A genome resource for the marine annelid Platynereis dumerilii.

The marine annelid Platynereis dumerilii is a model organism used in many research areas including evolution and development, neurobiology, ecology and regeneration. Here we present the genomes of P. dumerilii and of the closely related P. massiliensis and P. megalops , to facilitate comparative genomic approaches and help explore Platynereis biology. We used long-read sequencing technology and chromosomal-conformation capture along with extensive transcriptomic resources to obtain and annotate a draft genome assembly of ∼1.47 Gbp for P. dumerilii , of which more than half represent repeat elements. We predict around 29,000 protein-coding genes, with relatively large intron sizes, over 38,000 non-coding genes, and 580 miRNA loci. We further explore the high genetic variation (∼3% heterozygosity) within the Platynereis species complex. Gene ontology reveals the most variable loci to be associated with pigmentation, development and immunity. The current work sets the stage for further development of Platynereis genomic resources.

Guidelines for establishing a cytometry laboratory.

The purpose of this document is to provide guidance for establishing and maintaining growth and development of flow cytometry shared resource laboratories. While the best practices offered in this manuscript are not intended to be universal or exhaustive, they do outline key goals that should be prioritized to achieve operational excellence and meet the needs of the scientific community. Additionally, this document provides information on available technologies and software relevant to shared resource laboratories. This manuscript builds on the work of Barsky et al. 2016 published in Cytometry Part A and incorporates recent advancements in cytometric technology. A flow cytometer is a specialized piece of technology that require special care and consideration in its housing and operations. As with any scientific equipment, a thorough evaluation of the location, space requirements, auxiliary resources, and support is crucial for successful operation. This comprehensive resource has been written by past and present members of the International Society for Advancement of Cytometry (ISAC) Shared Resource Laboratory (SRL) Emerging Leaders Program https://isac-net.org/general/custom.asp?page=SRL-Emerging-Leaders with extensive expertise in managing flow cytometry SRLs from around the world in different settings including academia and industry. It is intended to assist in establishing a new flow cytometry SRL, re-purposing an existing space into such a facility, or adding a flow cytometer to an individual lab in academia or industry. This resource reviews the available cytometry technologies, the operational requirements, and best practices in SRL staffing and management.

Molecular and Pathological Characterization of Classical Swine Fever Virus Genotype 2 Strains Responsible for the 2013-2018 Outbreak in Colombia.

Classical swine fever (CSF) is a highly contagious transboundary viral disease of domestic and wild pigs. Despite mass vaccination and continuous eradication programs, CSF remains endemic in Asia, some countries in Europe, the Caribbean and South America. Since June 2013, Northern Colombia has reported 137 CSF outbreaks, mostly in backyard production systems with low vaccination coverage. The purpose of this study was to characterize the virus responsible for the outbreak. Phylogenetic analysis based on the full-length E2 sequence shows that the virus is closely related to CSF virus (CSFV) genotype 2.6 strains circulating in Southeast Asia. The pathotyping experiment suggests that the virus responsible is a moderately virulent strain. The 190 nucleotide stretch of the E2 hypervariable region of these isolates also shows high similarity to the CSFV isolates from Colombia in 2005 and 2006, suggesting a common origin for the CSF outbreaks caused by genotype 2.6 strains. The emergence of genotype 2.6 in Colombia suggests a potential transboundary spread of CSFV from Asia to the Americas, complicating the ongoing CSF eradication efforts in the Americas, and emphasizes the need for continuous surveillance in the region.

A three-colour stress biosensor reveals multimodal response in single cells and spatiotemporal dynamics of biofilms.

The plethora of stress factors that can damage microbial cells has evolved sophisticated stress response mechanisms. While existing bioreporters can monitor individual responses, sensors for detecting multimodal stress responses in living microorganisms are still lacking. Orthogonally detectable red, green, and blue fluorescent proteins combined in a single plasmid, dubbed RGB-S reporter, enable simultaneous, independent, and real-time analysis of the transcriptional response of Escherichia coli using three promoters which report physiological stress (PosmY for RpoS), genotoxicity (PsulA for SOS), and cytotoxicity (PgrpE for RpoH). The bioreporter is compatible with standard analysis and Fluorescent Activated Cell Sorting (FACS) combined with subsequent transcriptome analysis. Various stressors, including the biotechnologically relevant 2-propanol, activate one, two, or all three stress responses, which can significantly impact non-stress-related metabolic pathways. Implemented in microfluidic cultivation with confocal fluorescence microscopy imaging, the RGB-S reporter enabled spatiotemporal analysis of live biofilms revealing stratified subpopulations of bacteria with heterogeneous stress responses.

