Genome editing of human pancreatic beta cell models : problems, possibilities and outlook

Understanding the molecular mechanisms behind beta cell dysfunction is essential for the development of effective and specific approaches for diabetes care and prevention. Physiological human beta cell models are needed for this work. We review the possibilities and limitations of currently available human beta cell models and how they can be dramatically enhanced using genome-editing technologies. In addition to the gold standard, primary isolated islets, other models now include immortalised human beta cell lines and pluripotent stem cell-derived islet-like cells. The scarcity of human primary islet samples limits their use, but valuable gene expression and functional data from large collections of human islets have been made available to the scientific community. The possibilities for studying beta cell physiology using immortalised human beta cell lines and stem cell-derived islets are rapidly evolving. However, the functional immaturity of these cells is still a significant limitation. CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) has enabled precise engineering of specific genetic variants, targeted transcriptional modulation and genome-wide genetic screening. These approaches can now be exploited to gain understanding of the mechanisms behind coding and non-coding diabetes-associated genetic variants, allowing more precise evaluation of their contribution to diabetes pathogenesis. Despite all the progress, genome editing in primary pancreatic islets remains difficult to achieve, an important limitation requiring further technological development.Peer reviewe

Generation of a SOX2 reporter human induced pluripotent stem cell line using CRISPR/SaCas9

SOX2 is an important transcription factor involved in pluripotency maintenance, pluripotent reprogramming and differentiation towards neural lineages. Here we engineered the previously described HEL24.3 hiPSC to generate a SOX2 reporter by knocking-in a T2A fused nuclear tdTomato reporter cassette before the STOP codon of the SOX2 gene coding sequence. CRISPR/SaCas9-mediated stimulation of homologous recombination was utilized to facilitate faithful targeted insertion. This line accurately reports the expression of endogenous SOX2 and therefore constitutes a useful tool to study the SOX2 expression dynamics upon hiPSC culture, differentiation and somatic cell reprogramming. (C) 2017 The Authors. Published by Elsevier B.V.Non peer reviewe

Conditionally Stabilized dCas9 Activator for Controlling Gene Expression in Human Cell Reprogramming and Differentiation

CRISPR/Cas9 protein fused to transactivation domains can be used to control gene expression in human cells. In this study, we demonstrate that a dCas9 fusion with repeats of VP16 activator domains can efficiently activate human genes involved in pluripotency in various cell types. This activator in combination with guide RNAs targeted to the OCT4 promoter can be used to completely replace transgenic OCT4 in human cell reprogramming. Furthermore, we generated a chemically controllable dCas9 activator version by fusion with the dihydrofolate reductase (DHFR) destabilization domain. Finally, we show that the destabilized dCas9 activator can be used to control human pluripotent stem cell differentiation into endodermal lineages.Peer reviewe

Sermon predicado en el convento de las Carmelitas Descalças de Madrid en la octava que sus Magestades hizieron a la Santa Madre Teresa de Iesus al nuevo titulo de Patrona de España

Copia digital. Valladolid : Junta de Castilla y León. Consejería de Cultura y Turismo, 2012-2013Sign.: []2, A-D4Port. con esc. real xil

Análisis de riesgo en seguridad y salud ocupacional en una planta de derivados lácteos

