Negative modulation of the GABAAρ1 receptor function by l-cysteine

l-Cysteine is an endogenous sulfur-containing amino acid with multiple and varied roles in the central nervous system, including neuroprotection and the maintenance of the redox balance. However, it was also suggested as an excitotoxic agent implicated in the pathogenesis of neurological disorders such as Parkinson′s and Alzheimer′s disease. l-Cysteine can modulate the activity of ionic channels, including voltage-gated calcium channels and glutamatergic NMDA receptors, whereas its effects on GABAergic neurotransmission had not been studied before. In the present work, we analyzed the effects of l-cysteine on responses mediated by homomeric GABAAρ1 receptors, which are known for mediating tonic γ-aminobutyric acid (GABA) responses in retinal neurons. GABAAρ1 receptors were expressed in Xenopus laevis oocytes and GABA-evoked chloride currents recorded by two-electrode voltage-clamp in the presence or absence of l-cysteine. l-Cysteine antagonized GABAAρ1 receptor-mediated responses; inhibition was dose-dependent, reversible, voltage independent, and susceptible to GABA concentration. Concentration-response curves for GABA were shifted to the right in the presence of l-cysteine without a substantial change in the maximal response. l-Cysteine inhibition was insensitive to chemical protection of the sulfhydryl groups of the ρ1 subunits by the irreversible alkylating agent N-ethyl maleimide. Our results suggest that redox modulation is not involved during l-cysteine actions and that l-cysteine might be acting as a competitive antagonist of the GABAAρ1 receptors. (Figure presented.).Fil: Beltrán González, Andrea Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Vicentini, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Calvo, Daniel Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

Heuristic usability evaluation on games: a modular approach

Heuristic evaluation is the preferred method to assess usability in games when experts conduct this evaluation. Many heuristics guidelines have been proposed attending to specificities of games but they only focus on specific subsets of games or platforms. In fact, to date the most used guideline to evaluate games usability is still Nielsen’s proposal, which is focused on generic software. As a result, most evaluations do not cover important aspects in games such as mobility, multiplayer interactions, enjoyability and playability, etc. To promote the usage of new heuristics adapted to different game and platform aspects we propose a modular approach based on the classification of existing game heuristics using metadata and a tool, MUSE (Meta-heUristics uSability Evaluation tool) for games, which allows a rebuild of heuristic guidelines based on metadata selection in order to obtain a customized list for every real evaluation case. The usage of these new rebuilt heuristic guidelines allows an explicit attendance to a wide range of usability aspects in games and a better detection of usability issues. We preliminarily evaluate MUSE with an analysis of two different games, using both the Nielsen’s heuristics and the customized heuristic lists generated by our tool.Unión Europea PI055-15/E0

Benzodiazepinas

Antes de las irrupciones estelares del Prozac (1987) y el Viagra (1998) en la escena de la farmacología mundial, la promesa más grande de felicidad en cápsulas eran las ¨benzo¨, drogas sedantes utilizadas para tratar la ansiedad y el insomnio que reinaron en solitario durante tres décadas y que al día de hoy, pese a cierta mala prensa, parecen no haber perdido su sex appeal.Fil: Calvo, Daniel Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

An intracellular redox sensor for reactive oxygen species at the M3-M4 linker of GABAArho1 receptors

Background and Purpose: Reactive oxygen species (ROS) are normally involved in cell oxidative stress but also play a role as cellular messengers in redox signalling; for example, modulating the activity of neurotransmitter receptors and ion channels. However, the direct actions of ROS on GABAA receptors were not previously demonstrated. In the present work, we studied the effects of ROS on GABAAρ1 receptor function. Experimental Approach: GABAAρ1 receptors were expressed in oocytes and GABA-evoked responses electrophysiologically recorded in the presence or absence of ROS. Chemical protection of cysteines by selective sulfhydryl reagents and site-directed mutagenesis studies were used to identify protein residues involved in ROS actions. Key Results: GABAAρ1 receptor-mediated responses were significantly enhanced in a concentration-dependent and reversible manner by H2O2. Potentiating effects were attenuated by a free radical scavenger, lipoic acid or an inhibitor of the Fenton reaction, deferoxamine. Each ρ1 subunit contains only three cysteine residues, two extracellular at the Cys-loop (C177 and C191) and one intracellular (C364) at the M3-M4 linker. Mutant GABAAρ1 receptors in which C364 was exchanged by alanine were completely insensitive to modulation, implying that this site, rather than a cysteine in the Cys-loop, is essential for ROS modulation. Conclusion and Implications: Our results show that the function of GABAAρ1 receptors is enhanced by ROS and that the intracellular C364 is the sensor for ROS actions.Fil: Beltrán González, Andrea Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Calvo, Daniel Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Gasulla, Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentin

Allosteric modulation of retinal GABA receptors by ascorbic acid

Ionotropic GABA receptors (GABAA and GABAC) belong to the Cys-loop receptor family of ligand-gated ion channels. GABAC receptors are highly expressed in the retina, mainly localized at the axon terminals of bipolar cells. Ascorbic acid, an endogenous redox agent, modulates the function of diverse proteins, and basal levels of ascorbic acid in the retina are very high. However, the effect of ascorbic acid on retinal GABA receptors has not been studied. Here we show that the function of GABAC and GABAA receptors is regulated by ascorbic acid. Patch-clamp recordings from bipolar cell terminals in goldfish retinal slices revealed that GABAC receptor-mediated currents activated by tonic background levels of extracellular GABA, and GABAC currents elicited by local GABA puffs, are both significantly enhanced by ascorbic acid. In addition, a significant rundown of GABA puff-evoked currents was observed in the absence of ascorbic acid. GABA-evoked Cl- currents mediated by homomeric ρ1 GABAC receptors expressed in Xenopus laevis oocytes were also potentiated by ascorbic acid in a concentration-dependent, stereo-specific, reversible, and voltage-independent manner. Studies involving the chemical modification of sulfhydryl groups showed that the two Cys-loop cysteines and histidine 141, all located in the ρ1 subunit extracellular domain, each play a key role in the modulation of GABAC receptors by ascorbic acid. Additionally, we show that retinal GABAA IPSCs and heterologously expressed GABAA receptor currents are similarly augmented by ascorbic acid. Our results suggest that ascorbic acid may act as an endogenous agent capable of potentiating GABAergic neurotransmission in the CNS.Fil: Calero, Cecilia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Vickers, Evan. Oregon Health and Science University; Estados UnidosFil: Cid, Gustavo Moraga. Universidad de Concepción; ChileFil: Aguayo, Luis G.. Universidad de Concepción; ChileFil: von Gersdorff, Henrique. Oregon Health and Science University; Estados UnidosFil: Calvo, Daniel Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

An experimental evaluation of server performance in Networked Virtual Environments

Several works in the literature have recently addressed the study of different Networked Virtual Environments (NVE) due to their increasing popularity and widespread use in fields ranging from entertainment to e-Health. Open Wonderland is one of these NVEs which has been the subject of several studies mainly focused on the client side. This paper aims to cover the server-side performance issues to provide complementary results that can be useful for properly sizing Open Wonderland systems according to the number of expected users. An experimental testbed is used, which provides real data that shows that CPU and outgoing bandwidth are the most critical parameters when the number of clients increase.Ministerio de Ciencia e Innovación PROCUR@-IPT-2011-1038-900000Ministerio de Ciencia e Innovación TEC2009-10639-C04-0

A Hardware Implementation of a Run-Time Scheduler for Reconfigurable Systems

New generation embedded systems demand high performance, efficiency and flexibility. Reconfigurable hardware can provide all these features. However the costly reconfiguration process and the lack of management support have prevented a broader use of these resources. To solve these issues we have developed a scheduler that deals with task-graphs at run-time, steering its execution in the reconfigurable resources while carrying out both prefetch and replacement techniques that cooperate to hide most of the reconfiguration delays. In our scheduling environment task-graphs are analyzed at design-time to extract useful information. This information is used at run-time to obtain near-optimal schedules, escaping from local-optimum decisions, while only carrying out simple computations. Moreover, we have developed a hardware implementation of the scheduler that applies all the optimization techniques while introducing a delay of only a few clock cycles. In the experiments our scheduler clearly outperforms conventional run-time schedulers based on As-Soon-As-Possible techniques. In addition, our replacement policy, specially designed for reconfigurable systems, achieves almost optimal results both regarding reuse and performance

A Task-Graph Execution Manager for Reconfigurable Multi-tasking Systems

Reconfigurable hardware can be used to build multi tasking systems that dynamically adapt themselves to the requirements of the running applications. This is especially useful in embedded systems, since the available resources are very limited and the reconfigurable hardware can be reused for different applications. In these systems computations are frequently represented as task graphs that are executed taking into account their internal dependencies and the task schedule. The management of the task graph execution is critical for the system performance. In this regard, we have developed two dif erent versions, a software module and a hardware architecture, of a generic task-graph execution manager for reconfigurable multi-tasking systems. The second version reduces the run-time management overheads by almost two orders of magnitude. Hence it is especially suitable for systems with exigent timing constraints. Both versions include specific support to optimize the reconfiguration process