Severe and critical COVID-19 in a tertiary center in Colombia, a retrospective cross-sectional study.

BACKGROUND: Colombia has been one of the Latin American countries seriously affected by the covid-19 pandemic. Risk factors for severe disease and death in COVID 19 have been described across the world. Here we report the outcomes, clinical characteristics and risk factors for invasive mechanical ventilation and in-hospital death in a tertiary center in Palmira, Colombia. METHODS: This was a retrospective cross-sectional study involving one single center in Palmira, Colombia. People hospitalized with severe and critical covid-19, during the first pandemic wave, were included. The clinical characteristics and risk factors for in-hospital mortality and invasive mechanical ventilation were mean to be stablished by using a logistic regression analysis. RESULTS: One hundred and fifty-eight patients were analyzed. Most patients were male (70%) with a mean age of 63 years, invasive mechanical ventilation was provided to 39%, in-hospital mortality was 36%, mainly caused by refractory hypoxemia and septic shock, admission to intensive care was as high as 65%. The logistic regression analysis showed that the risk factors for in-hospital mortality were elevated levels of lactic dehydrogenase and high-sensitivity troponin I, acute renal failure, COPD, and > 10 points on the MuLBSTA score. The risk factors for invasive mechanical ventilation were high levels of C-reactive protein and very low lymphocyte counts, a PaO2/FiO2 < 70 and some clinical scores like CURB65, NEWS 2, and PSI/PORT. CONCLUSIONS: During the first pandemic wave in Colombia, for the experience of a tertiary center with a mainly elderly population, a high prevalence of severe ARDS was found, high requirement of intensive care, invasive ventilatory support, bacterial sepsis and an elevated mortality rate were found. The risk factors for in-hospital death and invasive mechanical ventilation were stablished.

High-speed fluorescence image-enabled cell sorting.

Fast and selective isolation of single cells with unique spatial and morphological traits remains a technical challenge. Here, we address this by establishing high-speed image-enabled cell sorting (ICS), which records multicolor fluorescence images and sorts cells based on measurements from image data at speeds up to 15,000 events per second. We show that ICS quantifies cell morphology and localization of labeled proteins and increases the resolution of cell cycle analyses by separating mitotic stages. We combine ICS with CRISPR-pooled screens to identify regulators of the nuclear factor κB (NF-κB) pathway, enabling the completion of genome-wide image-based screens in about 9 hours of run time. By assessing complex cellular phenotypes, ICS substantially expands the phenotypic space accessible to cell-sorting applications and pooled genetic screening.

Single-cell proteo-genomic reference maps of the hematopoietic system enable the purification and massive profiling of precisely defined cell states.

Single-cell genomics technology has transformed our understanding of complex cellular systems. However, excessive cost and a lack of strategies for the purification of newly identified cell types impede their functional characterization and large-scale profiling. Here, we have generated high-content single-cell proteo-genomic reference maps of human blood and bone marrow that quantitatively link the expression of up to 197 surface markers to cellular identities and biological processes across all main hematopoietic cell types in healthy aging and leukemia. These reference maps enable the automatic design of cost-effective high-throughput cytometry schemes that outperform state-of-the-art approaches, accurately reflect complex topologies of cellular systems and permit the purification of precisely defined cell states. The systematic integration of cytometry and proteo-genomic data enables the functional capacities of precisely mapped cell states to be measured at the single-cell level. Our study serves as an accessible resource and paves the way for a data-driven era in cytometry.

