Characterization of the "diabesity" gene HMG20A in pancreatic islets

Motivation: Type 2 Diabetes (T2D) accounts for 90-95% of diagnosed diabetic patients, which tendency in the next years is also expected to increase. Recent genomic wide association studies showed a correlation of an allelic variation of HMG20A with T2D in some ethnic groups. Up to date, there is no scientific evidence of the role of this gene in pancreatic tissue. But, in central nervous system, HMG20A regulates the expression of NeuroD, common in pancreas and nervous system morphogenesis. Here, our group makes an approach to characterize HMG20A in pancreatic islets, focusing on its involvement in glucose-stimulated insulin secretion (GSIS) and pancreatic islets development. We want to demonstrate that: 1) HMG20A is expressed in endocrine pancreas 2) HMG20A modifies expression of genes involved in pancreas development 3) silencing HMG20A affects expression levels of insulin secretion related genes and functionality.Methods: Qualitative expression of HMG20A is tested out in slides of pancreatic sections obtained from control mice. Co-localization with α or β cells is analyzed by immunofluorescence using anti-HMG20A, anti-insulin/glucagon antibodies and Dapi for nuclei. INS-1E cells are cultured and treated with a specific siRNA against HMG20A or a non-specific siRNA control during 72h. Genes involved in insulin secretion and endocrine pancreas development are assayed via qRT-PCR in INS1-E cells after siRNA treatment. Pdx1, Pax4, MafA and HMG20A expression levels are assessed following 2-ΔΔCt method. Finally, HMG20A silenced mouse islets and INS-1E are cultured at low glucose (2.8 mM) and high glucose medium (22 mM) following quantification of insulin secretion by ELISA.Results: Immunofluorescence confirmed co-localization of HMG20A with insulin (β-cell) and with glucagon (α-cells) producing cells in mouse pancreas. HMG20A expression diminished a 60% after treating INS1-E cells with a specific siRNA for HMG20A. Insulin secretion regulator gene, MafA, is downregulated significantly (50-60%) after HMG20A silencing. Pax4 expression significantly increased meanwhile Pdx1 showed a tendency to decrease. A 40% drop in insulin secretion is obtained in siHMG20A treated mouse islets compared to control.Conclusions: This data confirms HMG20A expression in pancreatic islets and impairment of insulin secretion when it is knocked down. Hence, concluding that HMG20A plays an important role in physiological GSIS and regulating pancreatic development related genes

Self-association of short DNA loops through minor groove C:G:G:C tetrads

In addition to the better known guanine-quadruplex, four-stranded nucleic acid structures can be formed by tetrads resulting from the association of Watson-Crick base pairs. When such association occurs through the minor groove side of the base pairs, the resulting structure presents distinctive features, clearly different from quadruplex structures containing planar G-tetrads. Although we have found this unusual DNA motif in a number of cyclic oligonucleotides, this is the first time that this DNA motif is found in linear oligonucleotides in solution, demonstrating that cyclization is not required to stabilize minor groove tetrads in solution. In this article, we have determined the solution structure of two linear octamers of sequence d(TGCTTCGT) and d(TCGTTGCT), and their cyclic analogue d, utilizing 2D NMR spectroscopy and restrained molecular dynamics. These three molecules self-associate forming symmetric dimers stabilized by a novel kind of minor groove C:G:G:C tetrad, in which the pattern of hydrogen bonds differs from previously reported ones. We hypothesize that these quadruplex structures can be formed by many different DNA sequences, but its observation in linear oligonucleotides is usually hampered by competing Watson-Crick duplexes

Influencia de la microestructura en las propiedades mecánicas de alta temperatura de los Eutécticos Al2O3/Y3Al5O12 crecidos por solidificación direccional

