RESUMEN
Aedes aegypti is an important vector of arboviruses, including dengue, chikungunya and Zika. The application of synthetic insecticides is a frequently used strategy to control this insect. Malathion is an organophosphate insecticide that was widely used in Brazil in the 1980s and 1990s to control the adult form of A. aegypti. In situations where resistance to currently used insecticides is detected, the use of malathion may be resumed as a control measure. Many studies have confirmed resistance to malathion, however, comparative studies of differential gene expression of the entire transcriptome of resistant and susceptible insects are scarce. Therefore, understanding the molecular basis of resistance to this insecticide in this species is extremely important. In this paper, we present the first transcriptomic description of susceptible and resistant strains of A. aegypti challenged with malathion. Guided transcriptome assembly resulted in 39,904 transcripts, where 2133 differentially expressed transcripts were detected, and three were validated by RT-qPCR. Enrichment analysis for these identified transcripts resulted in 13 significant pathways (padj < 0.05), 8 associated with down-regulated and 5 with up-regulated transcripts in treated resistant insects. It was possible to divide the transcripts according to the mechanism of action into three main groups: (i) genes involved in detoxification metabolic pathways; (ii) genes of proteins located in the membrane/extracellular region; and (iii) genes related to DNA integration/function. These results are important in advancing knowledge of genes related to resistance mechanisms in this insect, enabling the development of effective technologies and strategies for managing insecticide resistance.
Asunto(s)
Aedes , Resistencia a los Insecticidas , Insecticidas , Malatión , Transcriptoma , Malatión/farmacología , Animales , Aedes/genética , Aedes/efectos de los fármacos , Resistencia a los Insecticidas/genética , Insecticidas/farmacología , Transcriptoma/efectos de los fármacos , Transcriptoma/genética , Perfilación de la Expresión Génica/métodos , Mosquitos Vectores/genética , Mosquitos Vectores/efectos de los fármacos , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismoRESUMEN
BACKGROUND: B chromosomes are extra non-essential elements present in several eukaryotes. Unlike A chromosomes which are essential and present in all individuals of a species, B chromosomes are not necessary for normal functioning of an organism. Formerly regarded as genetically inactive, B chromosomes have been discovered to not only express their own genes, but also to exert influence on gene expression in A chromosomes. Recent studies have shown that, in some Psalidodon (Characiformes, Characidae) species, B chromosomes might be associated with phenotypic effects, such as changes in the reproductive cycle and gene expression. METHODS AND RESULTS: In this study, we aimed to establish stable reference genes for RT-qPCR experiments conducted on gonads of three fish species within Psalidodon genus, both in the presence and absence of B chromosomes. The stability of five selected reference genes was assessed using NormFinder, geNorm, BestKeeper, and RefFinder algorithms. We determined ppiaa and pgk1 as the most stable genes in P. fasciatus, whereas ppiaa and hmbsa showed the highest stability in P. bockmanni. For P. paranae, tbp and hprt1 were the most stable genes in females, and ppiaa and hprt1 were the most stable in males. CONCLUSIONS: We determined the most stable reference genes in gonads of three Psalidodon species considering the presence of B chromosomes. This is the first report of reference gene stability in the genus and provides valuable tools to better understand the effects of B chromosomes at gene expression level.
Asunto(s)
Cromosomas , Animales , Masculino , Femenino , Cromosomas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Estándares de Referencia , Perfilación de la Expresión Génica/métodos , Perfilación de la Expresión Génica/normas , Gónadas/metabolismo , Characidae/genética , Characiformes/genéticaRESUMEN
In samples of harmful algal blooms (HABs), seawater can contain a high abundance of microorganisms and elemental ions. Along with the hardness of the walls of key HAB dinoflagellates such as Prorocentrum triestinum, this makes RNA extraction very difficult. These components interfere with RNA isolation, causing its degradation, in addition to the complex seawater properties of HABs that could hinder RNA isolation for effective RNA sequencing and transcriptome profiling. In this study, an RNA isolation technique was established through the modification of the Trizol method by applying the Micropestle System on cell pellets of P. triestinum frozen at -20 °C, obtained from 400 mL of culture with a total of 107 cells/mL. The results of the modified Trizol protocol generated quality RNA samples for transcriptomics sequencing, as determined by their measurement in Analyzer Agilent 4150.
