Delirium en pacientes críticos con infección por SARS-CoV-2

INTRODUCCIÓN. Durante la pandemia en el año 2020, según los datos de John Hopkins University, más de 750000 pacientes con COVID-19 en todo el mundo estuvieron críticos y precisaron ventilación mecánica, exponiéndose a sufrir un mayor riesgo de disfunción cerebral aguda (coma y delirium). En este estudio se investigó la prevalencia y los factores de riesgo del delirium y coma en pacientes adultos críticos con COVID-19. METODOLOGIA. Estudio multicéntrico de cohortes retrospectivo, con la participación de 69 unidades de cuidados intensivos (UCI) de 14 países. Se incluyeron todos los pacientes adultos (≥ 18 años) con Síndrome de Distrés Respiratorio Agudo Severo por infección de Coronavirus-2 (SARS-CoV-2) admitidos en las UCIs participantes antes del 28 de Abril del 2020. Fueron excluidos: los que ingresaron moribundos y se limitaron en las primeras 24h de ingreso en UCI, los reclusos, pacientes con enfermedades mentales previas, trastornos neurodegenerativos, daño cerebral congénito o adquirido, coma hepático, intoxicación por drogas, intento de suicidio y aquellos que eran ciegos o sordos. Se recogieron, de manera anónima y en plataforma electrónica, datos demográficos, valoraciones y estrategias de manejo de delirium y coma, durante un período de 21 días y datos sobre soporte ventilatorio, estancia en UCI y estado vital durante 28 días. El objetivo principal era determinar la prevalencia de delirium y coma e investigar los factores de riesgo asociados. RESULTADOS. Durante el período del 20 de Enero al 28 de Abril del 2020, 2088 pacientes con COVID-19 de 69 UCIs fueron incluidos en el estudio. La edad media fue de 64 años (IQR 54-71) con un SAPS medio de 40.0 (30.0-53.0). 1397 pacientes (66,9%) necesitaron ventilación mecánica invasiva (VMI) el día del ingreso en UCI y 1827 (87,5%) VMI en algún momento. La infusión continua de sedación durante la ventilación mecánica fue para benzodiacepinas, 1337 pacientes (64%) una media de 7 días (de 4 a 12), y para propofol, 1481 (70,9%) pacientes una media de 7 días (de 4 a 11). La media para la escala de Richmond Agitation-Sedation Scale Score durante la ventilación mecánica (VM) fue de -4 (de -5 a -3). 1704 (81,6%) pacientes estuvieron comatosos durante una media de 10 días (de 6 a 15) y 1147 (54,9%) con delirium durante una media de 3 días (de 2 a 6 días). La VM, el uso de contenciones mecánicas, la infusión contínua de benzodiacepinas, opioides y drogas vasopresoras y los antipsicóticos estaban relacionados con un aumento de riesgo de delirium al día siguiente (todos p≤o,o4), mientras que las visitas familiares (presenciales o virtuales) estaban asociadas con un menor riesgo de delirium (p<0,0001). Durante el período de estudio de 21 días, los pacientes estuvieron vivos sin delirium o coma durante una media de 5 días (de 0 a 14). La edad avanzada, las puntuaciones SAPS más elevadas, sexo masculino, el consumo de tabaco o alcohol, el uso de vasopresores y la ventilación mecánica el primer día estuvieron asociados de manera independiente con menos días vivos y libres de delirium y coma (todos p<0,01). 601 (28,8%) pacientes murieron en los 28 días desde el ingreso, la mayoría en UCI. DISCUSIÓN. La disfunción cerebral aguda (coma y delirium) en los pacientes críticos adultos con COVID-19 fue altamente prevalente y prolongada. El uso de benzodiacepinas (70% máyor) y las restricciones en las visitas familiares (30% menor) se identificaron como factores de riesgo modificables y representan una oportunidad de mejora para reducir la disfunción cerebral aguda en los pacientes COVID-19, por tanto la supervivencia de estos pacientes

