Chapter 6: Characterization of TRPC Channels in a Heterologous System Using Calcium Imaging and the Patch-Clamp Technique

Transient receptor potential (TRP) ion channels are involved in a variety of fundamental physiological processes, and their malfunction produces numerous human diseases. Therefore, these proteins represent a class of attractive drug targets and a class of important off-targets for in vitro pharmacological profiling. In the past decades, the rapid progress in emerging functional assays and instrumentation has enabled to readily monitor thermoTRP channel activity, and to develop high throughput screening (HTS) assays for TPR drug discovery. Chronologically, functional methods for ion channels include the ligand binding assay, flux-based assay, electrophysiology, fluorescence-based assays, and, more recently, automated electrophysiological assays. Here we described the methodology used to monitor the functionality of two thermoTRPs, TRPV1 and TRPM8, based on Ca2+ microfluorography using a 96-well fluorescence plate reader that allows the implementation of a medium- to high-throughput format ideal for drug screening

Variable threshold of trigeminal cold-thermosensitive neurons is determined by a balance between TRPM8 and Kv1 potassium channels

Molecular determinants of threshold differences among cold thermoreceptors are unknown. Here we show that such differences correlate with the relative expression of IKD, a current dependent on Shaker-like Kv1 channels that acts as an excitability brake, and ITRPM8, a cold-activated excitatory current. Neurons responding to small temperature changes have high functional expression of TRPM8 (transient receptor potential cation channel, subfamily M, member 8) and low expression of IKD. In contrast, neurons activated by lower temperatures have a lower expression of TRPM8 and a prominent IKD. Otherwise, both subpopulations have nearly identical membrane and firing properties, suggesting that they belong to the same neuronal pool. Blockade of IKD shins the threshold of cold-sensitive neurons to higher temperatures and augments cold-evoked nocifensive responses in mice. Similar behavioral effects of IKD blockade were observed in TRPA1-/- mice. Moreover, only a small percentage of trigeminal cold-sensitive neurons were activated by TRPA1 agonists, suggesting that TRPA1 does not play a major role in the detection of low temperatures by uninjured somatic cold-specific thermosensory neurons under physiological conditions. Collectively, these findings suggest that innocuous cooling sensations and cold discomfort are signaled by specific low- and high-threshold cold thermoreceptor neurons, differing primarily in their relative expression of two ion channels having antagonistic effects on neuronal excitability. Thus, although TRPM8 appears to function as a critical cold sensor in the majority of peripheral sensory neurons, the expression of Kv1 channels in the same terminals seem to play an important role in the peripheral gating of cold-evoked discomfort and pain. Copyright © 2009 Society for Neuroscience.This work was supported by funds from the Spanish Ministry of Education and Science: Projects BFU2007-61855 to F.V., and BFU2005-08741 and CONSOLIDER-INGENIO 2010 CSD2007-00023 to C.B., and the Spanish Fundación Marcelino BotínPeer Reviewe

Validation of Six Commercial Antibodies for the Detection of Heterologous and Endogenous TRPM8 Ion Channel Expression

TRPM8 is a non-selective cation channel expressed in primary sensory neurons and other tissues, including the prostate and urothelium. Its participation in different physiological and pathological processes such as thermoregulation, pain, itch, inflammation and cancer has been widely described, making it a promising target for therapeutic approaches. The detection and quantification of TRPM8 seems crucial for advancing the knowledge of the mechanisms underlying its role in these pathophysiological conditions. Antibody-based techniques are commonly used for protein detection and quantification, although their performance with many ion channels, including TRPM8, is suboptimal. Thus, the search for reliable antibodies is of utmost importance. In this study, we characterized the performance of six TRPM8 commercial antibodies in three immunodetection techniques: Western blot, immunocytochemistry and immunohistochemistry. Different outcomes were obtained for the tested antibodies; two of them proved to be successful in detecting TRPM8 in the three approaches while, in the conditions tested, the other four were acceptable only for specific techniques. Considering our results, we offer some insight into the usefulness of these antibodies for the detection of TRPM8 depending on the methodology of choice

Medición de la actitud de los aspirantes a docentes en el examen de oposición a cátedra de una universidad pública