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Dinoflagelados , Dinoflagelados/genética , ARN/aislamiento & purificación , ARN/genética , Guanidinas/química , Análisis de Secuencia de ARN/métodos , Floraciones de Algas Nocivas , Perfilación de la Expresión Génica/métodos , Transcriptoma , Nucleótidos/genética , Nucleótidos/aislamiento & purificación , Agua de Mar , FenolesRESUMEN
Rodents are commonly used as animal models in studies investigating various experimental conditions, often requiring gene expression analysis. Quantitative real-time reverse transcription PCR (RT-qPCR) is the most widely used tool to quantify target gene expression levels under different experimental conditions in various biological samples. Relative normalization with reference genes is a crucial step in RT-qPCR to obtain reliable quantification results. In this work, the main reference genes used in gene expression studies among the three rodents commonly employed in scientific research-hamster, rat, and mouse-are analyzed and described. An individual literature search for each rodent was conducted using specific search terms in three databases: PubMed, Scopus, and Web of Science. A total of 157 articles were selected (rats = 73, mice = 79, and hamsters = 5), identifying various reference genes. The most commonly used reference genes were analyzed according to each rodent, sample type, and experimental condition evaluated, revealing a great variability in the stability of each gene across different samples and conditions. Classic genes, which are expected to be stably expressed in both samples and conditions analyzed, demonstrated greater variability, corroborating existing concerns about the use of these genes. Therefore, this review provides important insights for researchers seeking to identify suitable reference genes for their validation studies in rodents.
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Perfilación de la Expresión Génica , Estándares de Referencia , Roedores , Animales , Ratones , Ratas , Expresión Génica/genética , Perfilación de la Expresión Génica/métodos , Perfilación de la Expresión Génica/normas , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Roedores/genéticaRESUMEN
Gene expression through RT-qPCR can be performed by the relative quantification method, which requires the expression normalization through reference genes. Therefore, it is essential to validate, experimentally, the candidate reference genes. Thus, although there are several studies that are performed to identify the most stable reference genes, most them validate genes for very specific conditions, not exploring the whole potential of the research since not all possible combinations of treatments and/or conditions of the study are explored. For this reason, new experiments must be conducted by researchers that have interest in analyzing gene expression of treatments and/or conditions present, but not explored, in these studies. Here, we present the RGeasy tool, which aims to facilitate the selection of reference genes, allowing the user to choose genes for a greater number of combinations of treatments/conditions, compared to the ones present in the original articles, through just a few clicks. RGeasy was validated with RT-qPCR data from gene expression studies performed in two coffee species, Coffea arabica and Coffea canephora, and it can be used for any animal, plant or microorganism species. In addition to displaying a rank of the most stable reference genes for each condition or treatment, the user also has access to the primer pairs for the selected reference genes.
Asunto(s)
Perfilación de la Expresión Génica , Reacción en Cadena en Tiempo Real de la Polimerasa , Estándares de Referencia , Programas Informáticos , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Perfilación de la Expresión Génica/métodos , Perfilación de la Expresión Génica/normas , Genes de Plantas , Coffea/genética , Regulación de la Expresión Génica de las PlantasRESUMEN
Oxidative stress has long been postulated to play an essential role in aging mechanisms, and numerous forms of molecular damage associated with oxidative stress have been well documented. However, the extent to which changes in gene expression in direct response to oxidative stress are related to actual cellular aging, senescence, and age-related functional decline remains unclear. Here, we ask whether H2O2-induced oxidative stress and resulting gene expression alterations in prostate epithelial cells in vitro reveal gene regulatory changes typically observed in naturally aging prostate tissue and age-related prostate disease. While a broad range of significant changes observed in the expression of non-coding transcripts implicated in senescence-related responses, we also note an overrepresentation of gene-splicing events among differentially expressed protein-coding genes induced by H2O2. Additionally, the collective expression of these H2O2-induced DEGs is linked to age-related pathological dysfunction, with their protein products exhibiting a dense network of protein-protein interactions. In contrast, co-expression analysis of available gene expression data reveals a naturally occurring highly coordinated expression of H2O2-induced DEGs in normally aging prostate tissue. Furthermore, we find that oxidative stress-induced DEGs statistically overrepresent well-known senescence-related signatures. Our results show that oxidative stress-induced gene expression in prostate epithelial cells in vitro reveals gene regulatory changes typically observed in naturally aging prostate tissue and age-related prostate disease.