All in One High Quality Genomic DNA and Total RNA Extraction From Nematode Induced Galls for High Throughput Sequencing Purposes

Meloidogyne spp. are plant-parasitic nematodes that form a very complex pseudo-organ, called gall, which contains the giant cells (GCs) to nourish them. During the last decade, several groups have been studying the molecular processes accompanying the formation of these structures, combining both transcriptomics and cellular biology. Among others, it was confirmed that a generalized gene repression is a hallmark of early developing GCs formed by Meloidogyne javanica in Arabidopsis and tomato. One of the main mechanisms behind this gene repression involve small RNAs (sRNAs) directed gene silencing. This is supported not only by the described action of several microRNAs differentially expressed in galls, but by the differential abundance of 24-nucleotide sRNAs in early developing Arabidopsis galls, particularly those rasiRNAs which are mostly involved in RNA-directed DNA methylation. Their accumulation strongly correlates to the repression of several retrotransposons at pericentromeric regions of Arabidopsis chromosomes in early galls. However, the contribution of this global gene repression to GCs/galls formation and maintenance is still not fully understood. Further detailed studies, as the correlation between gene expression profiles and the methylation state of the chromatin in galls are essential to raise testable working hypotheses. A high quality of isolated DNA and RNA is a requirement to obtain non-biased and comprehensive results. Frequently, the isolation of DNA and RNA is performed from different samples of the same type of biological material. However, subtle differences on epigenetic processes are frequent even among independent biological replicates of the same tissue and may not correlate to those changes on the mRNA population obtained from different biological replicates. Herein, we describe a method that allows the simultaneous extraction and purification of genomic DNA and total RNA from the same biological sample adapted to our biological system. The quality of both nucleic acids from Arabidopsis galls formed by M. javanica was high and adequate to construct RNA and DNA libraries for high throughput sequencing used for transcriptomic and epigenetic studies, such as the analysis of the methylation state of the genomic DNA in galls (MethylC-seq) and RNA sequencing (RNAseq). The protocol presents guidance on the described procedure, key notes and troubleshooting

Human embryonic stem cells and derived contractile embryoid bodies are susceptible to Coxsakievirus B infection and respond to interferon Iβ treatment

We studied the susceptibility of human embryonic stem cells and derived contractile embryoid bodies from WAO9, HUES-5 and HUES-16 cell lines to Coxsackievirus B infection. After validating stem cell-like properties and cardiac phenotype, Coxsackievirus B receptors CAR and DAF, as well as type I interferon receptors were detected in all cell lines and differentiation stages studied. Real-time PCR analysis showed that CAR mRNA levels were 3.4-fold higher in undifferentiated cells, while DAF transcript levels were 2.78-fold more abundant in differentiated cultures (P5-106 plaque forming units (PFU)/ml, the highest titers were detected in undifferentiated cells. Cell viability detected by a colorimetric assay, showed inverse correlation with infectivity titers of cell culture supernatants. Treatment with 100 U of interferon Iβ significantly reduced viral replication and associated cell death during a 24-48 h observation period, as detected by reduced infectivity titers in the supernatants and increased cell viability by a colorimetric assay, respectively. We propose human embryonic stem cell and derived contractile embryoid bodies as a valid model to study cardiac Coxsackievirus B infection.Instituto de Biotecnologia y Biologia MolecularFacultad de Ciencias Exacta

Human embryonic stem cells and derived contractile embryoid bodies are susceptible to Coxsakievirus B infection and respond to interferon Iβ treatment

