Multidimensional chromatin profiling of zebrafish pancreas to uncover and investigate disease-relevant enhancers.

The pancreas is a central organ for human diseases. Most alleles uncovered by genome-wide association studies of pancreatic dysfunction traits overlap with non-coding sequences of DNA. Many contain epigenetic marks of cis-regulatory elements active in pancreatic cells, suggesting that alterations in these sequences contribute to pancreatic diseases. Animal models greatly help to understand the role of non-coding alterations in disease. However, interspecies identification of equivalent cis-regulatory elements faces fundamental challenges, including lack of sequence conservation. Here we combine epigenetic assays with reporter assays in zebrafish and human pancreatic cells to identify interspecies functionally equivalent cis-regulatory elements, regardless of sequence conservation. Among other potential disease-relevant enhancers, we identify a zebrafish ptf1a distal-enhancer whose deletion causes pancreatic agenesis, a phenotype previously found to be induced by mutations in a distal-enhancer of PTF1A in humans, further supporting the causality of this condition in vivo. This approach helps to uncover interspecies functionally equivalent cis-regulatory elements and their potential role in human disease.

Ptf1a function and transcriptional cis-regulation, a cornerstone in vertebrate pancreas development.

Vertebrate pancreas organogenesis is a stepwise process regulated by a complex network of signaling and transcriptional events, progressively steering the early endoderm toward pancreatic fate. Many crucial players of this process have been identified, including signaling pathways, cis-regulatory elements, and transcription factors (TFs). Pancreas-associated transcription factor 1a (PTF1A) is one such TF, crucial for pancreas development. PTF1A mutations result in dramatic pancreatic phenotypes associated with severe complications, such as neonatal diabetes and impaired food digestion due to exocrine pancreatic insufficiency. Here, we present a brief overview of vertebrate pancreas development, centered on Ptf1a function and transcriptional regulation, covering similarities and divergences in three broadly studied organisms: human, mouse and zebrafish.

Experience on how to implement a preanalytical and POCT unit in Madrid's IFEMA field hospital during this unprecedented COVID-19 emergency.

To fight the virus SARS-CoV-2 spread to Europe from China and to give support to the collapsed public health system, the Spanish Health Authorities developed a field hospital located in the facilities of Madrid exhibition centre (IFEMA) to admit and treat patients diagnosed with SARS-CoV-2 infectious disease (COVID-19). The Department of Laboratory Medicine of La Paz University Hospital in Madrid (LMD-HULP) was designated to provide laboratory services. Due to the emergency, the IFEMA field hospital had to be prepared for patient admission in less than 1 week and the laboratory professionals had to collaborate in a multidisciplinary group to assure that resources were available to start on time. The LMD-HULP participated together with the managers in the design of the tests portfolio and the integration of the healthcare information systems (IS) (hospital IS, laboratory IS and POCT management system). Laboratorians developed a strategy to quickly train clinicians and nurses on test requests, sample collection procedures and management/handling of the POCT blood gas analyser both by written materials and training videos. The IFEMA´s preanalytical unit managed 3782 requests, and more than 11,000 samples from March 27th to April 30th. Furthermore, 1151 samples were measured by blood gas analysers. In conclusion, laboratory professionals must be resilient and have to respond timely in emergencies as this pandemic. The lab's personnel selection, design and monitoring indicators to maintain and further improve the quality and value of laboratory services is crucial to support medical decision making and provide better patient care.

In Vivo Reporter Assays Uncover Changes in Enhancer Activity Caused by Type 2 Diabetes-Associated Single Nucleotide Polymorphisms.

Many single nucleotide polymorphisms (SNPs) associated with type 2 diabetes overlap with putative endocrine pancreatic enhancers, suggesting that these SNPs modulate enhancer activity and, consequently, gene expression. We performed in vivo mosaic transgenesis assays in zebrafish to quantitatively test the enhancer activity of type 2 diabetes-associated loci. Six out of 10 tested sequences are endocrine pancreatic enhancers. The risk variant of two sequences decreased enhancer activity, while in another two incremented it. One of the latter (rs13266634) locates in an SLC30A8 exon, encoding a tryptophan-to-arginine substitution that decreases SLC30A8 function, which is the canonical explanation for type 2 diabetes risk association. However, other type 2 diabetes-associated SNPs that truncate SLC30A8 confer protection from this disease, contradicting this explanation. Here, we clarify this incongruence, showing that rs13266634 boosts the activity of an overlapping enhancer and suggesting an SLC30A8 gain of function as the cause for the increased risk for the disease. We further dissected the functionality of this enhancer, finding a single nucleotide mutation sufficient to impair its activity. Overall, this work assesses in vivo the importance of disease-associated SNPs in the activity of endocrine pancreatic enhancers, including a poorly explored case where a coding SNP modulates the activity of an enhancer.

Amniotic membrane extract differentially regulates human peripheral blood T cell subsets, monocyte subpopulations and myeloid dendritic cells.

