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INTRODUCTION AND OBJECTIVES: Epigenetic changes represent a mechanism connecting external stresses with long-term modifications of gene expression programs. In solid organ transplantation, ischemia-reperfusion injury (IRI) appears to induce epigenomic changes in the graft, although the currently available data are extremely limited. The present study aimed to characterize variations in DNA methylation and their effects on the transcriptome in liver transplantation from brain-dead donors. PATIENTS AND METHODS: 12 liver grafts were evaluated through serial biopsies at different timings in the procurement-transplantation process: T0 (warm procurement, in donor), T1 (bench surgery), and T2 (after reperfusion, in recipient). DNA methylation (DNAm) and transcriptome profiles of biopsies were analyzed using microarrays and RNAseq. RESULTS: Significant variations in DNAm were identified, particularly between T2 and T0. Functional enrichment of the best 1000 ranked differentially methylated promoters demonstrated that 387 hypermethylated and 613 hypomethylated promoters were involved in spliceosomal assembly and response to biotic stimuli, and inflammatory immune responses, respectively. At the transcriptome level, T2 vs. T0 showed an upregulation of 337 and downregulation of 61 genes, collectively involved in TNF-α, NFKB, and interleukin signaling. Cell enrichment analysis individuates macrophages, monocytes, and neutrophils as the most significant tissue-cell type in the response. CONCLUSIONS: In the process of liver graft procurement-transplantation, IRI induces significant epigenetic changes that primarily act on the signaling pathways of inflammatory responses dependent on TNF-α, NFKB, and interleukins. Our DNAm datasets are the early IRI methylome literature and will serve as a launch point for studying the impact of epigenetic modification in IRI.
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Metilación de ADN , Epigénesis Genética , Perfilación de la Expresión Génica , Trasplante de Hígado , Hígado , Daño por Reperfusión , Trasplante de Hígado/efectos adversos , Daño por Reperfusión/genética , Daño por Reperfusión/metabolismo , Humanos , Hígado/metabolismo , Hígado/patología , Masculino , Persona de Mediana Edad , Femenino , Perfilación de la Expresión Génica/métodos , Transcriptoma , Adulto , AncianoRESUMEN
Resumen Los cambios epigenéticos juegan en el organismo un papel importante en el control de la expresión génica, durante el desarrollo y a lo largo de toda la vida, sobre todo durante el envejecimiento. En los últimos años se han acumulado evidencias que avalan la participación de los procesos epigenéticos en el desarrollo y evolución de diversas enfermedades como procesos tumorales, enfermedades genéticas, cardiovasculares y neurodegenerativas. Además, los marcadores epigenéticos (metilación del ADN, modificaciones en las histonas y los ARN no codificantes) podrían indicar la predisposición del individuo a determinados procesos patológicos. La administración de fármacos epigenéticos ha demostrado ser eficiente en el tratamiento de enfermedades tales como la aterosclerosis, neoplasias, procesos neurodegenerativos, enfermedades hepáticas, etc. En este artículo se abordarán algunos ejemplos de la contribución que las modificaciones epigenéticas dan a la patogenia de las enfermedades neurodegenerativas y cardiovasculares. En el futuro, la bioquímica clínica será frecuentemente utilizada en los análisis epigenéticos y ayudará al diseño de fármacos y estrategias terapéuticas dirigidas a modificar el epigenoma.
Abstract In the organism, epigenetic changes play an important role in the control of gene expression, during its development and throughout life, especially during ageing. In recent years, evidence has accumulated that supports the participation of epigenetic processes in the development and evolution of various diseases such as tumor processes, genetic, cardiovascular and neurodegenerative diseases. In addition, epigenetic markers (DNA methylation, histone modifications and non-coding RNAs) could indicate the predisposition to certain pathological processes. The administration of epigenetic drugs has proven to be efficient in the treatment of diseases such as atherosclerosis, neoplasms, neurodegenerative processes, liver diseases, etc. In this article we will address some examples of the contribution that epigenetic modifications give to the pathogenesis of neurodegenerative and cardiovascular diseases. In the future, clinical biochemistry will be frequently used in epigenetic analyzes and will help design drugs and therapeutic strategies aimed to modify the epigenome.