Valores de referencia del Senior Fitness Test en mujeres adultas mayores físicamente activas / Reference values of the Senior Fitness Test for physically active elderly women

Introducción: El proceso de envejecimiento en los adultos mayores puede conducir a la disminución de la condición física funcional. Por ello resulta importante su valoración para poder establecer programas de ejercicio físico que mejoren las capacidades físicas, así como la funcionalidad y calidad de vida en esta población. Objetivo: Establecer los valores de referencia de la condición física funcional en las mujeres físicamente activas pertenecientes a Centros Vida de la ciudad de Bucaramanga. Métodos: Estudio descriptivo transversal en 113 mujeres mayores entre 60-85 años. Se evalúo la condición física funcional con el Senior Fitness Test y medidas antropométricas de talla y peso. Los valores de referencia están en percentiles y distribuidos por rangos de edad. Resultados: Según los valores de referencia del Senior Fitness Test, las mujeres de nuestro estudio se encuentran por encima en los valores de fuerza de miembros inferiores y superiores. En la prueba de resistencia cardiovascular y flexibilidad en miembros inferiores y superiores se encuentran por debajo. En los valores de la prueba de agilidad el tiempo de ejecución es mayor. Conclusiones: Se observa una disminución en las capacidades físicas funcionales conforme avanza la edad, sin embargo, la capacidad física que menos disminuye es la fuerza(AU)

Introduction: The aging process may lead to a reduction of functional physical condition in the elderly. Its analysis is therefore necessary to implement physical exercise programs aimed at improving physical capacities, functionality and quality of life in this population. Objective: Establish the reference values for functional physical condition in physically active women from Life Centers in the city of Bucaramanga. Methods: A cross-sectional descriptive study was conducted of 113 elderly women aged 60-85 years. Functional physical condition was evaluated with the Senior Fitness Test and anthropometric measurements of height and weight. Reference values are expressed in percentiles and distributed by age ranges. Results: The women in our study are above the reference values of the Senior Fitness Test in upper and lower limb force. Upper and lower limb cardiovascular resistance and flexibility are below SFT reference values. Values for the agility test show a longer performance time. Conclusions: A reduction is observed in functional physical capacities as people grow older. However, force is the physical capacity that diminishes the least(AU)

Formulation of DNA Nanocomposites: Towards Functional Materials for Protein Expression.

DNA hydrogels are an emerging class of materials that hold great promise for numerous biotechnological applications, ranging from tissue engineering to targeted drug delivery and cell-free protein synthesis (CFPS). In addition to the molecular programmability of DNA that can be used to instruct biological systems, the formulation of DNA materials, e.g., as bulk hydrogels or microgels, is also relevant for specific applications. To advance the state of knowledge in this research area, the present work explores the scope of a recently developed class of complex DNA nanocomposites, synthesized by RCA polymerization of DNA-functionalized silica nanoparticles (SiNPs) and carbon nanotubes (CNTs). SiNP/CNT-DNA composites were produced as bulk materials and microgels which contained a plasmid with transcribable genetic information for a fluorescent marker protein. Using confocal microscopy and flow cytometry, we found that the materials are very efficiently taken up by various eukaryotic cell lines, which were able to continue dividing while the ingested material was evenly distributed to the daughter cells. However, no expression of the encoded protein occurred within the cells. While the microgels did not induce production of the marker protein even in a CFPS procedure with eukaryotic cell lysate, the bulk composites proved to be efficient templates for CFPS. This work contributes to the understanding of the molecular interactions between DNA composites and the functional cellular machinery. Implications for the use of such materials for CFPS procedures are discussed.

Single-cell transcriptomics reveals immune response of intestinal cell types to viral infection.

Human intestinal epithelial cells form a primary barrier protecting us from pathogens, yet only limited knowledge is available about individual contribution of each cell type to mounting an immune response against infection. Here, we developed a framework combining single-cell RNA-Seq and highly multiplex RNA FISH and applied it to human intestinal organoids infected with human astrovirus, a model human enteric virus. We found that interferon controls the infection and that astrovirus infects all major cell types and lineages and induces expression of the cell proliferation marker MKI67. Intriguingly, each intestinal epithelial cell lineage exhibits a unique basal expression of interferon-stimulated genes and, upon astrovirus infection, undergoes an antiviral transcriptional reprogramming by upregulating distinct sets of interferon-stimulated genes. These findings suggest that in the human intestinal epithelium, each cell lineage plays a unique role in resolving virus infection. Our framework is applicable to other organoids and viruses, opening new avenues to unravel roles of individual cell types in viral pathogenesis.