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Envejecimiento , Senescencia Celular , Células Epiteliales , Peróxido de Hidrógeno , Estrés Oxidativo , Próstata , Masculino , Humanos , Células Epiteliales/metabolismo , Próstata/metabolismo , Envejecimiento/genética , Envejecimiento/metabolismo , Senescencia Celular/genética , Regulación de la Expresión Génica , Perfilación de la Expresión Génica/métodosRESUMEN
Long noncoding RNAs (lncRNAs) are versatile RNA molecules recently identified as key regulators of gene expression in response to environmental stress. Our primary focus in this study was to develop a robust computational pipeline for identifying structurally identical lncRNAs across replicates from publicly available bulk RNA-seq datasets. In order to demonstrate the effectiveness of the pipeline, we utilized the transcriptome of the thermophilic fungus Thermothelomyces thermophilus and assessed the expression pattern of lncRNAs in conjunction with Heat Shock Proteins (HSP), a well-known protein family critical for the cell's response to high temperatures. Our findings demonstrate that the identification of structurally identical transcripts among replicates in this thermophilic fungus ensures the reliability and accuracy of RNA studies, contributing to the validity of biological interpretations. Furthermore, the majority of lncRNAs exhibited a distinct expression pattern compared to HSPs. Our study contributes to advancing the understanding of the biological mechanisms comprising lncRNAs in thermophilic fungi.
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Biología Computacional , ARN de Hongos , ARN Largo no Codificante , ARN Largo no Codificante/genética , ARN de Hongos/genética , ARN de Hongos/metabolismo , Biología Computacional/métodos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Regulación Fúngica de la Expresión Génica , Transcriptoma , Calor , Perfilación de la Expresión Génica/métodosRESUMEN
A recent paper shows that in gene expression space the manifold spanned by normal tissues and the manifold spanned by the corresponding tumors are disjoint. The statement is based on a two-dimensional projection of gene expression data. In the present paper, we show that, for the multi-dimensional vectors defining the centers of cloud samples: 1. The closest tumor to a given normal tissue is the tumor developed in that tissue, 2. Two normal tissues define quasi-orthogonal directions, 3. A tumor may have a projection onto its corresponding normal tissue, but it is quasi-orthogonal to all other normal tissues, and 4. The cancer manifold is roughly obtained by translating the normal tissue manifold along an orthogonal direction defined by a global cancer progression axis. These geometrical properties add a new characterization of normal tissues and tumors and may have biological significance. Indeed, normal tissues at the vertices of a high-dimensional simplex could indicate genotype optimization for given tissue functions, and a way of avoiding errors in embryonary development. On the other hand, the cancer progression axis could define relevant pan-cancer genes and seems to be consistent with the atavistic theory of tumors.
