Effect of Epas1 and Pcx inactivation in pancreatic β-cell formation and function.

According to the consensus model for GSIS (Glucose-Stimulated Insulin Secretion), glucose is rapidly metabolized and coupled to insulin secretion involving a substantial number of cofactors and metabolites intermediates such as ATP, NADPH and citrate, among many others (1). In particular, pyruvate carboxylase (PC) is a fundamental enzyme in redox cycling between NADH and NADPH and also participates in an intricate process known as “pyruvate cycling” which allows the anaplerotic entry of pyruvate in the krebs cycle (2).Pancreatic β-cells express abnormally high levels of pyruvate carboxylase (PC) and insignificant levels of phosphoenolpyruvate carboxylase, the enzyme necessary for gluconeogenesis. This implies that PC must play a different role in β-cells, such as insulin secretion, which is required for the "metabolic switch" from glycolytic to aerobic metabolism during β-cell maturation. It is also known that pyruvate carboxylase activity is elevated in mature β-cells but diminished under diabetic conditions. Recent studies have revealed that the Hypoxia Inducible factor (HIF) pathway plays an important role of in β-cell function (cita algun articulo nuestro). Both overexpression and inactivation of HIF-1α in β cells cause defects in insulin secretion. However, the role of HIF-2α in β-cell formation and function has been largely ignored despite been reported to be activated during diabetic conditions.In this project, we hypothesize that HIF-2α and pyruvate carboxylase activity during late pancreatic HIF-2α formation is critical for the metabolic switch that ocurrs in β-cell during early postnatal development and thus for proper β-cell function.In this study, we will analyze the expression of Epas1 (the gene encoding HIF-2α and Pcx) at different prenatal and postnatal stages by mRNA TaqMan essay. Using Cre/lox technology in mice, we will inactivate Epas1 and Pcx specifically in β-cells. Immunofluorescence and immunohistochemical assays will be carried out to determine specific markers of cell identity, vascularization, proliferation, and polarity in pancreatic tissue of Epas1- and Pcx-deficient mice. Finally, we will also evaluate the in vivo behavior of pancreatic β-cells in transgenic mice through glucose and insulin tolerance assays (GTT and ITT). This will help us understand the relationship between HIF-2 and PC activity and β-cell development and function, as well as whether HIF-2 and PC activity play a role in β-cell failure during diabetes

Healthcare workers hospitalized due to COVID-19 have no higher risk of death than general population. Data from the Spanish SEMI-COVID-19 Registry

Aim To determine whether healthcare workers (HCW) hospitalized in Spain due to COVID-19 have a worse prognosis than non-healthcare workers (NHCW). Methods Observational cohort study based on the SEMI-COVID-19 Registry, a nationwide registry that collects sociodemographic, clinical, laboratory, and treatment data on patients hospitalised with COVID-19 in Spain. Patients aged 20-65 years were selected. A multivariate logistic regression model was performed to identify factors associated with mortality. Results As of 22 May 2020, 4393 patients were included, of whom 419 (9.5%) were HCW. Median (interquartile range) age of HCW was 52 (15) years and 62.4% were women. Prevalence of comorbidities and severe radiological findings upon admission were less frequent in HCW. There were no difference in need of respiratory support and admission to intensive care unit, but occurrence of sepsis and in-hospital mortality was lower in HCW (1.7% vs. 3.9%; p = 0.024 and 0.7% vs. 4.8%; p<0.001 respectively). Age, male sex and comorbidity, were independently associated with higher in-hospital mortality and healthcare working with lower mortality (OR 0.211, 95%CI 0.067-0.667, p = 0.008). 30-days survival was higher in HCW (0.968 vs. 0.851 p<0.001). Conclusions Hospitalized COVID-19 HCW had fewer comorbidities and a better prognosis than NHCW. Our results suggest that professional exposure to COVID-19 in HCW does not carry more clinical severity nor mortality

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Study of gene sequences and Fluorescence In Situ Hybridization (FISH) in senegalese sole (Solea senegalensis)