Niveles de actividad física e indicadores antropométricos de riesgo cardiovascular en adultas mayores / Levels of physical activity and anthropometric indicators of cardiovascular risk in aged women

Introducción: A nivel mundial, la cifra de adultos mayores para el año 2030 será alrededor de 16,5 por ciento, llegando a ser alrededor de 21,5 por ciento para el año 2050. Actualmente en Colombia, la cifra de adultos mayores se encuentra en un 11 por ciento de la población total. La actividad física es una estrategia para mejorar la condición física funcional, calidad de vida, autonomía e independencia en adultos mayores. De igual manera, en esta población se incrementa el riesgo cardiovascular siendo mayor en mujeres que en hombres. Objetivo: Determinar el nivel de actividad física y valorar por medidas antropométricas el riesgo cardiovascular en mujeres mayores pertenecientes a Centros Vida de la ciudad de Bucaramanga. Métodos: Se realizó un estudio descriptivo transversal. La muestra estuvo conformada por 128 mujeres adultas mayores entre las edades de 60-85 años. Para determinar el nivel de actividad física se aplicó el Cuestionario Mundial de Actividad Física (GPAQ). Se valoró el riesgo cardiovascular por medio de mediciones antropométricas de talla, peso, IMC, porcentaje de grasa, perímetro de cintura, perímetro de cadera e índice de cintura/cadera. Resultados: El 89,84 por ciento de la población realiza actividad física de desplazamiento y el 48,43 por ciento realiza actividad física en tiempo libre. En indicadores antropométricos de riesgo cardiovascular, se obtuvo un IMC de 28,05 (25,08-31,65) kg/m2, un 43,60 (40,20-46,55) de porcentaje de grasa y un índice de cintura/cadera de 0,89 (0,85-0,91). Conclusiones: Las mujeres adultas mayores presentan riesgo cardiovascular a pesar de realizar actividad física a intensidad moderada(AU)

Introduction: Worldwide, the number of aged adults by 2030 will be around 16.5 percent, becoming around 21.5 percent by 2050. Currently, in Colombia, the number of aged adults is at 11 percent of the total population. Physical activity is a strategy to improve functional physical condition, quality of life, autonomy and independence in aged adults. Similarly, in this population cardiovascular risk increases, being higher in women than in men. Objective: To determine the level of physical activity and assess cardiovascular risk by anthropometric measures in aged women from Centros Vida in Bucaramanga city. Methods: A descriptive cross-sectional study was carried out. The sample consisted of 128 aged women, aging between 60-85 years. To determine the level of physical activity, the World Physical Activity Questionnaire (GPAQ) was applied. Cardiovascular risk was assessed through anthropometric measurements of height, weight, BMI, percentage of fat, waist circumference, hip circumference, and waist / hip ratio. Results: 89.84 percent of the population performs commuting physical activity and 48.43 percent performs free-time physical activity. In anthropometric indicators of cardiovascular risk, BMI of 28.05 (25.08-31.65) kg / m2, a 43.60 (40.20-46.55) percentage of fat and a waist index / hip of 0.89 (0.85-0.91). Conclusions: Aged women show cardiovascular risk despite being physical activity at moderate intensity(AU)

Factors associated with mortality in Infections caused by Carbapenem-resistant Enterobacteriaceae.

INTRODUCTION: There is little information about weigh of factors possibly associated with mortality, in infections caused by Carbapenem-resistant Enterobacteriaceae (CRE) in Latin America. METHODOLOGY: A case-controls study nested in a historical cohort was performed including all patients with CRE infections diagnosed between June 2013 and December 2018 at Hospital Universitario San Ignacio in Bogotá, Colombia. Univariate and multivariate analysis were performed to compare cases of mortality within the first month after the infection diagnosis with surviving patients. RESULTS: A total of 131 patients were included. The overall 30-day mortality rate was 38.17%. In the multivariate analysis, a direct association was found between mortality and septic shock (OR 26.7 CI6.6-107.3 p < 0.01), post-chemotherapy febrile neutropenia (OR 3.3 CI1.06-10.8 p = 0.04) and Charlson Index ≥ 3 (OR 5.5 CI 1.5-20.06 p < 0.01). An inverse association was found with interventions to control the infectious focus (OR 0.3 CI0.1-0.7 p < 0.01). The MIC of different antibiotics and the use of combined antibiotic therapy (triple therapy vs. double therapy or monotherapy) were not associated with mortality. CONCLUSIONS: In patients with CRE infections, septic shock, a Charlson comorbidity index ≥ 3, and post-chemotherapy febrile neutropenia are independently related to an increase in mortality. The control of the infectious focus is a protective factor. A rapid identification of these patients, and the implementation of measures to control infectious focus and to detect CRE colonization in patients who are going to be taken to myelosuppressive chemotherapy could impact positively the prognosis of these patients.