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Neoplasias , Humanos , Neoplasias/genética , Neoplasias/patología , Regulación Neoplásica de la Expresión Génica , Algoritmos , Perfilación de la Expresión Génica/métodos , Progresión de la EnfermedadRESUMEN
BACKGROUND: Testicular descent is a physiological process regulated by many factors. Eventually, disturbances in the embryological/fetal development path facilitate the occurrence of scrotal hernia, a congenital malformation characterized by the presence of intestinal portions within the scrotal sac due to the abnormal expansion of the inguinal ring. In pigs, some genes have been related to this anomaly, but the genetic mechanisms involved remain unclear. This study aimed to investigate the expression profile of a set of genes potentially involved with the manifestation of scrotal hernia in the inguinal ring tissue. METHODS AND RESULTS: Tissue samples from the inguinal ring/canal of normal and scrotal hernia-affected male pigs with approximately 30 days of age were used. Relative expression analysis was performed using qPCR to confirm the expression profile of 17 candidate genes previously identified in an RNA-Seq study. Among them, the Myosin heavy chain 1 (MYH1), Desmin (DES), and Troponin 1 (TNNI1) genes were differentially expressed between groups and had reduced levels of expression in the affected animals. These genes encode proteins involved in the formation of muscle tissue, which seems to be important for increasing the resistance of the inguinal ring to the abdominal pressure, which is essential to avoid the occurrence of scrotal hernia. CONCLUSIONS: The downregulation of muscular candidate genes in the inguinal tissue clarifies the genetic mechanisms involved with this anomaly in its primary site, providing useful information for developing strategies to control this malformation in pigs and other mammals.
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Regulación hacia Abajo , Escroto , Animales , Masculino , Porcinos/genética , Escroto/metabolismo , Escroto/anomalías , Escroto/patología , Regulación hacia Abajo/genética , Hernia Inguinal/genética , Hernia Inguinal/metabolismo , Hernia Inguinal/veterinaria , Perfilación de la Expresión Génica/métodos , Enfermedades de los Porcinos/genética , Enfermedades de los Porcinos/metabolismo , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismoRESUMEN
Omic tools have changed the way of doing research in experimental biology. The somatic embryogenesis (SE) study has not been immune to this benefit. The transcriptomic tools have been used to compare the genes expressed during the induction of SE with the genes expressed in zygotic embryogenesis or to compare the development of the different stages embryos go through. It has also been used to compare the expression of genes during the development of calli from which SE is induced, as well as many other applications. The protocol described here is employed in our laboratory to extract RNA and generate several transcriptomes for the study of SE on Coffea canephora.
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Coffea , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Técnicas de Embriogénesis Somática de Plantas , Transcriptoma , Coffea/genética , Coffea/embriología , Coffea/crecimiento & desarrollo , Técnicas de Embriogénesis Somática de Plantas/métodos , Perfilación de la Expresión Génica/métodos , Transcriptoma/genética , Semillas/genética , Semillas/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión GénicaRESUMEN
Myeloproliferative neoplasms (MPN) are hematological diseases associated with genetic driver mutations in the JAK2, CALR, and MPL genes and exacerbated oncoinflammatory status. Analyzing public microarray data from polycythemia vera (n = 41), essential thrombocythemia (n = 21), and primary myelofibrosis (n = 9) patients' peripheral blood by in silico approaches, we found that pro-inflammatory and monocyte-related genes were differentially expressed in MPN patients' transcriptome. Genes related to cell activation, secretion of pro-inflammatory and pro-angiogenic mediators, activation of neutrophils and platelets, coagulation, and interferon pathway were upregulated in monocytes compared to controls. Together, our results suggest that molecular alterations in monocytes may contribute to oncoinflammation in MPN.