AbstractWe studied the susceptibility of human embryonic stem cells and derived contractile embryoid bodies from WAO9, HUES-5 and HUES-16 cell lines to Coxsackievirus B infection. After validating stem cell-like properties and cardiac phenotype, Coxsackievirus B receptors CAR and DAF, as well as type I interferon receptors were detected in all cell lines and differentiation stages studied. Real-time PCR analysis showed that CAR mRNA levels were 3.4-fold higher in undifferentiated cells, while DAF transcript levels were 2.78-fold more abundant in differentiated cultures (P<0.05). All cell lines were susceptible to Coxsackievirus serotypes B1-5 infection as shown by RT-PCR detection of viral RNA, immunofluorescence detection of viral protein and infectivity titration of cell culture supernatants resulting in cell death. Supernatants infectivity titers 24-48h post-infection ranged from 105-106 plaque forming units (PFU)/ml, the highest titers were detected in undifferentiated cells. Cell viability detected by a colorimetric assay, showed inverse correlation with infectivity titers of cell culture supernatants. Treatment with 100 U of interferon Iβ significantly reduced viral replication and associated cell death during a 24–48 h observation period, as detected by reduced infectivity titers in the supernatants and increased cell viability by a colorimetric assay, respectively. We propose human embryonic stem cell and derived contractile embryoid bodies as a valid model to study cardiac Coxsackievirus B infection

Biological Effects of Maslinic Acid on Human Epithelial Cells Used in Tissue Engineering

In the present work, we evaluated the potential of maslinic acid (MA) to improve currently available keratinocyte culture methods for use in skin tissue engineering. Results showed that MA can increase cell proliferation and WST-1 activity of human keratinocytes after 24, 48, and 72 h, especially at the concentration of 5 μg/ml, without affecting cell viability. This effect was associated to a significant increase of KI-67 protein expression and upregulation of several genes associated to cell proliferation (PCNA) and differentiation (cytokeratins, intercellular junctions and basement membrane related genes). When human keratinocytes were isolated from skin biopsies, we found that MA at the concentration of 5 μg/ml significantly increased the efficiency of the explant and the cell dissociation methods. These results revealed the positive effects of MA to optimize human keratinocyte culture protocols for use in skin tissue engineering.PE-0395-2019 Consejería de Salud y Familias, Junta de Andalucía, SpainB-CTS-450-UGR20 (proyectos de I + D + i en el marco del Programa Operativo FEDER Andalucía 2014-2020, Universidad de Granada and Consejería de Transformación Económica, Industria, Conocimiento y Universidades)Spanish Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I + D + i) of the Spanish Ministry of Science and Innovation through grants FIS PI20/0317, FIS PI21/ 0980 and ICI19-00024 (BIOCLEFT) from Instituto de Salud Carlos III, co-financed by the European Regional Development Fun

Deep Learning Analyses to Delineate the Molecular Remodeling Process after Myocardial Infarction

Specific proteins and processes have been identified in post-myocardial infarction (MI) pathological remodeling, but a comprehensive understanding of the complete molecular evolution is lacking. We generated microarray data from swine heart biopsies at baseline and 6, 30, and 45 days after infarction to feed machine-learning algorithms. We cross-validated the results using available clinical and experimental information. MI progression was accompanied by the regulation of adipogenesis, fatty acid metabolism, and epithelial-mesenchymal transition. The infarct core region was enriched in processes related to muscle contraction and membrane depolarization. Angiogenesis was among the first morphogenic responses detected as being sustained over time, but other processes suggesting post-ischemic recapitulation of embryogenic processes were also observed. Finally, protein-triggering analysis established the key genes mediating each process at each time point, as well as the complete adverse remodeling response. We modeled the behaviors of these genes, generating a description of the integrative mechanism of action for MI progression. This mechanistic analysis overlapped at different time points; the common pathways between the source proteins and cardiac remodeling involved IGF1R, RAF1, KPCA, JUN, and PTN11 as modulators. Thus, our data delineate a structured and comprehensive picture of the molecular remodeling process, identify new potential biomarkers or therapeutic targets, and establish therapeutic windows during disease progression