En este trabajo se ha realizado un estudio del sistema eutéctico Al2O3/Y3Al5O12 crecido a distintas velocidades mediante solidificación direccional de zona flotante por calentamiento con láser. El análisis microestructural se ha realizado mediante Microscopía Electrónica de Barrido (MEB) y de Transmisión (MET), tanto en muestras tras el procesado con láser como en muestras sometidas a los distintos tratamientos termo-mecánicos. El estudio de las propiedades mecánicas se ha llevado a cabo mediante ensayos a velocidad de deformación constante y ensayos de fluencia -a carga de compresión constante- para diversos valores de tensión y de temperatura en atmósfera de aire. De los resultados obtenidos se deduce que la resistencia está íntimamente asociada a la microestructura de las muestras, la cual depende de la velocidad de solidificación. La presencia de dislocaciones es clara a altas velocidades de deformación, observándose los mecanismos activos de deformación típicos de la alúmina monocristalinas, como son la formación de bucles de dislocaciones para evitar obstáculos. Los valores de los exponentes de tensión y la energía de activación sugieren un comportamiento difusional para bajas velocidades de deformación, a las que no se observan dislocaciones.The Al2O3/Y3Al5O12 eutectic system melt grown by laser heating float zone in a range of pull rates has been studied. The microstructural analysis has been made by Scaning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) in as-fabricated and tested samples. The mechanical properties have been studied by compression tests at constant crosshead speed and compression creep for a range of stresses and temperatures in air. It has been determined that the resistance is intimately associated to processing. The presence of dislocations is clear after plastic deformation at high strain rates, being active the typical deformation mechanisms of alumina single crystals, like dislocation loop formation to avoid obstacles. The values of the stress exponents and the activation energy suggest a diffusional control for plastic deformation at low strain rates, where dislocations are not observed

Configuring the neighbourhood effect in irregular cellular automata based models

Cellular automata (CA) models have been widely employed to simulate urban growth and land use change. In order to represent urban space more realistically, new approaches to CA models have explored the use of vector data instead of traditional regular grids. However, the use of irregular CA-based models brings new challenges as well as opportunities. The most strongly affected factor when using an irregular space is neighbourhood. Although neighbourhood definition in an irregular environment has been reported in the literature, the question of how to model the neighbourhood effect remains largely unexplored. In order to shed light on this question, this paper proposed the use of spatial metrics to characterise and measure the neighbourhood effect in irregular CA-based models. These metrics, originally developed for raster environments, namely the enrichment factor and the neighbourhood index, were adapted and applied in the irregular space employed by the model. Using the results of these metrics, distance-decay functions were calculated to reproduce the push-and-pull effect between the simulated land uses. The outcomes of a total of 55 simulations (five sets of different distance functions and eleven different neighbourhood definition distances) were compared with observed changes in the study area during the calibration period. Our results demonstrate that the proposed methodology improves the outcomes of the urban growth simulation model tested and could be applied to other irregular CA-based models

Defective chaperone-mediated autophagy is a hallmark of joint disease in patients with knee osteoarthritis

[Abstract] Objective: Defects in autophagy contribute to joint aging and Osteoarthritis (OA). Identifying specific autophagy types could be useful for developing novel treatments for OA. Design: An autophagy-related gene array was performed in blood from non-OA and knee OA subjects from the Prospective Cohort of A Coruña (PROCOAC). The differential expression of candidate genes was confirmed in blood and knee cartilage and a regression analysis was performed adjusting for age and BMI. HSP90A, a chaperone mediated autophagy (CMA) marker was validated in human knee joint tissues, as well as, in mice with aging-related and surgically-induced OA. The consequences of HSP90AA1 deficiency were evaluated on OA pathogenesis. Finally, the contribution of CMA to homeostasis was studied by assessing the capacity to restore proteostasis upon ATG5-mediated macroautophagy deficiency and genetic HSP90AA1 overexpression. Results: 16 autophagy-related genes were significantly down-regulated in blood from knee OA subjects. Validation studies showed that HSP90AA1 was down-regulated in blood and human OA cartilage and correlated with risk incidence of OA. Moreover, HSP90A was reduced in human OA joints tissues and with aging and OA in mice. HSP90AA1 knockdown was linked to defective macroautophagy, inflammation, oxidative stress, senescence and apoptosis. However, macroautophagy deficiency increased CMA, highlighting the CMA-macroautophagy crosstalk. Remarkably, CMA activation was sufficient to protect chondrocytes from damage. Conclusions: We show that HSP90A is a key chaperone for chondrocyte homeostasis, while defective CMA contributes to joint damage. We propose that CMA deficiency is a relevant disease mechanism and could represent a therapeutic target for OA.Instituto de Salud Carlos III; PI17/02059Instituto de Salud Carlos III; PI20/0064

Genome-wide scan for five brain oscillatory phenotypes identifies a new qtl associated with theta eeg band

