Functional independence, frailty and perceived quality of life in patients who developed delirium during ICU stay: a prospective cohort study.

BACKGROUND: Survivors of critical illness are frequently left with a long-lasting disability. We hypothesised that patients who developed delirium during ICU stay, compared with patients who did not, would have worse health-related quality of life following a critical illness. METHODS: Prospective longitudinal observational and analytical study assessing functional independence, frailty and perceived quality of life measured with the Barthel Index, the Clinical Frailty Scale, and the SF-36, comparing patients who developed delirium during ICU stay and patients who did not. The questionnaires were used at different times during the follow-up (upon ICU admission, at ICU discharge, at hospital discharge and 2 years after hospital discharge). RESULTS: In a cohort of 1462 patients, we matched 93 patients who developed delirium (delirium group) with 93 patients who did not develop delirium (no-delirium group). Of 156 completed questionnaires (84.7%), we observed that (a) in each of the two groups of patients, the scores related to functional independence (Barthel Index) and frailty (Clinical Frailty Scale) tended to improve over time (p < 0.001), being consistently less favourable in the delirium group compared to the no-delirium group (p < 0.001); (b) the patients who developed delirium also presented lower scores on the SF-36 scale, these differences being statistically significant, and therefore evidencing a worse quality of life, with impact on both the psychological and social spheres (p < 0.001). CONCLUSIONS: Patients who developed delirium had significantly lower scores 2 years after hospital discharge on the three used questionnaires, displaying a clear negative impact on the physical, psychological, and social dimensions. The study's results reinforce the need to support and strengthen the care of ICU survivors.

RNA polymerase II pausing regulates chromatin organization in erythrocytes.

Chicken erythrocytes are nucleated cells often considered to be transcriptionally inactive, although the epigenetic changes and chromatin remodeling that would mediate transcriptional repression and the extent of gene silencing during avian terminal erythroid differentiation are not fully understood. Here, we characterize the changes in gene expression, chromatin accessibility, genome organization and chromatin nuclear disposition during the terminal stages of erythropoiesis in chicken and uncover complex chromatin reorganization at different genomic scales. We observe a robust decrease in transcription in erythrocytes, but a set of genes maintains their expression, including genes involved in RNA polymerase II (Pol II) promoter-proximal pausing. Erythrocytes exhibit a reoriented nuclear architecture, with accessible chromatin positioned towards the nuclear periphery together with the paused RNA Pol II. In erythrocytes, chromatin domains are partially lost genome-wide, except at minidomains retained around paused promoters. Our results suggest that promoter-proximal pausing of RNA Pol II contributes to the transcriptional regulation of the erythroid genome and highlight the role of RNA polymerase in the maintenance of local chromatin organization.

In-Nucleus Hi-C in Drosophila Cells.

The genome is organized into topologically associating domains (TADs) delimited by boundaries that isolate interactions between domains. In Drosophila, the mechanisms underlying TAD formation and boundaries are still under investigation. The application of the in-nucleus Hi-C method described here helped to dissect the function of architectural protein (AP)-binding sites at TAD boundaries isolating the Notch gene. Genetic modification of domain boundaries that cause loss of APs results in TAD fusion, transcriptional defects, and long-range topological alterations. These results provided evidence demonstrating the contribution of genetic elements to domain boundary formation and gene expression control in Drosophila. Here, the in-nucleus Hi-C method has been described in detail, which provides important checkpoints to assess the quality of the experiment along with the protocol. Also shown are the required numbers of sequencing reads and valid Hi-C pairs to analyze genomic interactions at different genomic scales. CRISPR/Cas9-mediated genetic editing of regulatory elements and high-resolution profiling of genomic interactions using this in-nucleus Hi-C protocol could be a powerful combination for the investigation of the structural function of genetic elements.

Risk factors associated with the development of delirium in general ICU patients. A prospective observational study.