El efecto agudo sobre niveles de glucemia en adultos mayores con el Entrenamiento Interválico de Alta Intensidad / Acute effect of High-Intensity Interval Training on glycemia levels in elderly people

Introducción: El Entrenamiento Interválico de Alta Intensidad durante los últimos años ha sido postulado como tratamiento no farmacológico para enfermedades como la diabetes tipo II en diferentes poblaciones, sin embargo, son pocos los estudios que se han realizado en adultos mayores. Objetivo: Determinar el efecto agudo de una sesión de Entrenamiento Interválico de Alta Intensidad sobre los niveles de glucosa en adultos mayores físicamente activos. Métodos: Se realizó un estudio experimental. La muestra estuvo conformada por 19 adultos mayores (60-85 años). El grupo fue sometido a una sesión de Entrenamiento Interválico de Alta Intensidad, en la que se combinaron ejercicios de fuerza con resistencia cardiovascular, y se realizaron 12 ejercicios con duración de 30 segundos de trabajo por 10 de descanso. La intensidad de la sesión fue controlada a través de la Escala de Percepción del Esfuerzo OMNI-GSE. Se midió la talla (cm), el peso (kg) e índice de masa corporal. Los niveles de glucosa en sangre fueron determinados antes y después de la sesión. Resultados: Se obtuvo una disminución significativa en los niveles de glucosa en sangre posterior a la realización de la sesión de Entrenamiento Interválico de Alta Intensidad (pre: 140,5 mg/dL y post: 116,1 mg/dL; p < 0,01) Conclusiones: El Entrenamiento Interválico de Alta Intensidad reduce en una sola sesión los niveles de glucosa en sangre en adultos mayores(AU)

Introduction: in recent years, High-Intensity Interval Training has been recognized as a non-pharmacological treatment for diseases like type II diabetes in a variety of populations. However, few studies about this topic have been conducted with elderly people. Objective: determine the acute effect of a High-Intensity Interval Training session on glucose levels in physically active elderly people. Methods: an experimental study was conducted. The sample was 19 elderly people aged 60-85 years. The group participated in a High-Intensity Interval Training session in which strength and cardiovascular resistance exercises were combined. The 12 exercises performed had a duration of 30 seconds' work and 10 seconds' rest. The intensity of the session was controlled with the OMNI-GSE Effort Perception Scale. Measurements were taken of the height (cm), weight (kg) and body mass index of participants. Blood glucose levels were gauged before and after the session. Results: a significant reduction in blood glucose levels was obtained after the conduct of the High-Intensity Interval Training session (pre: 140.5 mg/dl and post: 116.1 mg/dl; p < 0.01). Conclusions: in only one session, High-Intensity Interval Training reduces blood glucose levels in elderly people(AU)

Efecto de la suplementación con soja sobre la inflamación y ácido láctico inducido por ejercicio físico exhaustivo en ratas / Effect of supplementation with soy on inflammation and lactic acid induced by exhaustive physical exercise in rats