Positive modulation of the a9a10 nicotinic cholinergic receptor by ascorbic acid

Background and Purpose: The activation of α9α10 nicotinic cholinergic receptors (nAChRs) present at the synapse between efferent olivocochlear fibres and cochlear hair cells can prevent acoustic trauma. Hence, pharmacological potentiators of these receptors could be useful therapeutically. In this work, we characterize ascorbic acid as a positive modulator of recombinant α9α10 nAChRs. Experimental Approach: ACh-evoked responses were analysed under two-electrode voltage-clamp recordings in Xenopus laevis oocytes injected with α9 and α10 cRNAs. Key Results: Ascorbic acid potentiated ACh responses in X. laevis oocytes expressing α9α10 (but not α4β2 or α7) nAChRs, in a concentration-dependent manner, with an effective concentration range of 1–30 mM. The compound did not affect the receptor's current–voltage profile nor its apparent affinity for ACh, but it significantly enhanced the maximal evoked currents (percentage of ACh maximal response, 240 ± 20%). This effect was specific for the L form of reduced ascorbic acid. Substitution of the extracellular cysteine residues present in loop C of the ACh binding site did not affect the potentiation. Ascorbic acid turned into a partial agonist of α9α10 nAChRs bearing a point mutation at the pore domain of the channel (TM2 V13′T mutant). A positive allosteric mechanism of action rather than an antioxidant effect of ascorbic acid is proposed. Conclusions and Implications: The present work describes one of the few agents that activates or potentiates α9α10 nAChRs and leads to new avenues for designing drugs with potential therapeutic use in inner ear disorders.Fil: Boffi, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Wedemeyer, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Lipovsek, Maria Marcela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Katz, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Calvo, Daniel Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Elgoyhen, Ana Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Farmacología; Argentin

Oleoylethanolamide restores alcohol-induced inhibition of neuronal proliferation and microglial activity in striatum

Previous findings demonstrate a homeostatic role for oleoylethanolamide (OEA) signaling in the ethanol-related neuroinflammation and behavior. However, extensive research is still required in order to unveil the effects of OEA on a number of neurobiological functions such as adult neurogenesis, cell survival and resident neuroimmunity that become notably altered by alcohol. Daily consumption of ethanol (10%) for 2 weeks (6.3& #x202F;± 1.1 g/kg/day during last 5 days) caused hypolocomotor activity in rats. This effect appears to rely on central signaling mechanisms given that alcohol increased the OEA levels, the gene expression of OEA-synthesizing enzyme Nape-pld and the number of PPARα-immunoreactive neurons in the striatum. Ethanol-related neurobiological alterations such as a reduction in the number of microglial cells expressing iNOS (a cytokine-inducible immune defense) and in adult neural stem/progenitor cell (NSPC) proliferation (phospho-H3 and BrdU) and maturation (BrdU/β3-tubulin), as well as an increase in damage cell activity (FosB) and apoptosis (cleaved caspase 3) were also observed in the rat striatum. Pharmacological administration of OEA (10 mg/kg) for 5 days during ethanol exposure exacerbated ethanol-induced hypolocomotion and cell apoptosis in the striatum. Interestingly, OEA abrogated the impaired effects of ethanol on PPARα-positive cell population and NSPC proliferation and maturation. OEA also decreased astrocyte-related vimentin immunoreactivity and increased microglial cell population (Iba-1, iNOS) in the striatum. These results suggest that OEA-PPARα signaling modulates glial activation, cell apoptosis and NSPC proliferation and maturation in response to striatal-specific neurobiological alterations induced by prolonged ethanol intake in rats.This work was supported by RETICS Red de Trastornos Adictivos, Instituto de Salud Carlos III (ISCIII), Ministerio de Economía y Competitividad and European Regional Development Funds-European Union (ERDF-EU) (RD16/0017/0001); ISCIII, MINECO, ERDF-EU (JS: PI16/01374; FRF: PI16/01698; FJP: PI16/01953; AS: PI17/02026); Ministerio de Sanidad, Servicios Sociales e Igualdad and Plan Nacional sobre Drogas (JS: PNSD2015/047; AS: PND2017/043); Consejería de Economía, Innovación y Ciencia, Junta de Andalucía, ERDF-EU (FRF: CTS-8221); Consejería de Salud, Junta de Andalucía, ERDF-EU (FRF: SAS111224); German Research Foundation DFG (BL: FOR926, project CP1). FJP (CP14/00212) and AS (CP14/00173) are recipients of a research contract from “Miguel Servet” Program of ISCIII, ERDF-EU. JS holds a “Miguel Servet II” research contract from the National System of Health, ISCIII, ERDF-EU, FIMABIS (CPII17/00024). PR holds a “Sara Borrel” research contract from ISCIII, ERDF-EU (CD16/00067)