Expressão de TRF2 e GAPDH no envelhecimento do epitélio superficial do ovário in vitro

Se ha demostrado que la proteína GAPDH se puede unir al ADN telomérico de cadena sencilla, tanto in vitro como in vivo. Por lo tanto, se ha planteado la hipótesis de que la GAPDH juega un papel importante en la protección de los telómeros, papel que podría ser compartido con la TRF2, proteína que participa en una gran variedad de funciones relacionadas con la homeostasis telomérica. Objetivo: el objetivo de este estudio fue determinar si existe una correlación entre la expresión de ambos genes en el epitelio superficial del ovario in vitro. Materiales y métodos: la expresión relativa de cada gen fue establecida mediante qRT-PCR, en cultivos primarios de células del epitelio superficial del ovario provenientes de un grupo de 22 donantes colombianas mestizas sanas. Resultados: las pruebas no paramétricas de Kendall y Spearman permitieron establecer que existe una correlación significativa entre los niveles de expresión de GAPDH y TRF2 a lo largo de la historia replicativa de los cultivos, en forma independiente de la edad de las donantes. Conclusión: nuestros resultados sugieren un efecto sinérgico entre TRF2 y GAPDH, que podría estar orientado a contrarrestar la reducción de los telómeros in vitro.GAPDH can bind single-strand telomere DNA both in vitro and in vivo. Thus, it was hypothesisedthat GAPDH has an important role in protecting the telomeres, role that could be shared with TRF2, a well-known telomeric protein involved in a myriad of functions related to telomere homeostasis. Objective: The aim of this study was to determine if there was a correlation between the expression of these genes in the in vitro ovarian surface epithelium. Materials and methods: The relative expression of each gene was established by qRT-PCR in primary cell cultures of the ovarian surface epithelium from 22 healthy mestizo Colombian donors. Results: The Kendall and Spearman non-parametric tests established a significant correlation between the levels of expression in subsequent passages of the cell line, in an age-independent way. Conclusion: Our findings suggest a synergistic effect between TRF2 and GAPDH that could counter telomere shortening in vitro.Tem se demonstrado que GAPDH pode-se unir ao DNA telomérico de cadeia simples, tanto in vitro quanto in vivo. Portanto, tem se apresentado a hipótese de que GAPDH joga um papel importantena proteção dos telómeros, papel que poderia ser compartilhado com TRF2, proteína que participa em uma grande variedade de funções relacionadas com a homeostase telomérica. Objetivo. O objetivo deste estudo foi determinar se existe uma correlação entre a expressão de ambos os genes no epitélio superficial do ovário in vitro. Materiais e métodos: A expressão relativa de cada gene foi estabelecida mediante qRT-PCR em cultivos primários de células do epitélio superficial do ovário provenientes de um grupo de 22 doadoras colombianas mestiças sanas. Resultados. As provas não paramétricas de Kendall e Spearman permitiram estabelecer que existe uma correlação significativa entre os níveis de expressão de GAPDH e TRF2 ao longe da história replicativa dos cultivos, em forma independente da idade das doadoras. Conclusão. Nossos resultados sugerem um efeito sinérgico entre TRF2 e GAPDH que poderia estar orientado a contra-arrestar a redução dos telómeros in vitro

Integrative analysis of global gene expression identifies opposite patterns of reactive astrogliosis in aged human prefrontal cortex

The prefrontal cortex (PFC) is one of the brain regions with more prominent changes in human aging. The molecular processes related to the cognitive decline and mood changes during aging are not completely understood. To improve our knowledge, we integrated transcriptomic data of four studies of human PFC from elderly people (58–80 years old) compared with younger people (20–40 years old) using a meta-analytic approximation combined with molecular signature analysis. We identified 1817 differentially expressed genes, 561 up-regulated and 1256 down-regulated. Pathway analysis revealed down-regulation of synaptic genes with conservation of gene expression of other neuronal regions. Additionally, we identified up-regulation of markers of astrogliosis with transcriptomic signature compatible with A1 neurotoxic astrocytes and A2 neuroprotective astrocytes. Response to interferon is related to A1 astrocytes and the A2 phenotype is mediated in aging by activation of sonic hedgehog (SHH) pathway and up-regulation of metallothioneins I and genes of the family ERM (ezrin, radixin, and moesin). The main conclusions of our study are the confirmation of a global dysfunction of the synapses in the aged PFC and the evidence of opposite phenotypes of astrogliosis in the aging brain, which we report for the first time in the present article. © 2018 by the authors. Licensee MDPI, Basel, Switzerland

