Caracterización estructural y funcional de los promotores humanos SREBP1 y su regulador INSIG2

La homeostasis lipídica está regulada por factores de transcripción llamados Proteínas de unión a elementos de respuesta a esteroles ó SREBP. Estas porteínas activan a nivel transcripcional enzimas de las vías de síntesis de colesterol, ácidos grasos, triglicéridos y fosfolípidos. Además estos factores regulan la diferenciación adipocitaria y la expresión génica dependiente de insulina. Alteraciones funcionales en SREBP han sido relacionadas con el desarrollo de diversas patologías metabólicas humanas como la obesidad y la diabetes tipo 2. Existen tres miembros de la familia SREBP. Las variantes SREBP1a y SREBP1c, son producidas por splicing alternativo a partir de un único gen mientras que SREBP2 es codificada por un gen diferente. Las proteínas SREBP son sintetizadas como precursores inactivos anclados a la membrana del retículo endoplásmico. En condiciones de bajos niveles de esteroles, los precursores inactivos son transportados hasta el aparato de Golgi donde son activados mediante cascada proteolítica liberando la proteína nuclear activa. El precursor inactivo permanece anclado en el retículo debido a su interacción con otras dos proteínas de membrana: SCAP e INSIG (insulin-induced gene), ambas reguladas por esteroles. Se conocen dos isoformas de INSIG denominadas INSIG1 e INSIG2. En humanos, ambas proteínas tienen un 59% de homología y difieren principalmente en su forma de regulación. En ratones se han encontrado dos variantes de RNA mensajero de INSIG2 denominadas INSIG2a, específica de hígado y regulada negativamente por insulina, e INSIG2b, de expresión ubicua. Teniendo en cuenta que variaciones en la expresión de las proteínas SREBP se han relacionado con enfermedades complejas, así como la importancia que se está dando a las regiones reguladoras en la etiopatogenia de estas enfermedades, el objetivo de esta tesis ha sido caracterizar la regulación a nivel transcripcional de la vía SREBP con tres objetivos concretos: 1, caracterización del Promotor humano SREBP1a, 2, caracterización del Promotor humano SREBP1c, y 3, identificación del Promotor humano INSIG2.El estudio del promotor humano SREBP1a nos permitió identificar la región correspondiente al promotor proximal. Este promotor, al igual que el promotor murino, está comprendido en una pequeña zona del DNA de 75pb que se caracteriza por una región rica en GC. Esta región contiene 3 sitios superpuestos SP1/EGR1 capaces de unir ambos factores de transcripción tanto in vitro como in vivo, siendo SP1 un activador de la transcripción y EGR1 un inhibidor de su actividadHemos estudiado también la regulación del promotor humano SREBP1c tanto a nivel basal como la respuesta a factores nutricionales. Nuestros resultados demuestran que la regulación de los promotores de ambas especies presenta gran similitud. Los estudios realizados in vivo demostraron además que el receptor nuclear PPAR es capaz de unirse al promotor en presencia de RXR y que tanto el promotor SREBP1c humano como el de rata son activados por un agonista de este receptor. El cofactor PGC1 está también involucrado en la regulación de este promotor.Hemos clonado y caracterizado la región proximal del promotor humano INSIG2. Los resultados obtenidos demuestran por primera vez que proteínas de la familia de factores de transcripción Ets están involucradas en la regulación de este promotor. Se ha identificado la acción de la proteína SAP1a que controla su actividad basal en forma dependiente de su estado de fosforilación. Este promotor está además regulado por insulina en forma positiva. Este estudio funcional y estructural de los promotores humanos de SREBP1 y su regulador INSIG2 nos ha permitido delinear parte de la regulación trancripcional de la vía SREBP, así como similitudes y diferencias entre especies. Esta información nos permitirá analizar las alteraciones que puedan tener relevancia fisiológica así como los mecanismos indispensables para la correcta funcionalidad de estas vías metabólicas.ABSTRACSterol regulatory-element-binding proteins (SREBPs) are transcription factors with a central role in cholesterol and fatty acid metabolism. Functional alterations in these proteins have been implicated in the development of several human metabolic diseases such as obesity or type 2 diabetes. The SREBP family of transcription factors consists of three isoforms, SREBP1a and 1c, encoded by a unique gene by alternative splicing, and SREBP2 encoded by a separate gene. SREBPs are synthesized as inactive precursors embedded in ER membranes until they are transported to the Golgi apparatus where they are activated by proteolysis. The ER-to-Golgi migration of SREBPs is crucially dependent on its interaction with two other membrane proteins: SCAP (SREBP cleavage-activating protein) and INSIG (insulin-induced gene), both regulated by cell sterol levels. Two INSIG isoforms are known, designated INSIG1 and INSIG2. In humans, both proteins are 59% identical but they differ in their mode of regulation. Transcription of INSIG2 gene in mice give rise to alternative mRNA transcripts named INSIG2a which is liver specific and INSIG2b. In the present work we aimed to analyze the transcripcional regulation of the SREBP pathway focusing on the characterization of the functional al structural organization of SREBP1a, SREBP1c and INSG2 human promoters.Human SREBP1a promoter is characterized by three GC boxes responsible for its SP1 activation and EGR1 inactivation. Human SREBP1c is regulated by nutricional factors in a similar way as the mouse and rat promoter. A response mediated by PPAR nuclear receptor and its coactivator PGC1 is present in rat and human promoter. INSIG2 transcriptional regulation has not been previously studied. Human INSIG2 promoter was identified and cloned for analysis. The proximal promoter is comprised in a 300 pb fragment. The results show this region is regulated by Ets family of transcriptional factors with a particular participation of phosphorilated Sap1a protein. Human promoter is also positively regulated by insulin in primary culture hepatocytes.This study on human promoters of SREBP1 and INSIG2 allowed us to delineate part of the transcriptional regulation of SREBP pathway wich would be of great relevance for the analysis of physiologically relevant alterations related with metabolic pathology