La presente investigación tiene como objetivo identificar el tipo de actitud de los aspirantes a maestros a través de las variables independientes, dominio del tema, comunicación, empatía y presencia, en una Universidad Pública del Estado de Nuevo León, México, con el propósito de ser utilizada en los procesos de contratación y selección. Se aplicó un cuestionario conformado por 23 reactivos validados a través del alpha de cronbach a una población de 36 maestros que conformaron el jurado para calificar a los sustentantes. Se realizó la regresión lineal para ver la significancia de cada una de las variables independientes y los resultados obtenidos fueron satisfactorios. La Universidad, como institución de educación superior, requiere docentes altamente calificados para formar profesionales autónomos, creativos y comprometidos con su entorno y con la sociedad en general. ABSTRACT This research aims to identify the kind of attitude of aspiring teachers through independent variables, mastery, communication, empathy and presence in a Public University of Nuevo Leon, Mexico, in order to be used in the processes of recruitment and selection. A questionnaire which was composed of 23 items validated through Cronbach's alpha is applied a population of 36 teachers who formed the jury to assess prospective teachers. Linear regression was performed to check the significance of each of the independent variables and the results obtained were satisfactory. The University as an institution of higher education, requires highly qualified teachers to form autonomous, creative and committed to their professional environment and society in genera

TRPA1 modulation by Sigma-1 receptor prevents oxaliplatin-induced painful peripheral neuropathy

Chemotherapy-induced peripheral neuropathy is a frequent, disabling side effect of anticancer drugs. Oxaliplatin, a platinum compound used in the treatment of advanced colorectal cancer, often leads to a form of chemotherapy-induced peripheral neuropathy characterized by mechanical and cold hypersensitivity. Current therapies for chemotherapy-induced peripheral neuropathy are ineffective, often leading to the cessation of treatment. Transient receptor potential ankyrin 1 (TRPA1) is a polymodal, non-selective cation-permeable channel expressed in nociceptors, activated by physical stimuli and cellular stress products. TRPA1 has been linked to the establishment of chemotherapy-induced peripheral neuropathy and other painful neuropathic conditions. Sigma-1 receptor is an endoplasmic reticulum chaperone known to modulate the function of many ion channels and receptors. Sigma-1 receptor antagonist, a highly selective antagonist of Sigma-1 receptor, has shown effectiveness in a phase II clinical trial for oxaliplatin chemotherapy-induced peripheral neuropathy. However, the mechanisms involved in the beneficial effects of Sigma-1 receptor antagonist are little understood. We combined biochemical and biophysical (i.e. intermolecular Förster resonance energy transfer) techniques to demonstrate the interaction between Sigma-1 receptor and human TRPA1. Pharmacological antagonism of Sigma-1R impaired the formation of this molecular complex and the trafficking of functional TRPA1 to the plasma membrane. Using patch-clamp electrophysiological recordings we found that antagonists of Sigma-1 receptor, including Sigma-1 receptor antagonist, exert a marked inhibition on plasma membrane expression and function of human TRPA1 channels. In TRPA1-expressing mouse sensory neurons, Sigma-1 receptor antagonists reduced inward currents and the firing of actions potentials in response to TRPA1 agonists. Finally, in a mouse experimental model of oxaliplatin neuropathy, systemic treatment with a Sigma-1 receptor antagonists prevented the development of painful symptoms by a mechanism involving TRPA1. In summary, the modulation of TRPA1 channels by Sigma-1 receptor antagonists suggests a new strategy for the prevention and treatment of chemotherapy-induced peripheral neuropathy and could inform the development of novel therapeutics for neuropathic pain.Fil: Marcotti, Aída. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Farmacología Experimental de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Farmacología Experimental de Córdoba; Argentina. Universidad de Miguel Hernández; España. Consejo Superior de Investigaciones Científicas; EspañaFil: Fernández Trillo, Jorge. Universidad de Miguel Hernández; España. Consejo Superior de Investigaciones Científicas; EspañaFil: González, Alejandro. Consejo Superior de Investigaciones Científicas; España. Universidad de Miguel Hernández; EspañaFil: Vizcaíno Escoto, Marta. Universidad de Miguel Hernández; España. Consejo Superior de Investigaciones Científicas; EspañaFil: Ros Arlanzón, Pablo. Universidad de Miguel Hernández; España. Consejo Superior de Investigaciones Científicas; EspañaFil: Romero, Luz. Parc Cientific de Barcelona; EspañaFil: Vela, Jos Miguel. Parc Cientific de Barcelona; España. Consejo Superior de Investigaciones Científicas; EspañaFil: Gomis, Ana. Universidad de Miguel Hernández; España. Consejo Superior de Investigaciones Científicas; EspañaFil: Viana, Flix. Universidad de Miguel Hernández; España. Consejo Superior de Investigaciones Científicas; EspañaFil: De La Peña, Elvira. Universidad de Miguel Hernández; España. Consejo Superior de Investigaciones Científicas; Españ

Comprehensive description of clinical characteristics of a large systemic Lupus Erythematosus Cohort from the Spanish Rheumatology Society Lupus Registry (RELESSER) with emphasis on complete versus incomplete lupus differences