Sporadic Creutzfeldt-Jakob disease: Clinical, pathological and molecular study

Generalidades. Las encefalopatías espongiformes transmisibles son enfermedades neurodegenerativas ocasionadas por la acumulación anormal de una variante mal plegada de la proteína priónica, lo cual induce la formación de conglomerados proteicos resistentes a la degradación. Además, son responsables de la disfunción sináptica, daño neuronal y de la sintomatología clásica acompañante. Esta proteína de membrana es codificada por el exón 2 del gen PRNP, ubicado en el brazo corto del cromosoma 20 y parece estar involucrada en la trasmisión sináptica, la transducción de señales, la actividad antioxidante de la superoxidodismutasa, neuroplasticidad y sobrevida celular. Un polimorfismo en el codón 129 se asocia con una susceptibilidad diferencial a la enfermedad Creutzfeldt-Jakob esporádica. Objetivo. Estudio clínico, patológico y molecular de un caso de una mujer de 58 años con diagnóstico de enfermedad de Creutzfeldt- Jakob esporádica. Métodos y resultados. Se presenta el caso de una mujer en quien aparece un trastorno depresivo del afecto con demencia progresiva y sintomatología general. Al final de la enfermedad, el cuadro progresó a un déficit neurológico focalizado en el área visual. La RMN mostró hiperintensidades inespecíficas córtico-subcorticales en el núcleo estriado; en el EEG se encontró pérdida de ritmos de fondo, patrón de descargas periódicas generalizadas y complejos trifásicos; en la biopsia cerebral postmorten se evidenció pérdida severa de la población neuronal en todas las capas, vacuolas en el neuropil, en el soma neuronal y en la glía. El análisis de secuencia del gen PRNP, a partir de extracción de DNA de sangre periférica, identificó homocigosis para metionina en el codón 129. La paciente fallece a los 3 meses del inicio de la sintomatología. Conclusión. Por epidemiología, curso clínico y exámenes paraclínicos se confirma el diagnóstico de enfermedad de Creutzfeldt- Jakob esporádica.La determinación del genotipo para los polimorfismos de riesgo se convierte en una herramienta útil para complementar por medios moleculares el diagnóstico y para profundizar la comprensión de la fisiopatología de la enfermedad de Creutzfeldt-Jakob, tanto para formas esporádicas como para la nueva variante.Background: Transmissible spongiform encephalopathies are neurodegenerative diseases caused by abnormal accumulation of pathogenic isoform the prion protein, which induces the formation of conglomerates protein resistant to degradation. They are also responsible for synaptic dysfunction, neuronal damage and the classic symptoms of disease. This membrane protein is encoded by exon 2 of the gene PRNP, located on the short arm of chromosome 20 and appears to be involved in synaptic transmission, signal transduction, the antioxidant activity of the superoxid dismutasa, neuroplasticity and cell survival. One polymorphism at codon 129 is associated with differential susceptibility to disease sporadic Creutzfeldt-Jakob disease. Aim: Clinical, pathological and molecular report on an 58 year-old woman with pathological diagnosis of Creutzfeldt-Jakob sporadic disease. Methods and results. The clinic course appears with a behavior depressive disorder with progressive dementia and symptoms. At the end of the disease, the scenario progressed to a neurological deficit focused on the visual area. The MRI showed nonspecific hyperintensity in cortiço-subcortical nucleus in the striatum, the EEG showed patterns of recurrent generalized discharges and complex three-phase, the brain biopsy post-morten showed severe loss of the neuronal population in all the layers, vacuoles in the neuropil, in the neuronal soma and the glial. The analysis of sequence of the gene PRNP identified homozygotes for methionine at codon 129. The patient died at 3 months of the onset of symptoms. Conclusions: Epidemiology, clinical course and paraclinical examinations confirmed the diagnosis of Creutzfeldt-Jakob sporadic. The genotyping for polymorphisms of risk becomes useful tool to complement through molecular diagnosis and to deepen the understanding of the pathophysiology of Creutzfeldt- Jakob disease, both for sporadic forms and for the new variant

Co-expression network analysis identifies possible hub genes in aging of the human prefrontal cortex

