Browsing by Subject "High-Throughput Nucleotide Sequencing"
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Item A Comparison of Gene Expression Profiles between Glucocorticoid Responder and Non-Responder Bovine Trabecular Meshwork Cells Using RNA Sequencing(PLOS, 2017-01-09) Bermudez, Jaclyn Y.; Webber, Hannah C.; Brown, Bartley; Braun, Terry A.; Clark, Abbot F.; Mao, WeimingThe most common ocular side effect of glucocorticoid (GC) therapy is GC-induced ocular hypertension (OHT) and GC-induced glaucoma (GIG). GC-induced OHT occurs in about 40% of the general population, while the other 60% are resistant. This study aims to determine the genes and pathways involved in differential GC responsiveness in the trabecular meshwork (TM). Using paired bovine eyes, one eye was perfusion-cultured with 100nM dexamethasone (DEX), while the fellow eye was used to establish a bovine TM (BTM) cell strain. Based on maximum IOP change in the perfused eye, the BTM cell strain was identified as a DEX-responder or non-responder strain. Three responder and three non-responder BTM cell strains were cultured, treated with 0.1% ethanol or 100nM DEX for 7 days. RNA and proteins were extracted for RNA sequencing (RNAseq), qPCR, and Western immunoblotting (WB), respectively. Data were analyzed using the human and bovine genome databases as well as Tophat2 software. Genes were grouped and compared using Student's t-test. We found that DEX induced fibronectin expression in responder BTM cells but not in non-responder cells using WB. RNAseq showed between 93 and 606 differentially expressed genes in different expression groups between responder and non-responder BTM cells. The data generated by RNAseq were validated using qPCR. Pathway analyses showed 35 pathways associated with differentially expressed genes. These genes and pathways may play important roles in GC-induced OHT and will help us to better understand differential ocular responsiveness to GCs.Item Increased resolution screening of the pharmacogenetic gene CYP2D6 with microarray technology(2019-08) Davis, Carey P.; Budowle, Bruce; Luedtke, Robert R.; Hodge, Lisa M.; Phillips, Nicole R.; Woerner, August E.Autopsy is a primary methodology used for assessing cause and/or manner of death in medicolegal investigations. Some autopsies, however, do not resolve the cause of death unambiguously or there is no evident pathology to determine the cause of death. In addition, in some cases toxicology screens are negative or difficult to interpret because it is challenging to determine if a high concentration of a drug in the body derived from one large dose or has built up over time. Determining the genetic constitution of victims at specific target genes may clarify the cause of some of these unexplained deaths or at least indicate susceptibility to triggering effects. Cytochrome P450 (CYP450) is a super family of enzymes that detoxify foreign chemicals and are involved in the metabolism of drugs. One gene in this family that encodes CYP450 enzymes, CYP2D6, accounts for the metabolism of 25% of all drugs currently on the market. By examining the variability in the CYP2D6 gene, a SNP panel was developed and used to aid in personalized medicine with a long-term outcome of reducing risk in patients who partake in drug therapy. However, paralogs of the CYP2D6 gene can interfere with obtaining accurate typing results. The hypothesis of this dissertation is that it is possible to develop a targeted panel for clinically relevant variants in the CYP2D6 gene using array-based technology that can provide accurate and reliable genotyping results. The goal was first to demonstrate that high throughput sequencing, also known as next generation or massively parallel sequencing, could reliably sequence a complex target using a model system, i.e. the hypervariable regions of the human mitochondrial genome. Then, using this advanced sequencing capability define the baseline genetic variation of the CYP2D6 gene in a selected population and identify those genetic markers associated with metabolism capacity that would be verified against a database of actionable variants that cause reaction to drug exposure. The entire CYP2D6 gene was sequenced to identify SNPs at a population level. These SNPs were compared against a known database of clinically relevant samples with known metabolic responses of the same ethnic background to verify actionable variants. Once these variants were identified, a PCR assay workflow leveraging microarray technology was developed to quickly and efficiently screen individuals of interest by overcoming paralog interference. This assay can be used prior to administering drugs or post mortem to gain information about potential adverse drug reactions.Item IPAD: the Integrated Pathway Analysis Database for Systematic Enrichment Analysis(Springer Nature, 2012) Zhang, Fan; Drabier, ReneeBackground: Next-Generation Sequencing (NGS) technologies and Genome-Wide Association Studies (GWAS) generate millions of reads and hundreds of