Molecular determinants of disease in coxsackievirus B1 murine infection

To understand better how different genomic regions may confer pathogenicity for the coxsackievirus B (CVB), two intratypic CVB1 variants and a number of recombinant viruses were studied. Sequencing analysis showed 23 nucleotide changes between the parental non-pathogenic CVB1N and the pathogenic CVB1Nm. Mutations present in CVB1Nm were more conserved than those in CVB1N when compared to other CVB sequences. Inoculation in C3H/HeJ mice showed that the P1 region is critical for pathogenicity in murine pancreas and heart. The molecular determinants of disease for these organs partially overlap. Several P1 region amino acid differences appear to be located in the decay accelerating factor (DAF) footprint CVBs. CVB1N and CVB1Nm interacted with human CAR, but only CVB1N seemed to interact with human DAF, as determined using soluble receptors in a plaque reduction assay. However, the murine homologue Daf-1 did not interact with any virus assessed by haemagglutination. The results of this study suggest that an unknown receptor interaction with the virus play an important role in the pathogenicity of CVB1Nm. Further in vivo studies may clarify this issue.Instituto de Biotecnología y Biología Molecula

Aging-related tau astrogliopathy (ARTAG): not only tau phosphorylation in astrocytes

Aging-related tau astrogliopathy (ARTAG) is defined by the presence of two types of tau-bearing astrocytes: thorn-shaped astrocytes (TSAs) and granular/fuzzy astrocytes in the brain of old-aged individuals. The present study is focused on TSAs in rare forms of ARTAG with no neuronal tau pathology or restricted to entorhinal and transentorhinal cortices, to avoid bias from associated tauopathies. TSAs show 4Rtau phosphorylation at several specific sites and abnormal tau conformation, but they lack ubiquitin and they are not immunostained with tau-C3 antibodies which recognize truncated tau at Asp421. Astrocytes in ARTAG have atrophic processes, reduced glial fibrillary acidic protein (GFAP) and increased superoxide dismutase 2 (SOD2) immunoreactivity. Gel electrophoresis and western blotting of sarkosyl-insoluble fractions reveal a pattern of phospho-tau in ARTAG characterized by two bands of 68 and 64 kDa, and several middle bands between 35 and 50 kDa which differ from what is seen in AD. Phosphoproteomics of dissected vulnerable regions identifies an increase of phosphorylation marks in a large number of proteins in ARTAG compared with controls. GFAP, aquaporin 4, several serine-threonine kinases, microtubule associated proteins and other neuronal proteins are among the differentially phosphorylated proteins in ARTAG thus suggesting a hyper-phosphorylation background that affects several molecules, including many kinases and proteins from several cell compartments and various cell types. Finally, present results show for the first time that tau seeding is produced in neurons of the hippocampal complex, astrocytes, oligodendroglia and along fibers of the corpus callosum, fimbria and fornix following inoculation into the hippocampus of wild type mice of sarkosyl-insoluble fractions enriched in hyper-phosphorylated tau from selected ARTAG cases. These findings show astrocytes as crucial players of tau seeding in tauopathies

Human embryonic stem cells and derived contractile embryoid bodies are susceptible to Coxsakievirus B infection and respond to interferon Iβ treatment

We studied the susceptibility of human embryonic stem cells and derived contractile embryoid bodies from WAO9, HUES-5 and HUES-16 cell lines to Coxsackievirus B infection. After validating stem cell-like properties and cardiac phenotype, Coxsackievirus B receptors CAR and DAF, as well as type I interferon receptors were detected in all cell lines and differentiation stages studied. Real-time PCR analysis showed that CAR mRNA levels were 3.4-fold higher in undifferentiated cells, while DAF transcript levels were 2.78-fold more abundant in differentiated cultures (P5-106 plaque forming units (PFU)/ml, the highest titers were detected in undifferentiated cells. Cell viability detected by a colorimetric assay, showed inverse correlation with infectivity titers of cell culture supernatants. Treatment with 100 U of interferon Iβ significantly reduced viral replication and associated cell death during a 24-48 h observation period, as detected by reduced infectivity titers in the supernatants and increased cell viability by a colorimetric assay, respectively. We propose human embryonic stem cell and derived contractile embryoid bodies as a valid model to study cardiac Coxsackievirus B infection.Instituto de Biotecnologia y Biologia MolecularFacultad de Ciencias Exacta