Introducción: el envejecimiento es el principal factor de riesgo para el desarrollo de enfermedades crónicas como el cáncer, la diabetes, el Parkinson y el Alzheimer. El sistema nervioso central es particularmente susceptible al deterioro funcional progresivo asociado con la edad, entre las regiones cerebrales con mayor compromiso se encuentra la corteza prefrontal (cpf). Estudios de transcriptómica de esta región han identificado como características fundamentales del proceso de envejecimiento la disminución de la función sináptica y la activación de las células de la neuroglia. No es claro cuáles son las causas iniciales, ni los mecanismos moleculares subyacentes a estas alteraciones. El objetivo de este estudio fue identificar genes clave en la desregulación transcriptómica en el envejecimiento de la cpf para avanzar en el conocimiento de este proceso. Materiales y métodos: se hizo un análisis de coexpresión de genes de los transcriptomas de 45 personas entre 60 y 80 años con el de 38 personas entre 20 y 40 años. Las redes fueron visualizadas y analizadas usando Cytoscape, se usó citoHubba para determinar qué genes tenían las mejores características topológicas en las redes de coexpresión. Resultados: se identificaron cinco genes con características topológicas altas. Cuatro de ellos —hpca, cacng3, ca10, plppr4— reprimidos y uno sobreexpresado —cryab—. Conclusión: los cuatro genes reprimidos se expresan preferencialmente en neuronas y regulan la función sináptica y la plasticidad neuronal, mientras el gen sobreexpresado es típico de células de la glía y se expresa como respuesta a daño neuronal facilitando la mielinización y la regeneración neuronal.Introduction: Aging is the main risk factor for the development of chronic diseases such as cancer, diabetes, Parkinson’s disease, and Alzheimer’s disease. The central nervous system is particularly susceptible to progressive functional deterioration associated with age, among the brain regions the prefrontal cortex (PFC) has one of the highest involvements. Transcriptomics studies of this brain region have identified the decrease in synaptic function and activation of neuroglia cells as fundamental characteristics of the aging process. The aim of this study was to identify hub genes in the transcriptomic deregulation in the PFC aging to advance in the knowledge of this process. Materials and methods: A gene co-expression analysis was carried out for 45 people 60 to 80 years old compared with 38 people 20 to 40 years old. The networks were visualized and analyzed using Cytoscape; citoHubba was used to determine which genes had the best topological characteristics in the co-expression networks. Results: Five genes with high topological characteristics were identified. Four of them —HPCA, CACNG3, CA10, PLPPR4— were repressed and one was over-expressed —CRYAB—. Conclusion: The four repressed genes are expressed preferentially in neurons and regulate the synaptic function and the neuronal plasticity, while the overexpressed gene is typical of glial cells and is expressed as a response to neuronal damage, facilitating myelination and neuronal regeneration

Orthogonal analysis of mitochondrial function in Parkinson’s disease patients

The etiopathology of Parkinson’s disease has been associated with mitochondrial defects at genetic, laboratory, epidemiological, and clinical levels. These converging lines of evidence suggest that mitochondrial defects are systemic and causative factors in the pathophysiology of PD, rather than being mere correlates. Understanding mitochondrial biology in PD at a granular level is therefore crucial from both basic science and translational perspectives. In a recent study, we investigated mitochondrial alterations in fibroblasts obtained from PD patients assessing mitochondrial function in relation to clinical measures. Our findings demonstrated that the magnitude of mitochondrial alterations parallels disease severity. In this study, we extend these investigations to blood cells and dopamine neurons derived from induced pluripotent stem cells reprogrammed from PD patients. To overcome the inherent metabolic heterogeneity of blood cells, we focused our analyses on metabolically homogeneous, accessible, and expandable erythroblasts. Our results confirm the presence of mitochondrial anomalies in erythroblasts and induced dopamine neurons. Consistent with our previous findings in fibroblasts, we observed that mitochondrial alterations are reversible, as evidenced by enhanced mitochondrial respiration when PD erythroblasts were cultured in a galactose medium that restricts glycolysis. This observation indicates that suppression of mitochondrial respiration may constitute a protective, adaptive response in PD pathogenesis. Notably, this effect was not observed in induced dopamine neurons, suggesting their distinct bioenergetic behavior. In summary, we provide additional evidence for the involvement of mitochondria in the disease process by demonstrating mitochondrial abnormalities in additional cell types relevant to PD. These findings contribute to our understanding of PD pathophysiology and may have implications for the development of novel biomarkers and therapeutic strategies.</p

Differences and homologies of chromosomal alterations within and between breast cancer cell lines : A clustering analysis