Resumo As alterações epigenéticas têm no organismo um papel importante no controle da expressão gênica durante o desenvolvimento e ao longo de toda a vida, principalmente durante o envelhecimento. Nos últimos anos, foram acumuladas evidências que demonstram a participação dos processos epigenéticos no desenvolvimento e evolução de diversas doenças como, por exemplo, processos tumorais, doenças genéticas, cardiovasculares e neurodegenerativas. Além disso, os marcadores epigenéticos (metilação do DNA, modificações nas histonas e nos RNA não codificantes), poderiam indicar a predisposição do indivíduo a determinados processos patologicos. A administração de fármacos epigenéticos demonstrou ser eficiente no tratamento de doenças tais como a aterosclerose, neoplasias, processos neurodegenerativos, doenças hepáticas, etc. Neste estudo abordaremos alguns exemplos da contribuição que as alterações epigenéticas dão à patogenia das doenças neurodegenerativas e cardiovasculares. No futuro, a bioquímica clínica será frequentemente utilizada nas análises epigenéticas e ajudará ao desenho de medicamentos e estratégias terapêuticas dirigidas a modificar o epigenoma.
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Alternative splicing produces complex and dynamic changes in the protein isoforms that are necessary for the proper biological functioning of the metabolic pathways involved in liver development and hepatocyte homeostasis. Changes in the physiological state of alternatively spliced forms are increasingly linked to liver pathologies. This may occur when the expression or function of the set of proteins controlling the alternative splicing processes are altered by external effectors such as oxidative stress and other environmental variations. Studies addressing these modifications reveal a complex interplay between the expression levels of different proteins that regulate the alternative splicing process as well as the changes in alternative splicing. This interplay results in a cascade of different protein isoforms that correlate with the progression of non-alcoholic fatty liver disease, hepatocellular carcinoma, and alcoholic liver disease. However, research on the detailed molecular mechanism underlying the production of these isoforms is needed. It is imperative to identify the physiological processes affected by the differentially spliced isoforms and confirm their role on the onset and maintenance of the pathology. This is required to design potential therapeutic approaches targeting the key splicing changes to revert the pathological condition as well as identify prognostic markers. In this review, we describe the complexity of the splicing process through an example to encourage researchers to go down this path. Subsequently, rather than a catalog of splicing events we have hand-picked and discuss a few selected studies of specific liver pathologies and suggested ways to focus research on these areas.
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Empalme Alternativo/genética , Perfilación de la Expresión Génica/métodos , Hepatopatías/genética , ARN Mensajero/genética , Humanos , Hepatopatías/metabolismoRESUMEN
Los agregados de TDP-43 representan una de las característica histopatológicas más importantes de varias enfermedades neurodegenerativas, entre las que se incluye la Esclerosis Lateral Amiotrófica (ELA). TDP-43 está localizada principalmente en el núcleo. Sin embargo, los pacientes afectados por ELA presentan agregados de TDP-43 en el citoplasma de las neuronas comprometidas, con lo que se despoja al núcleo de TDP-43 funcional. Aún se desconoce si la degeneración causada por la agregación de TDP-43 es debida a una toxicidad intrínseca de los agregados o a la pérdida de función de TDP-43 como consecuencia del vaciamiento del núcleo. Varias investigaciones, incluidas las de estos autores, indican que la pérdida de función es el factor fundamental responsable de la neurodegeneración observada en presencia de inclusiones de TDP-43. Por otro lado, aún no existen tratamientos efectivos para la ELA. Por lo tanto, es de crucial importancia conocer las bases moleculares que conllevan al desarrollo de la enfermedad, con el objetivo de encontrar posibles estrategias terapéuticas. Para ello, estos autores han desarrollado un modelo celular capaz de imitar la agregación de TDP-43 y sus consecuencias. Finalmente, se ha utilizado este modelo para analizar el efecto de diferentes compuestos capaces de degradar los agregados de TDP-43 y se ha demostrado que esta podría ser una estrategia terapéutica válida para la ELA.
TDP-43 inclusions are important histopathological features of various neurodegenerative disorders, including Amyotrophic Lateral Sclerosis (ALS). TDP-43 is mainly a nuclear protein, but it shuffles from the nucleus to the cytoplasm. In patients’ brains, TDP-43 is retained in the cytoplasm of the affected motorneurons to form insoluble aggregates, which results in TDP-43 nuclear clearance. There is still no consensus whether TDP-43-mediated neurodegeneration results from a gain or loss of function of the protein or a combination of both. The work from several laboratories, including this, points towards a strong loss of function component. On the other hand, there is no effective treatment or cure for ALS. Thus, there is obviously a need to find new therapeutic strategies for ALS. In order to gain new insights into the molecular mechanism of the disease, and with the aim of looking for new methodologies that can revert it, a cellular model of TDP-43 aggregation that can mimic the phenotypic consequences found in ALS patients has been developed. Finally, this model was used to search for compounds that can dissolve these aggregates, and it was shown that the clearance of TDP-43 aggregates could be a therapeutic strategy for ALS.