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Monocitos , Trastornos Mieloproliferativos , Transcriptoma , Humanos , Monocitos/metabolismo , Monocitos/inmunología , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/sangre , Inflamación/genética , Inflamación/sangre , Perfilación de la Expresión Génica/métodos , Policitemia Vera/genética , Policitemia Vera/sangre , Janus Quinasa 2/genética , Mielofibrosis Primaria/genética , Mielofibrosis Primaria/sangre , Trombocitemia Esencial/genética , Trombocitemia Esencial/sangre , Receptores de Trombopoyetina/genética , Regulación Neoplásica de la Expresión GénicaRESUMEN
BACKGROUND: Real-time quantitative PCR (RT-qPCR) is one of the most widely used gene expression analyses for validating RNA-seq data. This technique requires reference genes that are stable and highly expressed, at least across the different biological conditions present in the transcriptome. Reference and variable candidate gene selection is often neglected, leading to misinterpretation of the results. RESULTS: We developed a software named "Gene Selector for Validation" (GSV), which identifies the best reference and variable candidate genes for validation within a quantitative transcriptome. This tool also filters the candidate genes concerning the RT-qPCR assay detection limit. GSV was compared with other software using synthetic datasets and performed better, removing stable low-expression genes from the reference candidate list and creating the variable-expression validation list. GSV software was used on a real case, an Aedes aegypti transcriptome. The top GSV reference candidate genes were selected for RT-qPCR analysis, confirming that eiF1A and eiF3j were the most stable genes tested. The tool also confirmed that traditional mosquito reference genes were less stable in the analyzed samples, highlighting the possibility of inappropriate choices. A meta-transcriptome dataset with more than ninety thousand genes was also processed successfully. CONCLUSION: The GSV tool is a time and cost-effective tool that can be used to select reference and validation candidate genes from the biological conditions present in transcriptomic data.
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Reacción en Cadena en Tiempo Real de la Polimerasa , Estándares de Referencia , Programas Informáticos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Animales , RNA-Seq/métodos , RNA-Seq/normas , Perfilación de la Expresión Génica/métodos , TranscriptomaRESUMEN
Common beans (Phaseolus vulgaris L.), besides being an important source of nutrients such as iron, magnesium, and protein, are crucial for food security, especially in developing countries. Common bean cultivation areas commonly face production challenges due to drought occurrences, mainly during the reproductive period. Dry spells last approximately 20 days, enough time to compromise production. Hence, it is crucial to understand the genetic and molecular mechanisms that confer drought tolerance to improve common bean cultivars' adaptation to drought. Sixty six RNASeq libraries, generated from tolerant and sensitive cultivars in drought time sourced from the R5 phenological stage at 0 to 20 days of water deficit were sequenced, generated over 1.5 billion reads, that aligned to 62,524 transcripts originating from a reference transcriptome, as well as 6673 transcripts obtained via de novo assembly. Differentially expressed transcripts were functionally annotated, revealing a variety of genes associated with molecular functions such as oxidoreductase and transferase activity, as well as biological processes related to stress response and signaling. The presence of regulatory genes involved in signaling cascades and transcriptional control was also highlighted, for example, LEA proteins and dehydrins associated with dehydration protection, and transcription factors such as WRKY, MYB, and NAC, which modulate plant response to water deficit. Additionally, genes related to membrane and protein protection, as well as water and ion uptake and transport, were identified, including aquaporins, RING-type E3 ubiquitin transferases, antioxidant enzymes such as GSTs and CYPs, and thioredoxins. This study highlights the complexity of plant response to water scarcity, focusing on the functional diversity of the genes involved and their participation in the biological processes essential for plant adaptation to water stress. The identification of regulatory and cell protection genes offers promising prospects for genetic improvement aiming at the production of common bean varieties more resistant to drought. These findings have the potential to drive sustainable agriculture, providing valuable insights to ensure food security in a context of climate change.