OBJECTIVE: We aimed to analyze risk factors related to the development of delirium, aiming for early intervention in patients with greater risk. MATERIAL AND METHODS: Observational study, including prospectively collected patients treated in a single general ICU. These were classified into two groups, according to whether they developed delirium or not (screening performed using CAM-ICU tool). Demographics and clinical data were analyzed. Multivariate logistic regression analyses were performed to quantify existing associations. RESULTS: 1462 patients were included. 93 developed delirium (incidence: 6.3%). These were older, scored higher on the Clinical Frailty Scale, on the risk scores on admission (SAPS-3 and SOFA), and had a greater number of organ failures (OF). We observed more incidence of delirium in patients who (a) presented more than two OF (20.4%; OR 4.9; CI95%: 2.9-8.2), and (b) were more than 74 years old albeit having <2 OF (8.6%; OR 2.1; CI95%: 1.3-3.5). Patients who developed delirium had longer ICU and hospital length-of-stays and a higher rate of readmission. CONCLUSIONS: The highest risk observed for developing delirium clustered in patients who presented more than 2 OF and patients over 74 years old. The detection of patients at high risk for developing delirium could imply a change in management and improved quality of care.

An Intronic Alu Element Attenuates the Transcription of a Long Non-coding RNA in Human Cell Lines.

Alu elements are primate-specific repeats and represent the most abundant type of transposable elements (TE) in the human genome. Genome-wide analysis of the enrichment of histone post-translational modifications suggests that human Alu sequences could function as transcriptional enhancers; however, no functional experiments have evaluated the role of Alu sequences in the control of transcription in situ. The present study analyses the regulatory activity of a human Alu sequence from the AluSx family located in the second intron of the long intergenic non-coding RNA Linc00441, found in divergent orientation to the RB1 gene. We observed that the Alu sequence acts as an enhancer element based on reporter gene assays while CRISPR-Cas9 deletions of the Alu sequence in K562 cells resulted in a marked transcriptional upregulation of Linc00441 and a decrease in proliferation. Our results suggest that an intragenic Alu sequence with enhancer activity can act as a transcriptional attenuator of its host lincRNA.

Epigenetic Effects of an Adenosine Derivative in a Wistar Rat Model of Liver Cirrhosis.

The pathological characteristic of cirrhosis is scarring which results in a structurally distorted and dysfunctional liver. Previously, we demonstrated that Col1a1 and Pparg genes are deregulated in CCl4 -induced cirrhosis but their normal expression levels are recovered upon treatment with IFC-305, an adenosine derivative. We observed that adenosine was able to modulate S-adenosylmethionine-dependent trans-methylation reactions, and recently, we found that IFC-305 modulates HDAC3 expression. Here, we investigated whether epigenetic mechanisms, involving DNA methylation processes and histone acetylation, could explain the re-establishment of gene expression mediated by IFC-305 in cirrhosis. Therefore, Wistar rats were CCl4 treated and a sub-group received IFC-305 to reverse fibrosis. Global changes in DNA methylation, 5-hydroxymethylation, and histone H4 acetylation were observed after treatment with IFC-305. In particular, during cirrhosis, the Pparg gene promoter is depleted of histone H4 acetylation, whereas IFC-305 administration restores normal histone acetylation levels which correlates with an increase of Pparg transcript and protein levels. In contrast, the promoter of Col1a1 gene is hypomethylated during cirrhosis but gains DNA methylation upon treatment with IFC-305 which correlates with a reduction of Col1a1 transcript and protein levels. Our results suggest a model in which cirrhosis results in a general loss of permissive chromatin histone marks which triggers the repression of the Pparg gene and the upregulation of the Col1a1 gene. Treatment with IFC-305 restores epigenetic modifications globally and specifically at the promoters of Pparg and Col1a1 genes. These results reveal one of the mechanisms of action of IFC-305 and suggest a possible therapeutic function in cirrhosis. J. Cell. Biochem. 119: 401-413, 2018. © 2017 Wiley Periodicals, Inc.