Brain waves, measured by electroencephalography (EEG), are a powerful tool in the investigation of neurophysiological traits and a noninvasive and cost-effective alternative in the diagnostic of some neurological diseases. In order to identify novel Quantitative Trait Loci (QTLs) for brain wave relative power (RP), we collected resting state EEG data in five frequency bands (d, ¿, a, ß1, and ß2) and genome-wide data in a cohort of 105 patients with late onset Alzheimer’s disease (LOAD), 41 individuals with mild cognitive impairment and 45 controls from Iberia, correcting for disease status. One novel association was found with an interesting candidate for a role in brain wave biology, CLEC16A (C-type lectin domain family 16), with a variant at this locus passing the adjusted genome-wide significance threshold after Bonferroni correction. This finding reinforces the importance of immune regulation in brain function. Additionally, at a significance cutoff value of 5 × 10-6, 18 independent association signals were detected. These signals comprise brain expression Quantitative Loci (eQTLs) in caudate basal ganglia, spinal cord, anterior cingulate cortex and hypothalamus, as well as chromatin interactions in adult and fetal cortex, neural progenitor cells and hippocampus. Moreover, in the set of genes showing signals of association with brain wave RP in our dataset, there is an overrepresentation of loci previously associated with neurological traits and pathologies, evidencing the pleiotropy of the genetic variation modulating brain function.This project is supported by “European Commission” and “European Regional Development Fund” under the project “Análisis y correlación entre el genoma completo y la actividad cerebral para la ayuda en el diagnóstico de la enfermedad de Alzheimer” (Project 1317_AD-EEGWA), (Cooperation Programme INTERREG V-A Spain-Portugal POCTEP 2014–2020) and the COMPETE 2020-Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020. Portuguese funds are supporting this work through FCT-Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274). SM, AML, NP and IG are funded by FCT: CEECIND/00684/2017, IF/01262/2014, SFRH/BPD/97414/2013 and CEECIND/02609/2017, respectively. MA is funded by the Grant RYC-2015-18241 from the Spanish Government. Spanish funds are supporting this work through “Ministerio de Ciencia e Innovación–Agencia Estatal de Investigación” and “European Regional Development Fund” under project PGC2018-098214-A-I00 and by “CIBER en Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN)” through “Instituto de Salud Carlos III” co-funded with “European Regional Development Fund” funds

Apoe variants in an iberian alzheimer cohort detected through an optimized sanger sequencing protocol

The primary genetic risk factor for late onset Alzheimer’s disease (LOAD) is the APOE4 allele of Apolipoprotein E (APOE) gene. The three most common variants of APOE are determined by single nucleotide polymorphisms (SNPs) rs429358 and rs7412. Our aim was to estimate allele and genotype frequencies of APOE variants in an Iberian cohort, thus helping to understand differences in APOE-related LOAD risk observed across populations. We analyzed saliva or buccal swab samples from 229 LOAD patients and 89 healthy elderly controls (=68 years old) from Northern Portugal and Castile and León region, Spain. The genotyping was performed by Sanger sequencing, optimized to overcome GC content drawbacks. Results obtained in our Iberian LOAD and control cohorts are in line with previous large meta-analyses on APOE frequencies in Caucasian populations; however, we found differences in allele frequencies between our Portuguese and Spanish subgroups of AD patients. Moreover, when comparing studies from Iberian and other Caucasian cohorts, differences in APOE2 and APOE4 frequencies and subsequent different APOE-related LOAD risks must be clarified. These results show the importance of studying genetic variation at the APOE gene in different populations (including analyses at a regional level) to increase our knowledge about its clinical significance.This work was supported by ‘European Commission’ and ‘European Regional Development Fund’ (FEDER) under the project “Análisis y correlación entre el genoma completo y la actividad cerebral para la ayuda en el diagnóstico de la enfermedad de Alzheimer” (Project 0378_AD_EEGWA_2_P), (Cooperation Programme INTERREG V-A Spain-Portugal POCTEP 2014– 2020) and the COMPETE 2020-Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020. Portuguese funds are supporting this work through FCT-Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274). IG, AML, SM, and NP are funded by FCT: CEECIND/02609/2017, IF/01262/2014, CEECIND/00684/2017, and through the Decreto-Lei n◦ 57/2016 de 29 de Agosto, respectively. Spanish funds are supporting this work through ‘Ministerio de Ciencia e Innovación–Agencia Estatal de Investigación’ and FEDER under project PGC2018-098214-A-I00 and by “CIBER en Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN)” through “Instituto de Salud Carlos III” co-funded with FEDER funds