Bases estructurales del mecanismo de reconocimiento y fosfotransferencia en los sistemas de señalización de dos componentes

Póster original presentado XXVII Congreso de la SEBBM, Sociedad Española de Bioquímica y Biología Molecular, celebrado en Lleida, 12-15 de septiembre, 2004.Los sistemas de dos componentes son el mecanismo principal de transducción de señal en microorganismos. Un sistema paradigmático se compone de dos proteínas: una histidina quinasa (HK) y un regulador de la respuesta (RR). El mecanismo de transducción de señal implica al menos tres reacciones (ver esquema): 1) Autofosforilación: la llegada de un estímulo al dominio extracelular sensor de la HK, induce la fosforilación en un residuo de His conservado en el dominio de dimerización (DHp) por parte del dominio de unión de ATP (CA). 2) Fosfotransferencia: este grupo fosforilo es transferido desde la His a un residuo de Asp del dominio regulador del correspondiente RR. 3) Defosforilación: el RR pierde el grupo fosforilo espontáneamente o mediado por la HK. El estado de fosforilación del dominio regulador en RR regula la actividad de su dominio efector, que es habitualmente un factor de transcripción.Financiado por la ayuda BIO2002-03709 del Ministerio de Ciencia y Tecnología. P. casino es becaria FPI del Ministerio de ciencia y TecnologíaPeer reviewe

SREBP-1a activation by HBx and the effect on hepatitis B virus enhancer II/core promoter

Hepatitis B virus (HBV) X protein (HBx) plays an important role in HBV pathogenesis by regulating gene expression. Sterol regulatory element binding protein-1a (SREBP-1a) is a key transcriptional factor for modulating fatty acid and cholesterol synthesis. Here we demonstrated that HBx increased mature SREBP-1a protein level in the nucleus and its activity as a transcription factor. We further showed that the up-regulation of SREBP-1a by HBx occurred at the transcriptional level after ectopic expression and in the context of HBV replication. Deletional analysis using SREBP-1a promoter revealed that the sequence from -436 to -398 in the promoter was required for its activation by HBx. This promoter region possesses the binding sequences for two basic leucine zipper (b-ZIP) transcription factors, namely C/EBP and E4BP4. Mutagenesis of the binding sequences on the SREBP-1a promoter and ectopic expression experiments demonstrated that C/EBPα enhanced SREBP-1a activation by HBx, while E4BP4 had an inhibitory effect. C/EBPα was able to significantly reverse the inhibitory activity of E4BP4 on SREBP-1a promoter. These results demonstrated that HBx activates SREBP-1a activity at the transcription level through a complex mechanism involving two bZIP transcription factors C/EBP and E4BP4 with C/EBP being the dominant positive factor. Finally, we showed that knocking down SREBP-1 abolishes HBV enhancer II/core promoter activation by HBx.Fil: Qiao, Ling. University Of Saskatchewan; CanadáFil: Wu, Qi. University Of Saskatchewan; CanadáFil: Lu, Xinya. University Of Saskatchewan; CanadáFil: Zhou, Yan. University Of Saskatchewan; CanadáFil: Fernández Alvarez, Ana Julia. Consejo Superior de Investigaciones Cientificas; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Ye, Lihong. Nankai University; ChinaFil: Zhang, Xiaodong. Nankai University; ChinaFil: Han, Jihong. Nankai University; ChinaFil: Casado, Marta. Consejo Superior de Investigaciones Cientificas; EspañaFil: Liu, Quiang. University Of Saskatchewan; Canad