INTRODUCCIÓN: El ejercicio físico exhaustivo genera marcadores inflamatorios y de ácido láctico. La suplementación con sustancias naturales es motivo de análisis debido a sus escasos efectos secundarios. OBJETIVO: Determinar la respuesta inflamatoria y el nivel de ácido láctico inducidos por ejercicio físico exhaustivo después de la ingesta de soja en modelo animal. MATERIALES Y MÉTODO: Se emplearon treinta ratas macho de raza Sprawley dawley de 180 a 200 g, sanos divididos en tres grupos: sedentario (C), con ingesta de soja a prueba (E+TP) y sin ingesta de soja a prueba (E). Los grupos E+TP y E, realizaron la prueba Morris Water Maze Test. Se determinaron marcadores inflamatorios como factor de necrosis tumoral alfa (TNF-a), interleuquina 1 beta (IL-1Beta), interleuquina 6 (IL-6) en plasma mediante técnica ELISA, enzima ciclooxigenasa 2 (COX-2), óxido nítrico sintaza (iNOS) y como marcador antiinflamatorio Receptor gamma activado por proliferador de peroxisoma (PPAR-γ), el cual, se midió en músculos cuádriceps mediante técnica de Western-blot y se midió el ácido láctico en sangre. RESULTADOS: Se obtuvo una disminución significativa en plasma de los niveles inflamatorios de TNF-alfa (600 vs 350 pg/ml), IL-1Beta (450 vs 150 pg/ml), e IL-6 (480 vs 100 pg/ml), COX-2 (52 vs 25 RDU) e iNOS (58 vs 8 RDU) en el grupo E+TP en comparación con el grupo E. Además se observó un aumento de la expresión de la proteína PPAR-γ (18 vs 65 RDU) en el grupo E+TP en comparación con el grupo E. Respecto a las mediciones de ácido láctico los grupos obtuvieron valores máximos de: E:35, C:22 y E+TP:28 Mmol/Lactato, lo cual, indica que el grupo E y E+TP a pesar que se sometieron a la misma prueba, los niveles de ácido láctico son heterogéneos. CONCLUSIÓN: La ingesta de soja mitiga los niveles de ácido láctico y de marcadores inflamatorios inducidos por el ejercicio fisico exhautivo en modelo animal

INTRODUCTION: Exhautive physical exercise generates inflammatory and lactic acid markers. The supplementation with natural substances is reason for analysis due to its limited side effects. OBJECTIVE: To determine the inflammatory response and the level of lactic acid induced by exhaustive physical exercise after the ingestion of soybean in animal model. MATERIALS AND METHOD: Thirty male Sprawley dawley rats from 180 to 200 g were used, healthy divided into three groups: sedentary (C), with soybean intake tested (E+TP) and without soybean intake tested (E). The E + TP and E groups performed the Morris Water Maze Test. Inflammatory markers were determined as tumor necrosis factor alpha (TNF-a), interleukin 1 beta (IL-1β), interleukin 6 (IL-6) in plasma by ELISA technique, enzyme cyclooxygenase 2 (COX-2), nitric oxide synthase (iNOS) and as anti-inflammatory marker Peroxisome proliferator-activated receptor gamma (PPAR-γ), which was measured in quadriceps muscles by Western-blot technique and measured lactic acid in blood. RESULTS: A significant decrease in plasma was obtained in the inflammatory levels of TNF-alpha (600 vs 350 pg/ml), IL-1Beta (450 vs 150 pg/ml), and IL-6 (480 vs 100 pg/ml), COX-2 (52 vs. 25 RDU) and iNOS (58 vs. 8 RDU) in the E+TP group compared to the E group. In addition an increase in the expression of the PPAR-γ protein was observed (18 vs 65 RDU) in the group E+TP compared to group E. Regarding the measurements of lactic acid, the groups obtained maximum values of: E: 35, C: 22 and E+TP: 28 Mmol/Lactate, which indicates that Group E and E+TP although they underwent the same test, lactic acid levels are heterogeneous. CONCLUSION: The intake of soy mitigates the levels of lactic acid and inflammatory markers induced by exhautive physical exercise in animal models

Cell-type-specific role of CHK2 in mediating DNA damage-induced G2 cell cycle arrest.

Cancer is a life-threatening disease that affects one in three people. Although most cases are sporadic, cancer risk can be increased by genetic factors. It remains unknown why certain genes predispose for specific forms of cancer only, such as checkpoint protein 2 (CHK2), in which gene mutations convey up to twofold higher risk for breast cancer but do not increase lung cancer risk. We have investigated the role of CHK2 and the related kinase checkpoint protein 1 (CHK1) in cell cycle regulation in primary breast and lung primary epithelial cells. At the molecular level, CHK1 activity was higher in lung cells, whereas CHK2 was more active in breast cells. Inhibition of CHK1 profoundly disrupted the cell cycle profile in both lung and breast cells, whereas breast cells were more sensitive toward inhibition of CHK2. Finally, we provide evidence that breast cells require CHK2 to induce a G2-M cell cycle arrest in response of DNA damage, whereas lung cells can partially compensate for the loss of CHK2. Our results provide an explanation as to why CHK2 germline mutations predispose for breast cancer but not for lung cancer.

Apoptotic Cell Exclusion and Bias-Free Single-Cell Selection Are Important Quality Control Requirements for Successful Single-Cell Sequencing Applications.