Os agregados proteicos TDP-43 são características histopatológicas importantes de muitas doenças neurodegenerativas, incluindo a Esclerose Lateral Amiotrófica (ALS). A proteína TDP-43 se localiza principalmente no núcleo, porém nos cérebros de indivíduos afetados, a proteína TDP-43 fica retida no citoplasma dos neurônios motores, o que leva a formação de agregados insolúveis, resultando em deposição nuclear. Ainda não existe um consenso se a neurodegeneração mediada por TDP43 é causada por ganho ou perda da função da proteína ou uma combinação de ambos. O trabalho de muitos laboratórios, bem como este trabalho, apontam para uma forte perda da função da proteína. Por outro lado, não existe um tratamento efetivo ou cura para a ALS. Portanto, existe uma grande necessidade de identificar novos tratamentos para a ALS. Para entender o mecanismo molecular da doença, e com o objetivo de identificar novas metodologias para reverter a doença, desenvolvemos o modelo celular de agregados de TDP-43, o qual mimetiza as consequências fenotípicas encontradas em pacientes com ALS. Por fim, utilizamos esse modelo para identificar compostos que podem dissolver os agregados, e demonstramos que a liberação de inclusões de TDP-43 poderiam ser usados como tratamentos para a ALS.
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Esclerosis Amiotrófica Lateral/terapia , Proteinopatías TDP-43/clasificación , Impacto Agregado , Esclerosis Amiotrófica Lateral/complicaciones , Proteinopatías TDP-43/terapiaRESUMEN
Alternative splicing plays a key role in generating protein diversity. Transfections with minigenes revealed coordination between two distant, alternatively spliced exons in the same gene. Mutations that either inhibit or stimulate inclusion of the upstream alternative exon deeply affect inclusion of the downstream one. However, similar mutations at the downstream alternative exon have little effect on the upstream one. This polar effect is promoter specific and is enhanced by inhibition of transcriptional elongation. Consistently, cells from mutant mice with either constitutive or null inclusion of a fibronectin alternative exon revealed coordination with a second alternative splicing region, located far downstream. Using allele-specific RT-PCR, we demonstrate that this coordination occurs in cis and is also affected by transcriptional elongation rates. Bioinformatics supports the generality of these findings, indicating that 25% of human genes contain multiple alternative splicing regions and identifying several genes with nonrandom distribution of mRNA isoforms at two alternative regions.
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Empalme Alternativo , Genes/genética , Alelos , alfa-Globulinas/genética , Animales , Antígenos Virales de Tumores/genética , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Biología Computacional , Proteínas de Unión al ADN/genética , Diclororribofuranosil Benzoimidazol/farmacología , Exones/genética , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibronectinas/genética , Humanos , Ratones , Ratones Noqueados , Modelos Genéticos , Proteínas Nucleares/genética , Regiones Promotoras Genéticas/genética , Isoformas de Proteínas/genética , ARN Polimerasa II/antagonistas & inhibidores , ARN Polimerasa II/metabolismo , Empalme del ARN , Proteínas de Unión al ARN/genética , Factores de Empalme Serina-Arginina , Factores de Transcripción/genética , TransfecciónRESUMEN
A novel serine-arginine-rich protein designated TcSR was identified in Trypanosoma cruzi. The deduced amino acid sequence reveals that TcSR is a member of the SR protein family of splicing factors that contains two RNA-binding domains at the N-terminal side and several serine-arginine repeats at the COOH-terminus. Over expression of either TcSR or the human SR-protein associated splicing factor/splicing factor 2 (ASF/SF2) in wild-type Schizosaccharomyces pombe, provoked an elongated phenotype similar to that of fission yeast over expressing the SR-containing splicing factor Prp2, a U2AF(65) orthologue. When a double mutant strain lacking two SR protein-specific protein kinases was used, expression of TcSR or human SR ASF/SF2 splicing factor reverted the mutant to a wild-type phenotype. Transient expression of TcSR in HeLa cells stimulated the inclusion of the EDI exon of human fibronectin in an in vivo functional alternative cis-splicing assay. Inclusion was dependent on a splicing enhancer sequence present in the EDI exon. In addition, TcSR and peptides carrying TcSR-RS domain sequences were phosphorylated by a human SR protein kinase. These results indicate that TcSR is a member of the SR splicing network and that some components common to the trans- and cis-splicing machineries evolved from the early origins of the eukaryotic lineage.