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multiple organ involvement and pronounced racial and ethnic heterogeneity. The aims of the present work were (1) to describe the cumulative clinical characteristics of those patients included in the Spanish Rheumatology Society SLE Registry (RELESSER), focusing on the differences between patients who fulfilled the 1997 ACR-SLE criteria versus those with less than 4 criteria (hereafter designated as incomplete SLE (iSLE)) and (2) to compare SLE patient characteristics with those documented in other multicentric SLE registries. RELESSER is a multicenter hospital-based registry, with a collection of data from a large, representative sample of adult patients with SLE (1997 ACR criteria) seen at Spanish rheumatology departments. The registry includes demographic data, comprehensive descriptions of clinical manifestations, as well as information about disease activity and severity, cumulative damage, comorbidities, treatments and mortality, using variables with highly standardized definitions. A total of 4.024 SLE patients (91% with ≥4 ACR criteria) were included. Ninety percent were women with a mean age at diagnosis of 35.4 years and a median duration of disease of 11.0 years. As expected, most SLE manifestations were more frequent in SLE patients than in iSLE ones and every one of the ACR criteria was also associated with SLE condition; this was particularly true of malar rash, oral ulcers and renal disorder. The analysis-adjusted by gender, age at diagnosis, and disease duration-revealed that higher disease activity, damage and SLE severity index are associated with SLE [OR: 1.14; 95% CI: 1.08-1.20 (P < 0.001); 1.29; 95% CI: 1.15-1.44 (P < 0.001); and 2.10; 95% CI: 1.83-2.42 (P < 0.001), respectively]. These results support the hypothesis that iSLE behaves as a relative stable and mild disease. SLE patients from the RELESSER register do not appear to differ substantially from other Caucasian populations and although activity [median SELENA-SLEDA: 2 (IQ: 0-4)], damage [median SLICC/ACR/DI: 1 (IQ: 0-2)], and severity [median KATZ index: 2 (IQ: 1-3)] scores were low, 1 of every 4 deaths was due to SLE activity. RELESSER represents the largest European SLE registry established to date, providing comprehensive, reliable and updated information on SLE in the southern European population

BIOVID: Micro espacios audiovisuales sobre biodiversidad

Se han realizado, editado y publicado microespacios de video alojados en la web www.ucm.es/segbiodiversidad/videos sobre aspectos relacionados con la biodiversidad, ecología y metodologías de seguimiento de aves, murciélagos, artrópodos y flora del campus de la Universidad Complutense de Madrid

The Mars Environmental Dynamics Analyzer, MEDA. A Suite of Environmental Sensors for the Mars 2020 Mission

86 pags., 49 figs., 24 tabs.NASA’s Mars 2020 (M2020) rover mission includes a suite of sensors to monitor current environmental conditions near the surface of Mars and to constrain bulk aerosol properties from changes in atmospheric radiation at the surface. The Mars Environmental Dynamics Analyzer (MEDA) consists of a set of meteorological sensors including wind sensor, a barometer, a relative humidity sensor, a set of 5 thermocouples to measure atmospheric temperature at ∼1.5 m and ∼0.5 m above the surface, a set of thermopiles to characterize the thermal IR brightness temperatures of the surface and the lower atmosphere. MEDA adds a radiation and dust sensor to monitor the optical atmospheric properties that can be used to infer bulk aerosol physical properties such as particle size distribution, non-sphericity, and concentration. The MEDA package and its scientific purpose are described in this document as well as how it responded to the calibration tests and how it helps prepare for the human exploration of Mars. A comparison is also presented to previous environmental monitoring payloads landed on Mars on the Viking, Pathfinder, Phoenix, MSL, and InSight spacecraft.This work has been funded by the Spanish Ministry of Economy and Competitiveness, through the projects No. ESP2014-54256-C4-1-R (also -2-R, -3-R and -4-R) and AYA2015-65041-P; Ministry of Science, Innovation and Universities, projects No. ESP2016-79612-C3-1-R (also -2-R and -3-R), ESP2016-80320-C2-1-R, RTI2018-098728-B-C31 (also -C32 and -C33) and RTI2018-099825-B-C31; Instituto Nacional de Técnica Aeroespacial; Ministry of Science and Innovation’s Centre for the Development of Industrial Technology; Grupos Gobierno Vasco IT1366-19; and European Research Council Consolidator Grant no 818602. The US co-authors performed their work under sponsorship from NASA’s Mars 2020 project, from the Game Changing Development program within the Space Technology Mission Directorate and from the Human Exploration and Operations Directorate