Single-cell sequencing experiments are a new mainstay in biology and have been advancing science especially in the biomedical field. The high pressure to integrate the technology into daily laboratory live requires solid knowledge with respect to potential limitations and precautions to be taken care of before applying it to complex research questions. In the past, we have identified two issues with quality measures neglected by the growing community involving SmartSeq and droplet or micro-well-based scRNASeq methods (1) how to ensure that only single cells are introduced without biasing on light scattering when handling complex cell mixtures and organ preparations or (2) how best to control for (pro-)apoptotic cell contaminations in single-cell sequencing approaches. Sighting of concurrent literature involving single-cell sequencing technologies revealed that these topics are generally neglected or simply approached in silico but not at the bench before generating single-cell data sets. We fear that those important quality aspects are overlooked due to reduced awareness of their importance for guaranteeing the quality of experiments. In this Cytometry rigor issue, we provide experimentally supported guidance on how to circumvent those critical shortcomings in order to promote a better use of the fantastic single-cell sequencing toolbox in biology. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Respuesta de la glucemia frente a dos intensidades de ejercicio físico realizado en ayunas en mujeres jóvenes / Blood glucose response to two intensities of physical exercise in young women during fasting

Introducción: La práctica de Ejercicio Físico (EF) en estado de ayuno ha sido controvertida; no obstante, algunas investigaciones evidencian mayor pérdida de grasa corporal y mejor control glucémico en quienes participan de entrenamiento aeróbico en estado de ayuno. Objetivo: Evaluar la respuesta de la glucemia después de una sesión de ejercicio físico de intensidad moderada o vigorosa realizado en ayunas en mujeres jóvenes. Materiales y métodos: Se realizó un ensayo clínico controlado aleatorizado. Veinticuatro mujeres (19 a 22 años) fueron asignadas de manera aleatoria a dos grupos de intervención. El primero fue sometido a una intensidad de ejercicio del 70% de la Frecuencia Cardíaca Máxima (FCM) durante 30 minutos y el segundo a una intensidad del 90% de la FCM durante 15 minutos. Se evaluaron la talla (cm), peso (Kg), índice de masa corporal (IMC), porcentaje de grasa y consumo máximo de oxígeno (VO2máx) mediante una prueba de esfuerzo. Los niveles de glucemia fueron determinados antes y después de la sesión de ejercicio de cada grupo. Resultados: No se encontraron cambios significativos en los niveles de glucosa en sangre post ejercicio en ningún grupo experimental, y las diferencias existentes no fueron estadísticamente significativas. Conclusión: El ejercicio físico moderado o vigoroso en estado de ayuno no mostró variaciones significativas en la glucemia posterior a su ejecución, lo que sugiere seguridad en el desarrollo del entrenamiento en ayuno en mujeres jóvenes saludables

Introduction: Physical exercise in the fasting state has been a controversial topic; however, some studies have shown a greater loss of body fat and better glycemic control in those who participate in aerobic training when fasting. Aim: To evaluate the glycemic response after a session of moderate or vigorous physical exercise in young women in the state of fasting. Material and method: A randomized clinical trial was carried out. Twenty-six women (19 to 22 years old) were randomly assigned to two intervention groups. The first group was trained at an intensity of 70% of maximum heart rate (MHR) for 30 minutes, and the second group at an intensity of 90% MHR for 15 minutes. Height (cm), weight (Kg), body mass index (BMI), fat percentage, and maximum oxygen consumption (VO2max) during a stress test were evaluated. Blood glucose levels were checked before and after the exercise session of each group. Results: No significant changes were found in post-exercise blood glucose levels in any experimental group, and the existing differences were not statistically significant. Conclusions: Moderate or vigorous physical exercise during fasting did not show significant variations in blood glucose, which suggests that it is safe for healthy young women to train when fasting

Inhibition of NGLY1 Inactivates the Transcription Factor Nrf1 and Potentiates Proteasome Inhibitor Cytotoxicity.