Epigenetic silencing of miR-181c by DNA methylation in glioblastoma cell lines.

BACKGROUND: Post-transcriptional regulation by microRNAs is recognized as one of the major pathways for the control of cellular homeostasis. Less well understood is the transcriptional and epigenetic regulation of genes encoding microRNAs. In the present study we addressed the epigenetic regulation of the miR-181c in normal and malignant brain cells. METHODS: To explore the epigenetic regulation of the miR-181c we evaluated its expression using RT-qPCR and the in vivo binding of the CCCTC-binding factor (CTCF) to its regulatory region in different glioblastoma cell lines. DNA methylation survey, chromatin immunoprecipitation and RNA interference assays were used to assess the role of CTCF in the miR-181c epigenetic silencing. RESULTS: We found that miR-181c is downregulated in glioblastoma cell lines, as compared to normal brain tissues. Loss of expression correlated with a notorious gain of DNA methylation at the miR-181c promoter region and the dissociation of the multifunctional nuclear factor CTCF. Taking advantage of the genomic distribution of CTCF in different cell types we propose that CTCF has a local and cell type specific regulatory role over the miR-181c and not an architectural one through chromatin loop formation. This is supported by the depletion of CTCF in glioblastoma cells affecting the expression levels of NOTCH2 as a target of miR-181c. CONCLUSION: Together, our results point to the epigenetic role of CTCF in the regulation of microRNAs implicated in tumorigenesis.

A novel chromatin insulator regulates the chicken folate receptor gene from the influence of nearby constitutive heterochromatin and the ß-globin locus.

The three-dimensional architecture of genomes provides new insights about genome organization and function, but many aspects remain unsolved at the local genomic scale. Here we investigate the regulation of two erythroid-specific loci, a folate receptor gene (FOLR1) and the ß-globin gene cluster, which are separated by 16kb of constitutive heterochromatin. We found that in early erythroid differentiation the FOLR1 gene presents a permissive chromatin configuration that allows its expression. Once the transition to the next differentiation state occurs, the heterochromatin spreads into the FOLR1 domain, concomitant with the dissociation of CTCF from a novel binding site, thereby resulting in irreversible silencing of the FOLR1 gene. We demonstrate that the sequences surrounding the CTCF-binding site possess classical insulator properties in vitro and in vivo. In contrast, the chicken cHS4 ß-globin insulator present on the other side of the heterochromatic segment is in a constitutive open chromatin configuration, with CTCF constantly bound from the early stages of erythroid differentiation. Therefore, this study demonstrates that the 16kb of constitutive heterochromatin contributes to silencing of the FOLR1 gene during erythroid differentiation.

Experimental strategies to manipulate the cellular levels of the multifunctional factor CTCF.

Cellular homeostasis is the result of an intricate and coordinated combinatorial of biochemical and molecular processes. Among them is the control of gene expression in the context of the chromatin structure which is central for cell survival. Interdependent action of transcription factors, cofactors, chromatin remodeling activities, and three-dimensional organization of the genome are responsible to reach exquisite levels of gene expression. Among such transcription factors there is a subset of highly specialized nuclear factors with features resembling master regulators with a large variety of functions. This is turning to be the case of the multifunctional nuclear factor CCCTC-binding protein (CTCF) which is involved in gene regulation, chromatin organization, and three-dimensional conformation of the genome inside the cell nucleus. Technically its study has turned to be challenging, in particular its posttranscriptional interference by small interference RNAs. Here we describe three main strategies to downregulate the overall abundance of CTCF in culture cell lines.

Ventilatory support in critically ill hematology patients with respiratory failure.