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Arginina/análisis , Proteínas Protozoarias/química , Proteínas Protozoarias/fisiología , Empalme del ARN , Serina/análisis , Trypanosoma cruzi/genética , Secuencia de Aminoácidos , Animales , Evolución Molecular , Células HeLa , Humanos , Proteínas Protozoarias/genética , Schizosaccharomyces/genética , Alineación de Secuencia , Trypanosoma cruzi/aislamiento & purificación , Trypanosoma cruzi/fisiologíaRESUMEN
En el presente trabajo se analizaron dos aspectos distintos del metabolismo lipoproteico. En el primero, en un estudio bioquímico clínico, se presentó un método rápido y simple de determinación de los polimorfismos de la apoE. La utilidad clínica de conocer el genotipo apoE fue evaluada correlacionando las isoformas de apoE con el espesor carotideo estimado con ecografía de ultrasonido. Los resultados mostraron que el genotipo apoE4 ejerce un efecto sobre la aterosclerosis carotídea, efecto que puede ser detectado antes que cualquier otro síntoma clínico. En la segunda parte de éste trabajo se ha analizado el metabolismo lipoproteico en ratones transgénicos expresando la apolipoproteína A-IV (apoA-IV) humana. Los resultados muestran que la apoA-IV tiene un rol pleiotrópico en el organismo, relacionado con la protección contra la aterosclerosis y con la regulación de la secreción gástrica (AU)
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Humanos , Cobayas , Persona de Mediana Edad , Apolipoproteínas , Apolipoproteínas E/genética , Apolipoproteínas A/genética , Lipoproteínas/metabolismo , Apolipoproteínas/fisiología , Apolipoproteínas E/fisiología , Apolipoproteínas E/sangre , Lipoproteínas/sangre , Isoformas de Proteínas , LDL-Colesterol/sangre , HDL-Colesterol/sangre , Expresión GénicaRESUMEN
En el presente trabajo se analizaron dos aspectos distintos del metabolismo lipoproteico. En el primero, en un estudio bioquímico clínico, se presentó un método rápido y simple de determinación de los polimorfismos de la apoE. La utilidad clínica de conocer el genotipo apoE fue evaluada correlacionando las isoformas de apoE con el espesor carotideo estimado con ecografía de ultrasonido. Los resultados mostraron que el genotipo apoE4 ejerce un efecto sobre la aterosclerosis carotídea, efecto que puede ser detectado antes que cualquier otro síntoma clínico. En la segunda parte de éste trabajo se ha analizado el metabolismo lipoproteico en ratones transgénicos expresando la apolipoproteína A-IV (apoA-IV) humana. Los resultados muestran que la apoA-IV tiene un rol pleiotrópico en el organismo, relacionado con la protección contra la aterosclerosis y con la regulación de la secreción gástrica
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Humanos , Cobayas , Persona de Mediana Edad , Apolipoproteínas A/genética , Apolipoproteínas E/genética , Apolipoproteínas , Lipoproteínas/metabolismo , Apolipoproteínas E/fisiología , Apolipoproteínas E/sangre , Apolipoproteínas/fisiología , HDL-Colesterol , LDL-Colesterol , Expresión Génica , Lipoproteínas/sangre , Isoformas de ProteínasRESUMEN
A peptide from hindguts of the Triatoma infestans, the hematophagous Chagas' insect vector, activates adenylyl cyclase activity in Trypanosoma cruzi epimastigote membranes and stimulates the in vitro differentiation of epimastigotes (proliferative and non-infectious forms) to metacyclic trypomastigotes (non-proliferative and infectious forms). The peptide was purified from hindguts of insects fed two days before with chicken blood. After purification, the peptide showed upon SDS-PAGE a single band of about 10 kDa. The sequence for 20 residues of the amino terminus of this peptide was: H2N-Met-Leu-Thr-Ala-Glu-Asp-Lys-Lys-Leu-Ile-Gln-Gln-Ala-Trp-Glu-Lys-Ala- Ala-Ser-His. This sequence corresponds to the amino terminus of chicken alpha D-globin. A synthetic peptide with the sequence of the 40 amino acids corresponding to the amino terminus of alpha D-globin, also stimulated T. cruzi adenylyl cyclase activity and promoted metacyclogenesis