Asunto(s)
Sequías , Regulación de la Expresión Génica de las Plantas , Phaseolus , Proteínas de Plantas , Phaseolus/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transcriptoma/genética , Estrés Fisiológico/genética , Adaptación Fisiológica/genética , Deshidratación/genética , Perfilación de la Expresión Génica/métodos , Resistencia a la SequíaRESUMEN
BACKGROUND: Neonatal health assessment is crucial for detecting and intervening in various disorders. Traditional gene expression analysis methods often require invasive procedures during sample collection, which may not be feasible or ideal for preterm infants. In recent years, saliva has emerged as a promising noninvasive biofluid for assessing gene expression. Another trend that has been growing is the use of "omics" technologies such as transcriptomics in the analysis of gene expression. The costs for carrying out these analyses and the difficulty of analysis make the detection of candidate genes necessary. These genes act as biomarkers for the maturation stages of the oral feeding issue. METHODOLOGY: Salivary samples (n = 225) were prospectively collected from 45 preterm (<34 gestational age) infants from five predefined feeding stages and submitted to RT-qPCR. A better description of the targeted genes and results from RT-qPCR analyses were included. The six genes previously identified as predictive of feeding success were tested. The genes are AMPK, FOXP2, WNT3, NPHP4, NPY2R, and PLXNA1, along with two reference genes: GAPDH and 18S. RT-qPCR amplification enabled the analysis of the gene expression of AMPK, FOXP2, WNT3, NPHP4, NPY2R, and PLXNA1 in neonatal saliva. Expression results were correlated with the feeding status during sample collection. CONCLUSIONS: In summary, the genes AMPK, FOXP2, WNT3, NPHP4, NPY2R, and PLXNA1 play critical roles in regulating oral feeding and the development of premature infants. Understanding the influence of these genes can provide valuable insights for improving nutritional care and support the development of these vulnerable babies. Evidence suggests that saliva-based gene expression analysis in newborns holds great promise for early detection and monitoring of disease and understanding developmental processes. More research and standardization of protocols are needed to fully explore the potential of saliva as a noninvasive biomarker in neonatal care.
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Recien Nacido Prematuro , Saliva , Humanos , Saliva/metabolismo , Recién Nacido , Femenino , Masculino , Perfilación de la Expresión Génica/métodos , Transcriptoma/genéticaRESUMEN
Alzheimer's disease (AD), a neurodegenerative disorder characterized by progressive cognitive decline, is the most common form of dementia. Currently, there is no single test that can diagnose AD, especially in understudied populations and developing countries. Instead, diagnosis is based on a combination of medical history, physical examination, cognitive testing, and brain imaging. Exosomes are extracellular nanovesicles, primarily composed of RNA, that participate in physiological processes related to AD pathogenesis such as cell proliferation, immune response, and neuronal and cardiovascular function. However, the identification and understanding of the potential role of long non-coding RNAs (lncRNAs) in AD diagnosis remain largely unexplored. Here, we clinically, cognitively, and genetically characterized a sample of 15 individuals diagnosed with AD (cases) and 15 controls from Barranquilla, Colombia. Advanced bioinformatics, analytics and Machine Learning (ML) techniques were used to identify lncRNAs differentially expressed between cases and controls. The expression of 28,909 lncRNAs was quantified. Of these, 18 were found to be differentially expressed and harbored in pivotal genes related to AD. Two lncRNAs, ENST00000608936 and ENST00000433747, show promise as diagnostic markers for AD, with ML models achieving > 95% sensitivity, specificity, and accuracy in both the training and testing datasets. These findings suggest that the expression profiles of lncRNAs could significantly contribute to advancing personalized AD diagnosis in this community, offering promising avenues for early detection and follow-up.
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Enfermedad de Alzheimer , ARN Largo no Codificante , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/genética , Humanos , ARN Largo no Codificante/genética , Femenino , Masculino , Anciano , Medicina de Precisión/métodos , Biomarcadores , Aprendizaje Automático , Anciano de 80 o más Años , Estudios de Casos y Controles , Perfilación de la Expresión Génica/métodos , Biología Computacional/métodosRESUMEN
Analyzing transcription data requires intensive statistical analysis to obtain useful biological information and knowledge. A significant portion of this data is affected by random noise or even noise intrinsic to the modeling of the experiment. Without robust treatment, the data might not be explored thoroughly, and incorrect conclusions could be drawn. Examining the correlation between gene expression profiles is one way bioinformaticians extract information from transcriptomic experiments. However, the correlation measurements traditionally used have worrisome shortcomings that need to be addressed. This paper compares five already published and experimented-with correlation measurements to the newly developed coincidence index, a similarity measurement that combines Jaccard and interiority indexes and generalizes them to be applied to vectors containing real values. We used microarray and RNA-Seq data from the archaeonHalobacterium salinarumand the bacteriumEscherichia coli, respectively, to evaluate the capacity of each correlation/similarity measurement. The utilized method explores the co-expressed metabolic pathways by measuring the correlations between the expression levels of enzymes that share metabolites, represented in the form of a weighted graph. It then searches for local maxima in this graph using a simulated annealing algorithm. We demonstrate that the coincidence index extracts larger, more comprehensive, and more statistically significant pathways for microarray experiments. In RNA-Seq experiments, the results are more limited, but the coincidence index managed the largest percentage of significant components in the graph.