Ciclo Optativo de Especialización y Profesionalización en Gestión de la Calidad y Auditoría AmbientalEl presente trabajo de investigación consistió en realizar el análisis de riesgo en Seguridad y Salud Ocupacional en la Planta Piloto de Leche de la Universidad Nacional Agraria La Molina, mediante la realización de la línea base en materia de salud y seguridad en el trabajo, identificación de los peligros y evaluación de los riegos en los procesos de producción de derivados lácteos con la finalidad de proponer y establecer medidas de control que prevengan la ocurrencia de accidentes y enfermedades ocupacionales. Asimismo, los peligros identificados en las áreas de la Planta Piloto de Leche se plasmaron en mapas de riesgos. Asimismo se fundamenta un análisis de riesgos en seguridad y salud ocupacional en la Planta Piloto de leche de la UNALM, logrando el cumplimento de la Ley N° 29783 y su reglamento D.S. N° 005-2012-TR. Se trabajó en la Planta Piloto de Leche donde se procesa principalmente la leche extraída de las vacas del establo de la Unidad Experimental de Zootecnia (UEZ) que se encuentra dentro de la Universidad Nacional Agraria La Molina (UNALM). La planta consta de las siguientes áreas: administración, producción, control de calidad; además cuenta con: la secretaria, sala de información, aulas, el almacén de insumos y mantenimiento. Para determinar el estado inicial de la Planta con respecto a Seguridad y Salud en el Trabajo, se elaboró la línea base previo al análisis de riesgo. La identificación de peligros y evaluación de riesgos realizada en la presente investigación, abarcó las siguientes actividades: reuniones de coordinación con personal de la planta piloto de leche, identificación de procesos a través de visitas guiadas a la planta, identificación de peligros a través de inspecciones, uso de listas de verificación y entrevistas al personal de planta; una vez identificados los peligros, se evaluó cada uno de los riesgos asociados, en base al método N° 2 (Identificación de Peligros y Evaluación de Riesgos Laborales) descrita en la R.M. N° 050-2013-TR. Se elaboró la Matriz IPER de la Planta Piloto de Leche (PPL), donde se obtuvieron resultados como: Los procesos de elaboración de yogurt (58), almacenamiento (39) y elaboración de mantequilla (31) fueron los que presentaron un mayor número de peligros identificados. Los tipos de peligros identificados en los procesos de la Planta Piloto de Leche (PPL) son: Disergonómico (31 %); físico (22 %); locativo (19 %); químico (14 %); mecánico (7 %); fisicoquímico (3 %); eléctrico (2 %); biológico (1 %); psicosociales (1 %). La frecuencia de los Niveles de riesgo dentro de la Matriz IPER son: Trivial (0%), Tolerable (21 %), Moderados (62 %) Importante (16 %), Intolerables (1 %). Los riesgos significativos (importantes e intolerables) son el 17 % del total de riesgos evaluados en la Planta Piloto de Leche. En la Matriz de Identificación de Peligros y Evaluación de Riesgos (IPER), además de la identificación de peligros y evaluación de riesgos se establecieron medidas de control para minimizar la ocurrencia de eventos no deseados que pudiesen generar pérdidas a la organización. Finalmente, en base a los resultados obtenidos se realizaron las conclusiones y recomendaciones del presente trabajo, además de la Línea Base en materia de Salud y Seguridad en el Trabajo y el Mapa de Riesgo de la Planta Piloto de Leche.The present research was to develop risk analysis in Occupational Health and Safety in the Milk Pilot Plant of UNALM, by developing the baseline on health and safety at work, identifying the hazards and assessment of risks in the production process of dairy products in order to propose and implement control measures to prevent the occurrence of accidents and occupational diseases. Also the hazards identified in the areas of Milk Pilot Plant were reflected in risk maps. Risk analysis in occupational health and safety in Milk Pilot Plant also builds UNALM, achieving compliance with Law N° 29783 and its regulations D.S. N° 005-2012-TR. It was worked at the Pilot Plant milk where cows milk extracted from the stable of the Experimental Husbandry Unit (UEZ) located inside the UNALM. The plant consists of the following areas: management, production, quality control; also has: secretary, information room, warehouse supplies and maintenance. For determining the initial state of the plant with regard to Safety and Health at Work, the previous baseline is prepared to risk analysis Hazard identification and risk assessment for the development of this project spanned the following activities: coordination meetings with staff from the pilot of milk, plant identification process through guided visits to the plant, through hazard identification inspection, use of checklists and interviews with plant personnel; once identified hazards, assessed each of the associated risks, based on the method # 2 (Hazard Identification and Assessment of Occupational Hazards) described in R.M. N° 050-2013-TR. The IPER Matrix of the Milk Pilot Plant, where results were obtained as elaborated: The yogurt-making processes (58), storage (39) and manufacture of butter (31) were those with a greater number of hazards identified. The types of hazards identified in the processes of Pilot Plant Milk are Disergonomic (31 %); physical (22 %); locative (19 %); chemical (14 %); mechanic (7 %); physicochemical (3 %); electric (2 %); biological (1 %); psychosocial (1 %). The frequency of risk levels within the Matrix IPER are: Trivial (0 %) Tolerable (21 %), Moderate (62 %) Important (16 %), Intolerable (1 %). The significant risks (Major and intolerable) are 17 % of total assessed risks at the Pilot Plant Milk. In the Matrix IPER addition to hazard identification and risk assessment control measures were established to minimize undesirable effects that could generate losses to the organization the occurrence of events. Finally, based on the results and recommendations of this study were performed in addition the Base Line of Safety and Health in the Job and the Risk Map of the Pilot Plant Milk.Tesi

Study of the microbiota associated to Ruditapes decussatus and Ruditapes philippinarum clams by 16S rRNA metabarcoding, dilution to extinction, and culture-based techniques

The study of the microbiota associated to clams is important not only to know their sanitary status but also to prevent pathobiology events. The use of different microbiological techniques can help to obtain a better picture of the bacterial diversity of clams as well as to isolate new bacterial taxa. In this study, two clam species, Ruditapes decussatus and R. philippinarum, were analyzed in two locations of Galicia (northwest of Spain) in April and October, by combining classic culturing, dilution-to-extinction approach, and 16S rRNA gene target sequencing. 16S rRNA gene target sequencing revealed a great diversity within the clam samples, shedding light into the vast microbial communities associated to these bivalves. All samples were dominated by the same bacterial genera in the different periods, namely Mycoplasma, Vibrio, and Cutibacterium. The α-diversity in the samples obtained during the month of October was lower and showed the dominance of rare bacterial taxa, such as Methylobacterium or Psychrobacter. Dilution-to-extinction technique demonstrated its usefulness to culture rare bacterial taxa that were not found in clams under the classic culturing techniques, including Rahnella, Brachybacterium, Micrococcus, Jantinobacter, and Lelliottia. Altogether, our study provides valuable information on the microbiota associated to R. decussatus and R. philippinarum, demonstrating the high complexity and dynamics of these microbial populationsOpen Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work was supported in part by grant AGL2013-42628-R and AGL2016-77539-R from the Ministerio de Economía y Competitividad (Spain)S

