were 220 (8·6%) patients in the control group and 239 (9·4%) in the remote ischaemic conditioning group (hazard ratio 1·10 [95% CI 0·91-1·32], p=0·32 for intervention versus control). No important unexpected adverse events or side effects of remote ischaemic conditioning were observed. INTERPRETATION: Remote ischaemic conditioning does not improve clinical outcomes (cardiac death or hospitalisation for heart failure) at 12 months in patients with STEMI undergoing PPCI. FUNDING: British Heart Foundation, University College London Hospitals/University College London Biomedical Research Centre, Danish Innovation Foundation, Novo Nordisk Foundation, TrygFonden

A High-Throughput Assay for Collagen Secretion Suggests an Unanticipated Role for Hsp90 in Collagen Production

Collagen overproduction is a feature of fibrosis and cancer, while insufficient deposition of functional collagen molecules and/or the secretion of malformed collagen is common in genetic disorders like osteogenesis imperfecta. Collagen secretion is an appealing therapeutic target in these and other diseases, as secretion directly connects intracellular biosynthesis to collagen deposition and biological function in the extracellular matrix. However, small molecule and biological methods to tune collagen secretion are severely lacking. Their discovery could prove useful not only in the treatment of disease, but also in providing tools for better elucidating mechanisms of collagen biosynthesis. We developed a cell-based, high-throughput luminescent assay of collagen type I secretion and used it to screen for small molecules that selectively enhance or inhibit that process. Among several validated hits, the Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG) robustly decreases the secretion of collagen-I by our model cell line and by human primary cells. In these systems, 17-AAG and other pan-isoform Hsp90 inhibitors reduce collagen-I secretion post-translationally and are not global inhibitors of protein secretion. Surprisingly, the consequences of Hsp90 inhibitors cannot be attributed to inhibition of the endoplasmic reticulum's Hsp90 isoform, Grp94. Instead, collagen-I secretion likely depends on the activity of cytosolic Hsp90 chaperones, even though such chaperones cannot directly engage nascent collagen molecules. Our results highlight the value of a cell-based high-throughput screen for selective modulators of collagen secretion and suggest an unanticipated role for cytosolic Hsp90 in collagen secretion.National Institutes of Health (U.S.) (Grant 1R03AR067503)National Institutes of Health (U.S.) (Grant 1R01AR071443)National Institutes of Health (U.S.). Ruth Kirschstein Pre-Doctoral Fellowship (1F31AR067615)National Cancer Institute (U.S.) (Cancer Center Support (core) Grant P30-CA14051

Comprehensive synthesis of various functionalized graphene nanoribbons

The contemporary semiconductor industry is experiencing problems with the miniaturization and efficiency of silicon-based components. After its first successful isolation in 2004, graphene has been widely researched due to the potential of surpassing silicon as the ubiquitous semiconductor material. The unprecedented electric conduction properties combined with possibilities of controlling the graphene fragment edges with atomic precision make it a true nano-scale building material that might revolutionize the whole logic circuit and semiconductor industries. Graphene is the carbon allotrope of single atomic layer of carbon atoms arranged into a hexagonally symmetrical honeycomb structure. Due to the confinement into a singular layer and the ultimate symmetry of the lattice, graphene exhibits highly unusually hybridized energy bands, which are the source of its coveted electromagnetic and mechanical properties. These properties can be tuned in a versatile manner by patterning graphene in nanometer scale. Especially the symmetric and well-defined graphene nanoribbons (GNRs) are of high interest, and their production has been under increasing research. The aim of this thesis was to assess various methods capable of producing nano-scale functionalized graphene structures. The most advanced contemporary method for this is the surfaceassisted self-assembly (SASA) of intelligently designed precursor molecules on transition metal surfaces. Five batches of different target molecules were synthesized in order to be used as precursor materials for GNR formation through the SASA approach. The synthesis details of these molecules have been presented in the experimental section of this work. The SASA approach is currently the only synthesis method capable of providing atomic scale control over the graphene domain edges. Other graphene synthesis methods include exfoliation from graphite, unzipping carbon nanotubes and epitaxial film synthesis from simple hydrocarbon gasses. The quality of graphene is highly affected by the synthesis transition metal catalyst, and the properties exhibited by graphene domains are directly influenced by the substrate. It is therefore of high importance to be aware of the properties and interactions of various substrate materials. Decoupling the graphene fragments from unfit surfaces and applying them on optimal substrates is a necessary step of the synthesis. Hexagonal boron nitride (hBN) is currently the best known substrate for graphene-based devices. While the exceptional nature of graphene has already been demonstrated by producing highly effective experimental devices, the challenges associated with scale-up and mass-production of well-defined graphene structures remain currently unsolved.Nykyaikaisessa puolijohdeteollisuudessa pii-peräisten komponenttien tehostukseen ja miniatyrisointiin liittyviä ongelmia on yritetty ratkaista etsimällä vaihtoehtoisia komponenttimateriaaleja. Grafeenia on tutkittu runsaasti vuoden 2004 jälkeen, jolloin ensimmäinen onnistunut yksikerroksisen grafeenin eristystapa esitettiin. Grafeenin erityislaatuiset sähkönjohtokykyyn liittyvät ominaisuudet yhdistettynä grafeenisaarekkeiden mahdolliseen atomitason reunakontrolliin tekevät siitä potentiaalisesti mullistavan nano-mittakaavan logiikkapiiri- ja puolijohdemateriaalin. Grafeeni on hiilen allotrooppi, jossa hiiliatomit järjestäytyvät yksikerroksiseksi, säännölliseksi, kuusikulmaiseksi hilaksi. Korkeasta symmetriasta ja yhden atomin paksuudesta johtuen grafeenin energiatasot ovat hybridisoituneet hyvin epätavallisen muotoisesti, mistä johtuen mm. sen sähkönjohtokyky on ilmiömäinen. Näiden energiatasojen (ja siten sähkömagneettisien ominaisuuksien) järjestäytymiseen voidaan vaikuttaa monipuolisesti leikkaamalla grafeenia nanometrien mittakaavassa. Erityisesti symmetristen ja atomitasolla kontrolloitujen grafeeninauhojen (GNR) onnistunut ja laajamittainen synteesi on suuresti kiinnostava ja tutkittu aihe. Tämän työn tarkoituksena oli perehtyä mahdollisiin keinoihin valmistaa nanometrien mittaisia, funktionalisoituja grafeenirakenteita. Edistynein nykyaikainen menetelmä tähän tarkoitukseen on ennalta suunniteltujen lähtöaineiden hallittu ja katalysoitu terminen hajottaminen ja yhdistyminen laadukkailla siirtymämetallipinnoilla (SASA). Tähän tarkoitukseen valmistettiin viisi eri tuotemolekyylierää, joiden synteesireitit on esitelty työn tutkimusosassa. Grafeenirakenteiden reunojen atomitason kontrolli ei ole toistaiseksi mahdollista muilla menetelmillä, joilla on puolestaan muita etuja käytännön grafeenikomponenttien valmistusta ajatellen. Näitä menetelmiä ovat mm. grafeenin mekaaninen kuoriminen grafiitista, nanoputkien kemiallinen aukaiseminen, sekä epitaksiaalisten grafeenifilmien valmistus yksinkertaisista hiilivedyistä. Koska grafeenin laatu ja havaittavat ominaisuudet riippuvat suuresti synteesissä käytetystä siirtymämetallista sekä pinnasta, jonka päälle se on sittemmin asetettu, on pintamateriaalien aiheuttamien interaktioiden tunteminen tärkeää. Synteesivaiheessa katalyyttisten ominaisuuksiensa takia käytetyt siirtymämetallit eivät sovellu alustoiksi mahdollisille grafeenikomponenteille. Tästä syystä grafeenin siirtomahdollisuudet tulee ottaa synteesissä huomioon. Heksagonaalinen boorinitridi (hBN) on tähän mennessä paras tunnettu pintamateriaali grafeenille. Tähän mennessä on jo näytetty toteen grafeenin erinomaisuus kokeellisten komponenttien muodossa, mutta massatuotantoon ja funktionalisoitujen grafeenirakenteiden ongelmattomaan synteesiin liittyvät haasteet ovat vielä toistaiseksi ratkaisematta