datasets, and there is an urgent need for a better way to accurately interpret and distill such large amounts of data. Extensive pathway and network analysis allow for the discovery of highly significant pathways from a set of disease vs. healthy samples in the NGS and GWAS. Knowledge of activation of these processes will lead to elucidation of the complex biological pathways affected by drug treatment, to patient stratification studies of new and existing drug treatments, and to understanding the underlying anti-cancer drug effects. There are approximately 141 biological human pathway resources as of Jan 2012 according to the Pathguide database. However, most currently available resources do not contain disease, drug or organ specificity information such as disease-pathway, drug-pathway, and organ-pathway associations. Systematically integrating pathway, disease, drug and organ specificity together becomes increasingly crucial for understanding the interrelationships between signaling, metabolic and regulatory pathway, drug action, disease susceptibility, and organ specificity from high-throughput omics data (genomics, transcriptomics, proteomics and metabolomics). Results: We designed the Integrated Pathway Analysis Database for Systematic Enrichment Analysis (IPAD, http://bioinfo.hsc.unt.edu/ipad), defining inter-association between pathway, disease, drug and organ specificity, based on six criteria: 1) comprehensive pathway coverage; 2) gene/protein to pathway/disease/drug/organ association; 3) inter-association between pathway, disease, drug, and organ; 4) multiple and quantitative measurement of enrichment and inter-association; 5) assessment of enrichment and inter-association analysis with the context of the existing biological knowledge and a "gold standard" constructed from reputable and reliable sources; and 6) cross-linking of multiple available data sources. IPAD is a comprehensive database covering about 22,498 genes, 25,469 proteins, 1956 pathways, 6704 diseases, 5615 drugs, and 52 organs integrated from databases including the BioCarta, KEGG, NCI-Nature curated, Reactome, CTD, PharmGKB, DrugBank, PharmGKB, and HOMER. The database has a web-based user interface that allows users to perform enrichment analysis from genes/proteins/molecules and inter-association analysis from a pathway, disease, drug, and organ. Moreover, the quality of the database was validated with the context of the existing biological knowledge and a "gold standard" constructed from reputable and reliable sources. Two case studies were also presented to demonstrate: 1) self-validation of enrichment analysis and inter-association analysis on brain-specific markers, and 2) identification of previously undiscovered components by the enrichment analysis from a prostate cancer study. Conclusions: IPAD is a new resource for analyzing, identifying, and validating pathway, disease, drug, organ specificity and their inter-associations. The statistical method we developed for enrichment and similarity measurement and the two criteria we described for setting the threshold parameters can be extended to other enrichment applications. Enriched pathways, diseases, drugs, organs and their inter-associations can be searched, displayed, and downloaded from our online user interface. The current IPAD database can help users address a wide range of biological pathway related, disease susceptibility related, drug target related and organ specificity related questions in human disease studies.Item Peripheral Circulating Exosomal miRNAs Potentially Contribute to the Regulation of Molecular Signaling Networks in Aging(MDPI, 2020-03-11) Zhang, Hongxia; Jin, KunlinPeople are living longer than ever. Consequently, they have a greater chance for developing a functional impairment or aging-related disease, such as a neurodegenerative disease, later in life. Thus, it is important to identify and understand mechanisms underlying aging as well as the potential for rejuvenation. Therefore, we used next-generation sequencing to identify differentially expressed microRNAs (miRNAs) in serum exosomes isolated from young (three-month-old) and old (22-month-old) rats and then used bioinformatics to explore candidate genes and aging-related pathways. We identified 2844 mRNAs and 68 miRNAs that were differentially expressed with age. TargetScan revealed that 19 of these miRNAs are predicated to target the 766 mRNAs. Pathways analysis revealed signaling components targeted by these miRNAs: mTOR, AMPK, eNOS, IGF, PTEN, p53, integrins, and growth hormone. In addition, the most frequently predicted target genes regulated by these miRNAs were EIF4EBP1, insulin receptor, PDK1, PTEN, paxillin, and IGF-1 receptor. These signaling pathways and target genes may play critical roles in regulating aging and lifespan, thereby validating our analysis. Understanding the causes of aging and the underlying mechanisms may lead to interventions that could reverse certain aging processes and slow development of aging-related diseases.