The discovery of the immunoregulatory potential of human amniotic membrane (hAM) propelled several studies focusing on its application for the treatment of immunological disorders. However, there is little information regarding the effects of hAM on distinct activation and differentiation stages of immune cells. Here, we aim to investigate the effect of human amniotic membrane extract (hAME) on the pattern of cytokine production by T cells, monocytes and myeloid dendritic cells (mDCs). For this purpose, peripheral blood mononuclear cells (PBMCs) from eight healthy individuals were stimulated in vitro in the presence or absence of hAME. Mitogen-induced proliferation of PBMCs and cytokine production among the distinct T cell functional compartments, monocyte subpopulations and mDCs were evaluated. hAME displayed an anti-proliferative effect and decreased the frequency of T cells producing tumor necrosis factor (TNF)α, interferon (IFN)γ and interleukin (IL)-2, for all T cell functional compartments. The frequency of IL-17 and IL-9-producing T cells was also reduced. The inhibition of mRNA expression of granzyme B, perforin and NKG2D by CD8+ T cells and γδ T cells and the augment of FoxP3 and IL-10 in CD4+ T cells and IL-10 in regulatory T cells were also observed. Furthermore, hAME inhibited IFNγ-induced protein (IP)-10 expression by classical and non-classical monocytes, without hampering the production of TNFα and IL-6 by monocytes and mDCs. These results suggest that hAME exerts an anti-inflammatory effect on T cells, still at a different extent for distinct T cell functional compartments.

Compliance with a sepsis bundle and its effect on intensive care unit mortality in surgical septic shock patients.

BACKGROUND: The Surviving Sepsis Campaign was launched in 2002, aiming at a 25% reduction in mortality in sepsis during a 5-year period. We hypothesized that the compliance with an adapted sepsis bundle would improve intensive care unit (ICU) survival in a cohort of surgical septic shock patients. METHODS: A retrospective, observational study was performed in surgical ICUs from two University hospitals. Seven quality indicators were considered to study the compliance with the sepsis bundle in 182 patients: (1) administration of antibiotics within 6 hours from diagnosis of septic shock, (2) initial effective antibiotic treatment, (3) adequate resuscitation within 6 hours after the diagnosis of septic shock, (4) administration of steroids, (5) use of activated protein C, (6) glucose control, and (7) protective ventilation. Univariate and multivariate logistic regression analyses were performed to make a predictive model to study the probability of survival according to the number of therapeutic guidelines fulfilled and to adjust for other predictive factors. RESULTS: Compliance with individual guidelines was considered adequate in more than 60% of the cases, except in the case of glucose control. For all quality indicators, ICU survival was higher in the bundle-compliant patients. Survival (61%) was associated with the fulfilment of increasing number of therapeutic guidelines (odds ratio, 1.64; 95% confidence interval, 1.28-2.1; p < 0.001). CONCLUSIONS: In surgical septic shock patients, the outcome was significantly related to the number of fulfilled therapeutic guidelines included in a sepsis bundle.

Elaboración de tejido conectivo artificial autólogo de mucosa oral y evaluación de su desempeño como cobertura biológica en lesiones mucosas inducidas en conejos / Elaboration of artificial autologous connective tissue of oral mucosa and evaluation of its performance as a biological coverage in induced mucosal lesions in rabbits

Para superar la escasez de tejido disponible para injerto en los tratamientos quirúrgicos convencionales de las lesiones de la mucosa bucal, se ha propuesto elaborar sustitutos de mucosa empleando las técnicas desarrolladas por la ingeniería tisular. En Colombia, este campo es incipiente y hasta ahora se conocen los primeros esfuerzos para elaborar injertos artificiales que sustituyan al tejido propio cuando éste es escaso. En investigaciones previas, nuestros grupo estandarizó un método de cultivo de fibroblastos primarios (aislados de piel humana) sobre un soporte de colágeno I. Los análisis histológicos y moleculares de los cultivos realizados mostraron la producción de matriz extracelular característica de la dermis. En el presente trabajo estandarizamos los métodos requeridos para elborar tejido conjuntivo oral, a partir de mucosa qureratinizada humana. La metodología establecida fue aplicada para desarrollar tejido conectivo oral de conejo, el cual se evaluó como injerto autólogo en heridas inducidas en los conejos fuentes de las células utilizadas en su preparación. El comportamiento de los tejidos artificiales in vitro e in vivo, en todas las etapas de su desarrollo, fue seguido histológica y molecularmente. Para los análisis histológicos se emplearon coloraciones convencionales con hematoxilina-eosina y azul de alciano. Para los estudios moleculares, se llevaron a cabo análisis de la expresión de genes de proteínas constitutivas de la matriz extracelular mediante transcripción reversa y reacción en cadena de polimerasa (RT-PCR). El conjunto de nuestros resultados indica que el tejido autólogo desarrollado in vitro funciona como cobertura biológica en heridas mucosas. Durante los procedimientos quirúrgicos se observó que los injertos artificiales se posicionan en el sitio del defecto con facilidad, favorecen la hemostasia y cubren adecuadamente la herida. A nivel histológico, a las 4 y 8 semanas, se observó presencia de epitelio plano crónico no queratinizado con una capa basal activa, proliferación fibroblástica y en algunos casos, la presencia de infiltrado inflamatorio crónico leve en el tejido conectivo subyacente

Hemofilia: ejercicio y actividades deportivas / Haemophilia: exercises and sport

Este folleto realizado por el Grupo de Hemofilia de la Fundación Santafé de Bogotá presenta conceptos básicos sobre las actividades deportivas para pacientes con hemofilia y un programa de ejercicios que se puede realizar en casa con ayuda de la familia. Está diseñado para tratar los problemas específicos asi como para mejorar la condición física general. Para lograr este propósito los autores recomiendan realizar los ejercicios con una frecuencia mínima de tres veces por semana y tener una actividad deportiva una o dos veces por semana, sin demandar esfuerzo del organismo