Background: The MCF7 (ER+/HER2-), T47D (ER+/HER2-), BT474 (ER+/HER2+) and SKBR3 (ER-/HER2+) breast cancer cell lines are widely used in breast cancer research as paradigms of the luminal and HER2 phenotypes. Although they have been subjected to cytogenetic analysis, their chromosomal abnormalities have not been carefully characterized, and their differential cytogenetic profiles have not yet been established. In addition, techniques such as comparative genomic hybridization (CGH), microarray-based CGH and multiplex ligation-dependent probe amplification (MLPA) have described specific regions of gains, losses and amplifications of these cell lines; however, these techniques cannot detect balanced chromosomal rearrangements (e.g., translocations or inversions) or low frequency mosaicism. Results: A range of 19 to 26 metaphases of the MCF7, T47D, BT474 and SKBR3 cell lines was studied using conventional (G-banding) and molecular cytogenetic techniques (multi-color fluorescence in situ hybridization, M-FISH). We detected previously unreported chromosomal changes and determined the content and frequency of chromosomal markers. MCF7 and T47D (ER+/HER2-) cells showed a less complex chromosomal make up, with more numerical than structural alterations, compared to BT474 and SKBR3 (HER2+) cells, which harbored the highest frequency of numerical and structural aberrations. Karyotype heterogeneity and clonality were determined by comparing all metaphases within and between the four cell lines by hierarchical clustering. The latter analysis identified five main clusters. One of these clusters was characterized by numerical chromosomal abnormalities common to all cell lines, and the other four clusters encompassed cell-specific chromosomal abnormalities. T47D and BT474 cells shared the most chromosomal abnormalities, some of which were shared with SKBR3 cells. MCF7 cells showed a chromosomal pattern that was markedly different from those of the other cell lines. Conclusions: Our study provides a comprehensive and specific characterization of complex chromosomal aberrations of MCF7, T47D, BT474 and SKBR3 cell lines.The chromosomal pattern of ER+/HER2- cells is less complex than that of ER+/HER2+ and ER-/HER2+ cells. These chromosomal abnormalities could influence the biologic and pharmacologic response of cells. Finally, although gene expression profiling and aCGH studies have classified these four cell lines as luminal, our results suggest that they are heterogeneous at the cytogenetic level. © 2014Rondón-Lagos et al.; licensee BioMed Central Ltd

Deciphering the RNA landscape by RNAome sequencing

Current RNA expression profiling methods rely on enrichment steps for specific RNA classes, thereby not detecting all RNA species in an unperturbed manner. We report strand-specific RNAome sequencing that determines expression of small and large RNAs from rRNA-depleted total RNA in a single sequence run. Since current analysis pipelines cannot reliably analyze small and large RNAs simultaneously, we developed TRAP, Total Rna Analysis Pipeline, a robust interface that is also compatible with existing RNA sequencing protocols. RNAome sequencing quantitatively preserved all RNA classes, allowing cross-class comparisons that facilitates the identification of relationships between different RNA classes. We demonstrate the strength of RNAome sequencing in mouse embryonic stem cells treated with cisplatin. MicroRNA and mRNA expression in RNAome sequencing significantly correlated between replicates and was in concordance with both existing RNA sequencing methods and gene expression arrays generated from the same samples. Moreover, RNAome sequencing also detected additional RNA classes such as enhancer RNAs, anti-sense RNAs, novel RNA species and numerous differentially expressed RNAs undetectable by other methods. At the level of complete RNA classes, RNAome sequencing also identified a specific global repression of the microRNA and microRNA isoform classes after cisplatin treatment whereas all other classes such as mRNAs were unchanged. These characteristics of RNAome sequencing will significantly improve expression analysis as well as studies on RNA biology not covered by existing methods

Identification of transcriptomic responses related to normal, healthy and accelerated aging