Hepatic cyclooxygenase-2 expression protects against diet-induced steatosis, obesity and insulin resistance

Resumen del póster presentado a la Conferencia: FASEB SRC: Liver Biology: Fundamental Mechanisms and Translational Applications, celebrada en Keystone-Colorado (US) del 6 al 11 de julio de 2014.[Background and Aims: Accumulation evidence links obesity-induced inflammation as an important contributor to the induction of insulin resistance. Moreover, insulin resistance plays a key role in the pathophysiology of obesity-related diseases such as type 2 diabetes and non alcoholic fatty liver disease. Cyclooxygenase-1 and -2 catalyze the first step in prostanoid biosynthesis. Since adult hepatocytes fail to induce COX-2 expression regardless of the pro-inflammatory factors used, we have evaluated whether this lack of expression under mild pro-inflammatory conditions might constitute a permissive condition for the onset of insulin resistance. [Methods]: We evaluated the role of COX-2 expression in hepatocytes in a model of insulin resistance and altered energy homeostasis induced by high fat diet by metabolic parameters in transgenic mice constitutively expressing human COX-2 in hepatocytes. [Results]: COX-2 expression in hepatocytes protects from high fat diet-induced hepatic steatosis, obesity and hence insulin resistance, as demonstrated by a decreased hepatic steatosis, adiposity and adipocyte area, an enhanced insulin sensitivity and glucose tolerance, decreased plasmatic and hepatic triglycerides and free fatty acids levels, increased adiponectin/leptin ratio and decreased levels of pro-inflammatory cytokines. COX-2 transgenic mice exhibited increased whole body energy expenditure and fatty acid oxidation. Moreover, when hepatic insulin signaling was analyzed, an increase in insulin receptor-mediated Akt phosphorylation was found in hCOX-2 transgenic mice. Similar results were obtained in human and murine hepatic cells expressing a COX-2 transgene. [Conclusion]: Constitutively expression of COX-2 in hepatocytes protects against adiposity, inflammation and hepatic insulin resistance in mice under high fat diet.Peer Reviewe

Methodology and fieldwork logistics of a multilevel research study on the influence of neigbourdhood's characteristics on natives and Ecuadorian's mental health in Spain

The methodological design, characteristics and fieldwork stage of a multilevel research study on the impact of the environmental characteristics on mental health in an autochthonous and immigrant population are described in this paper. Individual data were obtained using a core questionnaire 40 minutes length from home interviews of Spanish and Ecuadorian adults from September 2006 to January 2007. A random sample of 1186 people aged 18-55, with equal distribution of gender and nationality was obtained from Civil Registers of 33 areas (municipalities or neighbourhoods) of Madrid, Alicante, Almeria and Murcia, chosen by ethnic density and socioeconomic criteria. Previously, a pilot study was carried out. Socioeconomic indicators of neighbourhoods and selected communities were obtained from Municipal Registers and other secondary sources. Finally, 1144 people were interviewed (96%). Each person was contacted at home at two different times. The global response rate was 61%, higher among Ecuadorians (69%), who presented more problems of localisation (34%). Analyzing methods and fieldwork process the conclusion is that sample strategies for this type of population studies should be evaluated using feasibility criteria given time and money constraints, against the need to obtain representative samples of the target populations. There were serious shortcomings in the availability of social integration indicators at the neighbourhood level. Se realizó una investigación multinivel sobre el impacto de las características ambientales de la zona de residencia en la salud mental de población autóctona e inmigrante. El objetivo de este artículo es describir el planteamiento metodológico de la investigación, el trabajo de campo, las tasas de respuesta correspondientes y discutir el diseño metodológico y las dificultades derivadas de su puesta en práctica. Los datos individuales se obtuvieron aplicando un cuestionario estructurado de aproximadamente 40 minutos, mediante entrevista domiciliaria a personas españolas y ecuatorianas de 18 a 55 años. El trabajo se realizó de septiembre de 2006 a enero de 2007 en una muestra estimada de 1.186 personas equiparada por sexo y nacionalidad, obtenida aleatoriamente de los Padrones Municipales de 33 áreas (municipios o barrios) de Madrid, Alicante, Almería y Murcia, seleccionadas según criterios de densidad étnica y socioeconómicos. Previamente se realizó un estudio piloto (n=113) Los indicadores sociodemográficos de las áreas se obtuvieron a partir de fuentes secundarias. Se entrevistó a 1.144 personas (96%). La tasa de respuesta global fue del 61%, superior entre ecuatorianos (69%), colectivo que presentó más problemas de localización (34%). Las negativas a colaborar fueron más altas entre españoles (21%). Se concluye que en este tipo de estudios sería conveniente revisar las estrategias de muestreo para combinar criterios de eficiencia con la necesidad de obtener una muestra representativa de la población diana. Se constata la dificultad de obtener datos inframunicipales de integración social