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Escherichia coli , Halobacterium salinarum , Redes y Vías Metabólicas , Escherichia coli/genética , Escherichia coli/metabolismo , Halobacterium salinarum/metabolismo , Halobacterium salinarum/genética , Perfilación de la Expresión Génica/métodos , Biología Computacional , Transcriptoma , Algoritmos , RNA-SeqRESUMEN
Banana (Musa spp.) is the most widely consumed fruit globally. Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is a highly threatening disease to banana production. Resistance genes to Foc exist in wild Musa genotypes such as Musa acuminata subsp. burmannicoides var. Calcutta 4. Whilst real-time PCR (RT-qPCR) is appropriate for accurate analysis of gene expression in pathways involved in host defence responses, reference genes with stable expression under specific biotic stress conditions and host tissue types are necessary for normalization of sample variation. In this context, the stability in potential host reference genes ACT1, APT, EF1α, GAPDH, αTUB, RAN, UBIQ1, UBIQ2, ßTUB1, ßTUB3, L2 and ACTA1 was evaluated in total RNA samples from root tissues in Calcutta 4 (resistant) and Musa sp. cultivar Prata-anã (susceptible) extracted during interaction with Foc subtropical race 4 (STR4). Expression stability was calculated using the algorithms geNorm, NormFinder and BestKeeper. ßTUB3 and L2 were identified as the most stable in Calcutta 4, with ACTA1 and GAPDH the most stable in Prata-anã. These reference genes for analysis of gene expression modulation in the Musa-Foc STR4 pathosystem are fundamental for advancing understanding of host defence responses to this important pathogen.
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Resistencia a la Enfermedad , Fusarium , Genotipo , Musa , Enfermedades de las Plantas , Reacción en Cadena en Tiempo Real de la Polimerasa , Fusarium/genética , Musa/microbiología , Musa/genética , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Resistencia a la Enfermedad/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Estándares de Referencia , Perfilación de la Expresión Génica/métodosRESUMEN
Choosing appropriate reference genes or internal controls to normalize RT-qPCR data is mandatory for the interexperimental reproducibility of gene expression data obtained by RT-qPCR in most studies, including those on endometriosis. Particularly for miRNAs, the choice for reference genes is challenging because of their physicochemical and biological characteristics. Moreover, the retrograde menstruation theory, mesenchymal stem cells in menstrual blood (MenSCs), and changes in post-transcriptional regulatory processes through miRNAs have gained prominence in the scientific community as important players in endometriosis. Therefore, we originally explored the stability of 10 miRNAs expressions as internal control candidates in conditions involving the two-dimensional culture of MenSCs from healthy women and patients with endometriosis. Here, we applied multiple algorithms (geNorm, NormFinder, Bestkeeper, and delta Ct) to screen reference genes and assessed the comprehensive stability classification of miRNAs using RefFinder. Pairwise variation calculated using geNorm identified three miRNAs as a sufficient number of reference genes for accurate normalization. MiR-191-5p, miR-24-3p, and miR-103a-3p were the best combination for suitable gene expression normalization. This study will benefit similar research, but is also attractive for regenerative medicine and clinics that use MenSCs, miRNA expression, and RT-qPCR.