Disruption of the sea bass skin-scale barrier by antidepressant fluoxetine and estradiol: in vivo and in vitro evidence

Trabajo presentado en la Joint 30th Conference of the European Society for Comparative Endocrinology and of the 9th International Society for Fish Endocrinology, celebrada en Faro (Portugal) del 04 al 08 de septiembre de 2022.Fluoxetine (FLX) is a highly prescribed selective inhibitor of serotonin-reuptake and an emerging pollutant affecting fish behaviour, stress and reproduction, but little is known about possible actions and mechanisms in barrier tissues. We combined in vivo and in vitro approaches to demonstrate multi-level impacts of FLX on the sea bass (Dicentrarchus labrax) skin-scale barrier and on the estrogenic system. Juvenile sea bass intraperitoneally injected with FLX had significantly increased levels of FLX and its metabolite nor-FLX. In contrast to the natural estrogen E2, FLX did not increase plasma calcium, phosphorus (P) or vitellogenin, although a slight decrease in scale P content was detected. Quantitative SWATH-MS proteomics of the scales identified 134 proteins that were affected by FLX. Modified proteins were mainly related to extracellular matrix and protein turnover and energy production, 31 of which were also affected by E2. Multiple estrogen receptors and genes related to serotonin activity, transport and degradation were expressed in sea bass scales and transcript abundance of some of them was modulated by E2 and/or FLX. Using a minimally invasive in vitro bioassay with cultured sea bass scales and adhering epithelia we showed direct effects of FLX exposure on enzymatic activity associated with mineral mobilization, while the expression of estrogen receptors was not significantly affected. In in vitro receptor-reporter assays, FLX alone did not activate any of the three sea bass nuclear estrogen receptors but had antiestrogenic effects on Esr1/2b when in co-treatment with E2, and directly activated both plasma membrane Gprotein-coupled estrogen receptors. The combination of in vitro and in vivo assays substantiated the notion that FLX disrupted scale physiology through several different processes, with probable consequences for fish health, and revealed that some of the mechanisms of disruption can result from direct interaction with multiple estrogen .Projects UIDB/04326/2020, PTDC/AAG-GLO/4003/2012 and DL57/2016/CP1361/CT0015 from FCT (Pt); EU Interreg FR-UK project RedPol; grant AGL2015-67477-C2-1- R (Sp)

El ciclo celular

ResumenEl proceso conocido como ciclo celular es de gran importacia para la célula ya que tiene como función la formación completa de una nueva célula, evitando en lo posible la creación de células con múltiples errorres, lo cual le permite al organismo permanecer en un constante equilibrio, previeniendo así aquellos desórdenes que pueden perjudicar su salud; de esta manera, todas las célular están controladas por proteínas que no permiten que se presenten situaciones desastrosas para un ser vivo.[Lomanto LD, Ortiz OL, Bretón CO, Gómez AL, Mesa VM. El ciclo celular. MedUNAB 2003; 6(16):21-29].Palabras clave: Ciclo celular, factor promotor de mitosis, proteína RB o 104, clinicas, cinasas

Characterisation of HRas local signal transduction networks using engineered site-specific exchange factors.

Ras GTPases convey signals from different types of membranes. At these locations, different Ras isoforms, interactors and regulators generate different biochemical signals and biological outputs. The study of Ras localisation-specific signal transduction networks has been hampered by our inability to specifically activate each of these Ras pools. Here, we describe a new set of site-specific tethered exchange factors, engineered by fusing the RasGRF1 CDC25 domain to sub-localisation-defining cues, whereby Ras pools at specific locations can be precisely activated. We show that the CDC25 domain has a high specificity for activating HRas but not NRas and KRas. This unexpected finding means that our constructs mainly activate endogenous HRas. Hence, their use enabled us to identify distinct pathways regulated by HRas in endomembranes and plasma membrane microdomains. Importantly, these new constructs unveil different patterns of HRas activity specified by their subcellular localisation. Overall, the targeted GEFs described herein constitute ideal tools for dissecting spatially-defined HRas biochemical and biological functions