El envejecimiento es la reducción de las capacidades fisiológicas y adaptativas del organismo con el paso del tiempo. La acumulación de daño en el ADN podría ser el evento central desencadenante del proceso de envejecimiento. Los síndromes progeroides causados por una deficiencia de la sub-vía de reparación de escisión de nucleótidos acoplada a transcripción (TCR-NER) presentan un vínculo directo entre daño en el ADN y envejecimiento. Hay un paralelo entre la respuesta transcripcional de ratones progeroides y ratones sometidos a restricción dietética (DR) (una intervención que prolonga la vida). La DR aumenta la resistencia a diferentes formas de estrés. Ratones deficientes en TCR-NER también son menos susceptibles a un tipo de estrés agudo. El paralelo entre las respuestas transcriptómicas de los animales en dos extremos de esperanza de vida se ha explicado por la existencia de una respuesta de supervivencia programada. Esta tesis aborda varias cuestiones relacionadas con la respuesta de supervivencia utilizando principalmente el análisis de datos transcriptómicos. Primero, se estableció que los ratones viejos activan una respuesta de supervivencia incompleta después de tres días de DR, en segundo lugar, se describió un mecanismo común de activación de la respuesta protectora. En tercer lugar, se proporcionó una conexión entre la acumulación de daño en el ADN y la neurodegeneración. Finalmente, por medio de una metodología de análisis integrador sobre datos de transcriptómica de cerebro humano se encontró que en envejecimiento normal los astrocitos desarrollan dos fenotipos opuestos.Aging is defined as the reduction in the physiological and adaptive capabilities of organisms with the passage of time. The accumulation of DNA damage could be the central event on which other factors related to the aging process coalesce. One of the links connecting DNA damage to aging are the progeroid syndromes caused by a deficiency of DNA transcription-coupled nucleotide excision repair (TCR-NER) subpathway. There is a parallel between the transcriptional response of progeroid mice and mice on a dietary restriction (DR) regimen (an intervention that extend the lifespan). DR increased resistance to different forms of acute stress. Corroborating that TCR-NER deficiency induces activation of similar protective mechanisms, Csb-/- and Csa-/- mice are less susceptible to renal ischemia-reperfusion injury. The parallel between the transcriptomic responses of animals at two life expectancy extremes, in addition to the shared increase in resistance to ischemia-reperfusion injury, has been explained by the existence of a programmed survival response. This thesis addresses several questions related with the survival response, the mechanism of neurodegeneration and normal aging using mainly analysis of transcriptomic data. First, it was established that old mice activate an incomplete survival response after three days of DR, second, a common mechanism of activation of the protective response was described. Third, a connection between the accumulation of DNA damage and neurodegeneration was provided. Finally, an integrative methodology of analysis was used over brain human transcriptomic data, the result was the identification of an opposite activation of astrocytes in the human aged prefrontal cortex

Identification of transcriptomic responses related to normal, healthy and accelerated aging

El envejecimiento es la reducción de las capacidades fisiológicas y adaptativas del organismo con el paso del tiempo. La acumulación de daño en el ADN podría ser el evento central desencadenante del proceso de envejecimiento. Los síndromes progeroides causados por una deficiencia de la sub-vía de reparación de escisión de nucleótidos acoplada a transcripción (TCR-NER) presentan un vínculo directo entre daño en el ADN y envejecimiento. Hay un paralelo entre la respuesta transcripcional de ratones progeroides y ratones sometidos a restricción dietética (DR) (una intervención que prolonga la vida). La DR aumenta la resistencia a diferentes formas de estrés. Ratones deficientes en TCR-NER también son menos susceptibles a un tipo de estrés agudo. El paralelo entre las respuestas transcriptómicas de los animales en dos extremos de esperanza de vida se ha explicado por la existencia de una respuesta de supervivencia programada. Esta tesis aborda varias cuestiones relacionadas con la respuesta de supervivencia utilizando principalmente el análisis de datos transcriptómicos. Primero, se estableció que los ratones viejos activan una respuesta de supervivencia incompleta después de tres días de DR, en segundo lugar, se describió un mecanismo común de activación de la respuesta protectora. En tercer lugar, se proporcionó una conexión entre la acumulación de daño en el ADN y la neurodegeneración. Finalmente, por medio de una metodología de análisis integrador sobre datos de transcriptómica de cerebro humano se encontró que en envejecimiento normal los astrocitos desarrollan dos fenotipos opuestos.Aging is defined as the reduction in the physiological and adaptive capabilities of organisms with the passage of time. The accumulation of DNA damage could be the central event on which other factors related to the aging process coalesce. One of the links connecting DNA damage to aging are the progeroid syndromes caused by a deficiency of DNA transcription-coupled nucleotide excision repair (TCR-NER) subpathway. There is a parallel between the transcriptional response of progeroid mice and mice on a dietary restriction (DR) regimen (an intervention that extend the lifespan). DR increased resistance to different forms of acute stress. Corroborating that TCR-NER deficiency induces activation of similar protective mechanisms, Csb-/- and Csa-/- mice are less susceptible to renal ischemia-reperfusion injury. The parallel between the transcriptomic responses of animals at two life expectancy extremes, in addition to the shared increase in resistance to ischemia-reperfusion injury, has been explained by the existence of a programmed survival response. This thesis addresses several questions related with the survival response, the mechanism of neurodegeneration and normal aging using mainly analysis of transcriptomic data. First, it was established that old mice activate an incomplete survival response after three days of DR, second, a common mechanism of activation of the protective response was described. Third, a connection between the accumulation of DNA damage and neurodegeneration was provided. Finally, an integrative methodology of analysis was used over brain human transcriptomic data, the result was the identification of an opposite activation of astrocytes in the human aged prefrontal cortex