Randomized Controlled Trial Assessing the Impact of Tacrolimus Versus Cyclosporine on the Incidence of Posttransplant Diabetes Mellitus

Despite the high incidence of posttransplant diabetes mellitus (PTDM) among high-risk recipients, no studies have investigated its prevention by immunosuppression optimization. We conducted an open-label, multicenter, randomized trial testing whether a tacrolimus-based immunosuppression and rapid steroid withdrawal (SW) within 1 week (Tac-SW) or cyclosporine A (CsA) with steroid minimization (SM) (CsA-SM), decreased the incidence of PTDM compared with tacrolimus with SM (Tac-SM). All arms received basiliximab and mycophenolate mofetil. High risk was defined by age >60 or >45 years plus metabolic criteria based on body mass index, triglycerides, and high-density lipoprotein-cholesterol levels. The primary endpoint was the incidence of PTDM after 12 months. The study comprised 128 de novo renal transplant recipients without pretransplant diabetes (Tac-SW: 44, Tac-SM: 42, CsA-SM: 42). The 1-year incidence of PTDM in each arm was 37.8% for Tac-SW, 25.7% for Tac-SM, and 9.7% for CsA-SM (relative risk [RR] Tac-SW vs. CsA-SM 3.9 [1.2-12.4; P = 0.01]; RR Tac-SM vs. CsA-SM 2.7 [0.8-8.9; P = 0.1]). Antidiabetic therapy was required less commonly in the CsA-SM arm (P = 0.06); however, acute rejection rate was higher in CsA-SM arm (Tac-SW 11.4%, Tac-SM 4.8%, and CsA-SM 21.4% of patients; cumulative incidence P = 0.04). Graft and patient survival, and graft function were similar among arms. In high-risk patients, tacrolimus-based immunosuppression with SM provides the best balance between PTDM and acute rejection incidence

Identification of novel risk loci, causal insights, and heritable risk for Parkinson's disease: a meta-analysis of genome-wide association studies

Background Genome-wide association studies (GWAS) in Parkinson's disease have increased the scope of biological knowledge about the disease over the past decade. We aimed to use the largest aggregate of GWAS data to identify novel risk loci and gain further insight into the causes of Parkinson's disease. Methods We did a meta-analysis of 17 datasets from Parkinson's disease GWAS available from European ancestry samples to nominate novel loci for disease risk. These datasets incorporated all available data. We then used these data to estimate heritable risk and develop predictive models of this heritability. We also used large gene expression and methylation resources to examine possible functional consequences as well as tissue, cell type, and biological pathway enrichments for the identified risk factors. Additionally, we examined shared genetic risk between Parkinson's disease and other phenotypes of interest via genetic correlations followed by Mendelian randomisation. Findings Between Oct 1, 2017, and Aug 9, 2018, we analysed 7·8 million single nucleotide polymorphisms in 37 688 cases, 18 618 UK Biobank proxy-cases (ie, individuals who do not have Parkinson's disease but have a first degree relative that does), and 1·4 million controls. We identified 90 independent genome-wide significant risk signals across 78 genomic regions, including 38 novel independent risk signals in 37 loci. These 90 variants explained 16–36% of the heritable risk of Parkinson's disease depending on prevalence. Integrating methylation and expression data within a Mendelian randomisation framework identified putatively associated genes at 70 risk signals underlying GWAS loci for follow-up functional studies. Tissue-specific expression enrichment analyses suggested Parkinson's disease loci were heavily brain-enriched, with specific neuronal cell types being implicated from single cell data. We found significant genetic correlations with brain volumes (false discovery rate-adjusted p=0·0035 for intracranial volume, p=0·024 for putamen volume), smoking status (p=0·024), and educational attainment (p=0·038). Mendelian randomisation between cognitive performance and Parkinson's disease risk showed a robust association (p=8·00 × 10−7). Interpretation These data provide the most comprehensive survey of genetic risk within Parkinson's disease to date, to the best of our knowledge, by revealing many additional Parkinson's disease risk loci, providing a biological context for these risk factors, and showing that a considerable genetic component of this disease remains unidentified. These associations derived from European ancestry datasets will need to be followed-up with more diverse data. Funding The National Institute on Aging at the National Institutes of Health (USA), The Michael J Fox Foundation, and The Parkinson's Foundation (see appendix for full list of funding sources)