El lenguado senegalés (Solea senegalensis) es un teleósteo perteneciente al orden de los Pleuronectiformes y a la familia Soleidae. Se trata de una especie muy apreciada gastronómicamente y de alto valor comercial, que actualmente se cultiva a gran escala en piscifactorías de toda Europa. A pesar de que la producción del lenguado senegalés ha seguido una tendencia ascendente con los años, aún existen una serie de dificultades en su cultivo en cautividad, como la susceptibilidad a enfermedades, la pérdida del instinto de cortejo de los machos, o anomalías en la pigmentación y la metamorfosis. Al mismo tiempo, el desconocimiento de las bases genéticas del lenguado, dificultan el desarrollo de programas de mejora genética que permitan aumentar el rendimiento de los cultivos. El presente trabajo contribuye a aumentar la información genómica de S. senegalensis disponible hasta la fecha, incrementando la densidad del mapa genético actual. Con ese objetivo, se realizó el estudio de las secuencias génicas contenidas en 5 BACs (Cromosoma Bacteriano Artificial) rastreados a partir de una genoteca BAC de S. senegalensis. Estos BACs contenían genes relevantes (c8a, sfsr3a, bpg, aanat1 y rps6kb1) relacionados con el sistema inmune y la metamorfosis del lenguado senegalés. Tras secuenciar cada uno de los BACs, se realizó la anotación de los genes que contenían y se determinó su ontología. Un total de 35 nuevos genes fueron descritos, destacando 15 genes implicados en caracteres relevantes en acuicultura, como el desarrollo del sistema nervioso (myh10 y ptprg), la diferenciación y proliferación celular (ube2o, ppp4r2, oard1 y pgap6), el desarrollo y funcionamiento del sistema inmune (foxp1b, c8a, c8b, fyb2 y tfrc), la regulación del estrés (fkbp5) y el control del ciclo circadiano y desarrollo de la metamorfosis (aanat1, srsf3a y rps6kb1). Mediante el análisis de la microsintenia, se determinó el orden relativo de los genes anotados en S. senegalensis utilizando como referencia las especies filogenéticamente más cercanas, observándose una alta conservación con las regiones génicas estudiadas. La aplicación de la hibridación in situ de fluorescencia permitió determinar la morfología de los cromosomas sobre los que hibrida cada BAC, situándose el BAC 10F5 (c8a) en la pareja de cromosomas metacéntricos mayor (cromosoma 1) y el resto de BACs en cromosomas acrocéntricos, sin determinar de forma exacta su localización. Finalmente, unificando la información genética obtenida del estudio de las secuencias y la hibridación in situ de fluorescencia, se aumentó la densidad del mapa genético integrado de S. senegalensis.Senegalese sole (S. senegalensis) is a teleost belonging to the order of the Pleuronectiformes and the family Soleidae. It is considered a highly valued species, both gastronomically and for its high comercial value, that is currently being cultivated on a large scale in plenty of fish farms across Europe. Despite the senegalese sole production has followed an increasing tendency over the last years, there still exist certain difficulties in its cultivation in captivity, such as disease susceptibility, the loss of courtship instinct in males or pigmentation and metamorphosis abnormalities. At the same time, the lack of knowledge of genetic bases of the sole, make it difficult to develop genetic improvement programs that allow to increase the culture yields. This work contributes to increase the genomic information in S. senegalensis that is available until date, allowing to augment the current genetic map density. With this objective, the study of gene sequences contained in 5 BACs (Bacterial Artificial Chromosomes), tracked from a BAC library of S.senegalensis, was carried out. These BACs contained relevant genes (c8a, sfsr3a, bpg, aanat1 and rps6kb1) related to immune system and metamorphosis of senegalese sole. After sequencing, both annotation and ontology determination of the genes contained in the BACs were carried out. A total of 35 genes were described, standing out 15 genes that were involved in important traits in aquaculture, such as development of nervous system (myh10 and ptprg), cell proliferation and diferenciation (ube2o, ppp4r2, oard1 and pgap6), development and operation of immune system (foxp1b, srsf3a, c8a, c8b, fyb2 and tfrc), stress regulation (fkbp5) and control of circadian cycle and metamorphosis (aanat1 and rps6kb1). Analyzing the microsinteny, the relative order of the annotated genes in S. senegalensis, using as a reference the phylogenetically closest species, was determined, observing a high conservation with the studied gene regions. The fluorescence in situ hybridization was applied to determine the morphology of the chromosomes on wich every BAC hybridize. The BAC 10F5 (c8a) was located on the biggest metacentric couple of chromosomes (chromosome 1) and the rest of the BACs in acrocentric chromosomes, not specifying its exact location. Eventually, unifying genetic information from the study of sequences and fluorescence in situ hybridization, the density of the integrated genetic map of S. senegalensis was increased

Physico-chemical characterization of the tumour microenvironment of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive lethal malignancy that accounts for more than 90% of pancreatic cancer diagnoses. Our research is focused on the physico-chemical properties of the tumour microenvironment (TME), including its tumoural extracellular matrix (tECM), as they may have an important impact on the success of cancer therapies. PDAC xenografts and their decellularized tECM offer a great material source for research in terms of biomimicry with the original human tumour. Our aim was to evaluate and quantify the physico-chemical properties of the PDAC TME. Both cellularized (native TME) and decellularized (tECM) patient-derived PDAC xenografts were analyzed. A factorial design of experiments identified an optimal combination of factors for effective xenograft decellularization. Our results provide a complete advance in our understanding of the PDAC TME and its corresponding stroma, showing that it presents an interconnected porous architecture with very low permeability and small pores due to the contractility of the cellular components. This fact provides a potential therapeutic strategy based on the therapeutic agent size