Research capacity. Enabling the genomic revolution in Africa.

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Establishing core outcome domains in pediatric kidney disease: report of the Standardized Outcomes in Nephrology—Children and Adolescents (SONG-KIDS) consensus workshops

Trials in children with chronic kidney disease do not consistently report outcomes that are critically important to patients and caregivers. This can diminish the relevance and reliability of evidence for decision making, limiting the implementation of results into practice and policy. As part of the Standardized Outcomes in Nephrology—Children and Adolescents (SONG-Kids) initiative, we convened 2 consensus workshops in San Diego, California (7 patients, 24 caregivers, 43 health professionals) and Melbourne, Australia (7 patients, 23 caregivers, 49 health professionals). This report summarizes the discussions on the identification and implementation of the SONG-Kids core outcomes set. Four themes were identified; survival and life participation are common high priority goals, capturing the whole child and family, ensuring broad relevance across the patient journey, and requiring feasible and valid measures. Stakeholders supported the inclusion of mortality, infection, life participation, and kidney function as the core outcomes domains for children with chronic kidney disease

Health-status outcomes with invasive or conservative care in coronary disease

BACKGROUND In the ISCHEMIA trial, an invasive strategy with angiographic assessment and revascularization did not reduce clinical events among patients with stable ischemic heart disease and moderate or severe ischemia. A secondary objective of the trial was to assess angina-related health status among these patients. METHODS We assessed angina-related symptoms, function, and quality of life with the Seattle Angina Questionnaire (SAQ) at randomization, at months 1.5, 3, and 6, and every 6 months thereafter in participants who had been randomly assigned to an invasive treatment strategy (2295 participants) or a conservative strategy (2322). Mixed-effects cumulative probability models within a Bayesian framework were used to estimate differences between the treatment groups. The primary outcome of this health-status analysis was the SAQ summary score (scores range from 0 to 100, with higher scores indicating better health status). All analyses were performed in the overall population and according to baseline angina frequency. RESULTS At baseline, 35% of patients reported having no angina in the previous month. SAQ summary scores increased in both treatment groups, with increases at 3, 12, and 36 months that were 4.1 points (95% credible interval, 3.2 to 5.0), 4.2 points (95% credible interval, 3.3 to 5.1), and 2.9 points (95% credible interval, 2.2 to 3.7) higher with the invasive strategy than with the conservative strategy. Differences were larger among participants who had more frequent angina at baseline (8.5 vs. 0.1 points at 3 months and 5.3 vs. 1.2 points at 36 months among participants with daily or weekly angina as compared with no angina). CONCLUSIONS In the overall trial population with moderate or severe ischemia, which included 35% of participants without angina at baseline, patients randomly assigned to the invasive strategy had greater improvement in angina-related health status than those assigned to the conservative strategy. The modest mean differences favoring the invasive strategy in the overall group reflected minimal differences among asymptomatic patients and larger differences among patients who had had angina at baseline