A global experiment on motivating social distancing during the COVID-19 pandemic

Finding communication strategies that effectively motivate social distancing continues to be a global public health priority during the COVID-19 pandemic. This cross-country, preregistered experiment (n = 25,718 from 89 countries) tested hypotheses concerning generalizable positive and negative outcomes of social distancing messages that promoted personal agency and reflective choices (i.e., an autonomy-supportive message) or were restrictive and shaming (i.e., a controlling message) compared with no message at all. Results partially supported experimental hypotheses in that the controlling message increased controlled motivation (a poorly internalized form of motivation relying on shame, guilt, and fear of social consequences) relative to no message. On the other hand, the autonomy-supportive message lowered feelings of defiance compared with the controlling message, but the controlling message did not differ from receiving no message at all. Unexpectedly, messages did not influence autonomous motivation (a highly internalized form of motivation relying on one’s core values) or behavioral intentions. Results supported hypothesized associations between people’s existing autonomous and controlled motivations and self-reported behavioral intentions to engage in social distancing. Controlled motivation was associated with more defiance and less long-term behavioral intention to engage in social distancing, whereas autonomous motivation was associated with less defiance and more short- and long-term intentions to social distance. Overall, this work highlights the potential harm of using shaming and pressuring language in public health communication, with implications for the current and future global health challenges

Differential cross section measurements for the production of a W boson in association with jets in proton–proton collisions at √s = 7 TeV

Measurements are reported of differential cross sections for the production of a W boson, which decays into a muon and a neutrino, in association with jets, as a function of several variables, including the transverse momenta (pT) and pseudorapidities of the four leading jets, the scalar sum of jet transverse momenta (HT), and the difference in azimuthal angle between the directions of each jet and the muon. The data sample of pp collisions at a centre-of-mass energy of 7 TeV was collected with the CMS detector at the LHC and corresponds to an integrated luminosity of 5.0 fb[superscript −1]. The measured cross sections are compared to predictions from Monte Carlo generators, MadGraph + pythia and sherpa, and to next-to-leading-order calculations from BlackHat + sherpa. The differential cross sections are found to be in agreement with the predictions, apart from the pT distributions of the leading jets at high pT values, the distributions of the HT at high-HT and low jet multiplicity, and the distribution of the difference in azimuthal angle between the leading jet and the muon at low values.United States. Dept. of EnergyNational Science Foundation (U.S.)Alfred P. Sloan Foundatio

Optimasi Portofolio Resiko Menggunakan Model Markowitz MVO Dikaitkan dengan Keterbatasan Manusia dalam Memprediksi Masa Depan dalam Perspektif Al-Qur`an

Risk portfolio on modern finance has become increasingly technical, requiring the use of sophisticated mathematical tools in both research and practice. Since companies cannot insure themselves completely against risk, as human incompetence in predicting the future precisely that written in Al-Quran surah Luqman verse 34, they have to manage it to yield an optimal portfolio. The objective here is to minimize the variance among all portfolios, or alternatively, to maximize expected return among all portfolios that has at least a certain expected return. Furthermore, this study focuses on optimizing risk portfolio so called Markowitz MVO (Mean-Variance Optimization). Some theoretical frameworks for analysis are arithmetic mean, geometric mean, variance, covariance, linear programming, and quadratic programming. Moreover, finding a minimum variance portfolio produces a convex quadratic programming, that is minimizing the objective function ðð¥with constraintsð ð 𥠥 ðandð´ð¥ = ð. The outcome of this research is the solution of optimal risk portofolio in some investments that could be finished smoothly using MATLAB R2007b software together with its graphic analysis