A Strong Contractile Actin Fence and Large Adhesions Direct Human Pluripotent Colony Morphology and Adhesion

Cell-type-specific functions and identity are tightly regulated by interactions between the cell cytoskeleton and the extracellular matrix (ECM). Human pluripotent stem cells (hPSCs) have ultimate differentiation capacity and exceptionally low-strength ECM contact, yet the organization and function of adhesion sites and associated actin cytoskeleton remain poorly defined. We imaged hPSCs at the cell-ECM interface with total internal reflection fluorescence microscopy and discovered that adhesions at the colony edge were exceptionally large and connected by thick ventral stress fibers. The actin fence encircling the colony was found to exert extensive Rho-ROCK-myosin-dependent mechanical stress to enforce colony morphology, compaction, and pluripotency and to define mitotic spindle orientation. Remarkably, differentiation altered adhesion organization and signaling characterized by a switch from ventral to dorsal stress fibers, reduced mechanical stress, and increased integrin activity and cell-ECM adhesion strength. Thus, pluripotency appears to be linked to unique colony organization and adhesion structure.Peer reviewe

Differentiating functional human islet-like aggregates from pluripotent stem cells

Publisher Copyright: © 2022 The Author(s)We present here a robust and reliable protocol by which to differentiate pancreatic islet-like aggregates (SC-islets) from human pluripotent stem cells. The 7-stage protocol mimics developmental patterning factors that induce endocrine lineage formation and spans monolayer, microwell, and aggregate suspension culture. The SC-islets demonstrate dynamic glucose-sensitive insulin secretion and an endocrine cell composition similar to those of primary human islets. SC-islets generated using this optimized protocol are suitable for in vitro modeling of islet cell pathophysiology and therapeutic applications. For complete details on the use and execution of this protocol, please refer to Balboa et al. (2022).Peer reviewe

Frequency of lower extremity artery disease in type 2 diabetic patients using pulse oximetry and the ankle-brachial index

Observational study[Abstract] Objectives: To determine the of undiagnosed lower extremity artery disease using the pulse oximetry in a type 2 diabetic population sample. Methods: Observational, cross-sectional, descriptive study that included 594 type 2 diabetic patients, with no previous history of lower extremity artery disease. Medical history, physical examination, determination of the ankle-brachial index (portable Doppler) and measurement of oxygen saturation in upper and lower extremities (pulse oximeter) were performed. Results: Frequency of lower extremity artery disease determined by ankle-brachial index was 18.4%. No significant correlations were detected between oxygen saturation and the ankle-brachial index except for the relationship between ankle-brachial index vs. oxygen saturation at 30 cm lower limb elevation vs. the supine position at no elevation (0 cm) in subjects under the age of 40. Pulse oximetry showed little diagnostic value in the screening of lower extremity artery disease. A relationship between lower extremity artery disease and age has been found. Its diagnosis was associated with a lower body mass index and lower systolic blood pressure in the lower extremities and higher in the upper extremities. Conclusions: We conclude that pulse oximetry is not useful in the screening for asymptomatic lower extremity artery disease in type 2 diabetics

Kaposi’s Sarcoma-Associated Herpesvirus Reactivation by Targeting of a dCas9-Based Transcription Activator to the ORF50 Promoter

CRISPR activation (CRISPRa) has revealed great potential as a tool to modulate the expression of targeted cellular genes. Here, we successfully applied the CRISPRa system to trigger the Kaposi’s sarcoma-associated herpesvirus (KSHV) reactivation in latently infected cells by selectively activating ORF50 gene directly from the virus genome. We found that a nuclease-deficient Cas9 (dCas9) fused to a destabilization domain (DD) and 12 copies of the VP16 activation domain (VP192) triggered a more efficient KSHV lytic cycle and virus production when guided to two different sites on the ORF50 promoter, instead of only a single site. To our surprise, the virus reactivation induced by binding of the stable DD-dCas9-VP192 on the ORF50 promoter was even more efficient than reactivation induced by ectopic expression of ORF50. This suggests that recruitment of additional transcriptional activators to the ORF50 promoter, in addition to ORF50 itself, are needed for the efficient virus production. Further, we show that CRISPRa can be applied to selectively express the early lytic gene, ORF57, without disturbing the viral latency. Therefore, CRISPRa-based systems can be utilized to facilitate virus–host interaction studies by controlling the expression of not only cellular but also of specific KSHV genes