Proteasome inhibitors are used to treat blood cancers such as multiple myeloma (MM) and mantle cell lymphoma. The efficacy of these drugs is frequently undermined by acquired resistance. One mechanism of proteasome inhibitor resistance may involve the transcription factor Nuclear Factor, Erythroid 2 Like 1 (NFE2L1, also referred to as Nrf1), which responds to proteasome insufficiency or pharmacological inhibition by upregulating proteasome subunit gene expression. This "bounce-back" response is achieved through a unique mechanism. Nrf1 is constitutively translocated into the ER lumen, N-glycosylated, and then targeted for proteasomal degradation via the ER-associated degradation (ERAD) pathway. Proteasome inhibition leads to accumulation of cytosolic Nrf1, which is then processed to form the active transcription factor. Here we show that the cytosolic enzyme N-glycanase 1 (NGLY1, the human PNGase) is essential for Nrf1 activation in response to proteasome inhibition. Chemical or genetic disruption of NGLY1 activity results in the accumulation of misprocessed Nrf1 that is largely excluded from the nucleus. Under these conditions, Nrf1 is inactive in regulating proteasome subunit gene expression in response to proteasome inhibition. Through a small molecule screen, we identified a cell-active NGLY1 inhibitor that disrupts the processing and function of Nrf1. The compound potentiates the cytotoxicity of carfilzomib, a clinically used proteasome inhibitor, against MM and T cell-derived acute lymphoblastic leukemia (T-ALL) cell lines. Thus, NGLY1 inhibition prevents Nrf1 activation and represents a new therapeutic approach for cancers that depend on proteasome homeostasis.

The transcriptional repressor Gfi1 prevents lupus autoimmunity by restraining TLR7 signaling.

The transcriptional repressor growth factor independence 1 (Gfi1) is important in myeloid and lymphoid differentiation. In the current study we evaluated the involvement of Gfi1 in systemic lupus erythematosus (SLE). We found that Genista mice, which carry a hypomorphic mutation in the gfi1 gene or Gfi1-deficient (Gfi1-/- ) mice develop signs of spontaneous lupus autoimmunity, including increased serum levels of IgM and IgG2a, autoantibodies against RNA and DNA, glomerular immunodeposits and increased frequencies of plasmablasts, germinal center (GC) B cells and age-associated B cells (ABCs). On the contrary, Genista mice deprived of TLR7 did not show any of these phenotypes, suggesting that the observed lupus autoimmunity in Genista mice is TLR7-dependent. Moreover, Genista mice showed an increased activation of dendritic cells (DCs), B and T cells that was dependent on TLR7 for DCs and B cells, but not for T cells. Upon TLR7 or TLR4 stimulation Genista DCs produced increased amounts of TNF, IL-6 and IFN-ß and showed increased NF-κB phosphorylation and IRF7 nuclear translocation, suggesting that Gfi1 controls the NF-κB and type I IFN signaling pathway downstream of TLRs. Our data reveal that Gfi1 plays a critical role in the prevention of spontaneous lupus autoimmunity by negatively regulating TLR7 signaling.

Effects of Ranolazine on Astrocytes and Neurons in Primary Culture.

Ranolazine (Rn) is an antianginal agent used for the treatment of chronic angina pectoris when angina is not adequately controlled by other drugs. Rn also acts in the central nervous system and it has been proposed for the treatment of pain and epileptic disorders. Under the hypothesis that ranolazine could act as a neuroprotective drug, we studied its effects on astrocytes and neurons in primary culture. We incubated rat astrocytes and neurons in primary cultures for 24 hours with Rn (10-7, 10-6 and 10-5 M). Cell viability and proliferation were measured using trypan blue exclusion assay, MTT conversion assay and LDH release assay. Apoptosis was determined by Caspase 3 activity assay. The effects of Rn on pro-inflammatory mediators IL-ß and TNF-α was determined by ELISA technique, and protein expression levels of Smac/Diablo, PPAR-γ, Mn-SOD and Cu/Zn-SOD by western blot technique. In cultured astrocytes, Rn significantly increased cell viability and proliferation at any concentration tested, and decreased LDH leakage, Smac/Diablo expression and Caspase 3 activity indicating less cell death. Rn also increased anti-inflammatory PPAR-γ protein expression and reduced pro-inflammatory proteins IL-1 ß and TNFα levels. Furthermore, antioxidant proteins Cu/Zn-SOD and Mn-SOD significantly increased after Rn addition in cultured astrocytes. Conversely, Rn did not exert any effect on cultured neurons. In conclusion, Rn could act as a neuroprotective drug in the central nervous system by promoting astrocyte viability, preventing necrosis and apoptosis, inhibiting inflammatory phenomena and inducing anti-inflammatory and antioxidant agents.