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Endometriosis , Menstruación , Células Madre Mesenquimatosas , MicroARNs , Reacción en Cadena en Tiempo Real de la Polimerasa , Humanos , Femenino , MicroARNs/genética , Endometriosis/genética , Células Madre Mesenquimatosas/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Menstruación/genética , Adulto , Perfilación de la Expresión Génica/métodos , Estándares de Referencia , Reproducibilidad de los Resultados , AlgoritmosRESUMEN
INTRODUCTION AND OBJECTIVES: Liver fibrosis remains a complication derived from a chronic Hepatitis C Virus (HCV) infection even when it is resolved, and no liver antifibrotic drug has been approved. Molecular mechanisms on hepatocytes and activation of hepatic stellate cells (HSCs) play a central role in liver fibrogenesis. To elucidate molecular mechanisms, it is important to analyze pathway regulation during HSC activation and HCV infection. MATERIALS AND METHODS: We evaluate the fibrosis-associated molecular mechanisms during a co-culture of human HSCs (LX2), with human hepatocytes (Huh7) that express HCV NS5A or Core protein. We evaluated LX2 activation induced by HCV NS5A or Core expression in Huh7 cells during co-culture. We determined a fibrosis-associated gene expression profile in Huh7 that expresses NS5A or Core proteins during the co-culture with LX2. RESULTS: We observed that NS5A induced 8.3-, 6.7- and 4-fold changes and that Core induced 6.5-, 1.8-, and 6.2-fold changes in the collagen1, TGFß1, and timp1 gene expression, respectively, in LX2 co-cultured with transfected Huh7. In addition, NS5A induced the expression of 30 genes while Core induced 41 genes and reduced the expression of 30 genes related to fibrosis in Huh7 cells during the co-culture with LX2, compared to control. The molecular pathways enriched from the gene expression profile were involved in TGFB signaling and the organization of extracellular matrix. CONCLUSIONS: We demonstrated that HCV NS5A and Core protein expression regulate LX2 activation. NS5A and Core-induced LX2 activation, in turn, regulates diverse fibrosis-related gene expression at different levels in Huh7, which can be further analyzed as potential antifibrotic targets during HCV infection.
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Técnicas de Cocultivo , Colágeno Tipo I , Hepacivirus , Células Estrelladas Hepáticas , Hepatocitos , Cirrosis Hepática , Inhibidor Tisular de Metaloproteinasa-1 , Factor de Crecimiento Transformador beta1 , Proteínas del Núcleo Viral , Proteínas no Estructurales Virales , Humanos , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Células Estrelladas Hepáticas/metabolismo , Cirrosis Hepática/metabolismo , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Cirrosis Hepática/virología , Hepacivirus/genética , Hepatocitos/metabolismo , Hepatocitos/virología , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/genética , Regulación de la Expresión Génica , Transducción de Señal , Cadena alfa 1 del Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I/metabolismo , Perfilación de la Expresión Génica/métodos , Línea Celular Tumoral , ARN Polimerasa Dependiente del ARNRESUMEN
BACKGROUND: Cuproptosis is known to regulate diverse physiological functions in many diseases, but its role in regulating Myocardial Ischemia-Reperfusion Injury (MI/RI) remains unclear. METHODS: For this purpose, the MI/RI microarray datasets GSE61592 were downloaded from the Gene Expression Omnibus (GEO) database, and the Differently Expressed Genes (DEGs) in MI/RI were identified using R software. Moreover, the MI/RI mice model was established to confirm further the diagnostic value of Pyruvate Dehydrogenase B (Pdhb), Dihydrolipoamide S-acetyltransferase (Dlat), and Pyruvate dehydrogenase E1 subunit alpha 1 (Pdhα1). RESULTS: The analysis of microarray datasets GSE61592 revealed that 798 genes were upregulated and 768 were downregulated in the myocardial tissue of the ischemia-reperfusion injury mice. Furthermore, Dlat, Pdhb, Pdhα1, and cuproptosis-related genes belonged to the downregulated genes. The receiver operating characteristics curve analysis results indicated that the Dlat, Pdhb, and Pdhα1 levels were downregulated in MI/RI and were found to be potential biomarkers for MI/RI diagnosis and prognosis. Similarly, analysis of Dlat, Pdhb, and Pdhα1 levels in the MI/RI mice revealed Pdhb being the key diagnostic marker. CONCLUSIONS: This study demonstrated the prognostic value of cuproptosis-related genes (Dlat, Pdhb, and Pdhα1), especially Pdhb, MI/RI, providing new insight into the MI/RI treatment.