INTRODUCTION: Hematology patients admitted to the ICU frequently experience respiratory failure and require mechanical ventilation. Noninvasive mechanical ventilation (NIMV) may decrease the risk of intubation, but NIMV failure poses its own risks. METHODS: To establish the impact of ventilatory management and NIMV failure on outcome, data from a prospective, multicenter, observational study were analyzed. All hematology patients admitted to one of the 34 participating ICUs in a 17-month period were followed up. Data on demographics, diagnosis, severity, organ failure, and supportive therapies were recorded. A logistic regression analysis was done to evaluate the risk factors associated with death and NIVM failure. RESULTS: Of 450 patients, 300 required ventilatory support. A diagnosis of congestive heart failure and the initial use of NIMV significantly improved survival, whereas APACHE II score, allogeneic transplantation, and NIMV failure increased the risk of death. The risk factors associated with NIMV success were age, congestive heart failure, and bacteremia. Patients with NIMV failure experienced a more severe respiratory impairment than did those electively intubated. CONCLUSIONS: NIMV improves the outcome of hematology patients with respiratory insufficiency, but NIMV failure may have the opposite effect. A careful selection of patients with rapidly reversible causes of respiratory failure may increase NIMV success.

The beta subunit of the heterotrimeric G protein triggers the Kluyveromyces lactis pheromone response pathway in the absence of the gamma subunit.

The Kluyveromyces lactis heterotrimeric G protein is a canonical Galphabetagamma complex; however, in contrast to Saccharomyces cerevisiae, where the Ggamma subunit is essential for mating, disruption of the KlGgamma gene yielded cells with almost intact mating capacity. Expression of a nonfarnesylated Ggamma, which behaves as a dominant-negative in S. cerevisiae, did not affect mating in wild-type and DeltaGgamma cells of K. lactis. In contrast to the moderate sterility shown by the single DeltaKlGalpha, the double DeltaKlGalpha DeltaKlGgamma mutant displayed full sterility. A partial sterile phenotype of the DeltaKlGgamma mutant was obtained in conditions where the KlGbeta subunit interacted defectively with the Galpha subunit. The addition of a CCAAX motif to the C-end of KlGbeta, partially suppressed the lack of both KlGalpha and KlGgamma subunits. In cells lacking KlGgamma, the KlGbeta subunit cofractionated with KlGalpha in the plasma membrane, but in the DeltaKlGalpha DeltaKlGgamma strain was located in the cytosol. When the KlGbeta-KlGalpha interaction was affected in the DeltaKlGgamma mutant, most KlGbeta fractionated to the cytosol. In contrast to the generic model of G-protein function, the Gbeta subunit of K. lactis has the capacity to attach to the membrane and to activate mating effectors in absence of the Ggamma subunit.

"Dipeptoids": from the chemical structure of the endogenous peptide to the design of peptidomimetics.

The present review details the rational multi-step process followed for the discovery of a family of non-peptide CCK receptor ligands ("dipeptoids"), starting from the structure of the endogenous peptide, CCK(8). Emphasis will be made on the N- and C-terminal modifications, on the singular effects of the stereochemical changes and the incorporation of conformational constraints into the structure of "dipeptoids", and on the modifications directed to improve the pharmacological profile of these compounds to afford valuable clinical candidates.

Hallazgos histopatológicos en biopsias de ganglios linfáticos en el departamento de patología-HEODRA, enero 1999 a diciembre 2003 / Findings histopatology in biopsias of lymphatic ganglia in the pathology department HEODRA, January 1999 to December 2003

Presenta estudio sobre las patologías de ganglios linfáticos mas frecuentes. Los mas frecuentes fueron las benignas, representadas por la hiperplasia linfoide reactiva. En cuanto a procesos malignos predominó el carcino metastásico, seguido por los linfomas. El grupo poblaconal mas afectado fue de 10-19 años en los preocesos benignos y de 40 a 60 años en los malignos, predominando en ambos el sexo femenino y de procedencia urbana. Las linfadenopatias biopsiadas tuvieron por localización más común la región cervical y en la mayoría de los casos el tamaño fue de 1 a 1.9 cm en las condiciones benignas y en los procesos malignos fue igual o superior a 2 cms