Identifying Glioblastoma Gene Networks Based on Hypergeometric Test Analysis
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ABSTRACT: Patient specific therapy is emerging as an important possibility for many cancer patients. However, to identify such therapies it is essential to determine the genomic and transcriptional alterations present in one tumor relative to control samples. This presents a challenge since use of a single sample precludes many standard statistical analysis techniques. We reasoned that one means of addressing this issue is by comparing transcriptional changes in one tumor with those observed in a large cohort of patients analyzed by The Cancer Genome Atlas (TCGA). To test this directly, we devised a bioinformatics pipeline to identify differentially expressed genes in tumors resected from patients suffering from the most common malignant adult brain tumor, glioblastoma (GBM). We performed RNA sequencing on tumors from individual GBM patients and filtered the results through the TCGA database in order to identify possible gene networks that are overrepresented in GBM samples relative to controls. Importantly, we demonstrate that hypergeometric-based analysis of gene pairs identifies gene networks that validate experimentally. These studies identify a putative workflow for uncovering differentially expressed patient specific genes and gene networks for GBM and other cancers. RNAseq on 2 gliosblastoma samples and 2 epileptic samples
Project description:Recombinant attenuated Salmonella are believed to act as powerful live vaccine carrier able to elicit protection against many infectious agents. âaroA exhibits auxotrophy for aromatic amino acids and is commonly used for attenuation. Interestingly, deletion of aroA dramatically increased virulence of the Salmonella. Detailed analyses demonstrate that Salmonellaâs physiology is significantly altered, most likely due to osmotic imbalance and stress. The result is a serious influence of lipid and amino acid turn over, possible increasing sensitivity to penicillin, complement and phagocytic uptake. In addition, in concert with other immunomodulating mutations, the phase of flagella expression is changed. Furthermore, virulence associated genes like arnT or ansB were affected. These alterations could explain the increased virulence. Thus, intravenous application of âaroA Salmonella to mice significantly increased induction of pro-inflammatory cytokines. We also employed such bacteria in therapy against tumors and realized that âaroA strains display an improved anti-tumor activity. The samples of Salmonella Typhimurium Wild type strain and derivates (SF100 (ÎlpxR9 ÎpagL7 ÎpagP8), SF101 (ÎaroA), SF102 (ÎlpxR9 ÎpagL7 ÎpagP8 ÎaroA)) were analyzed by RNA-seq. The control samples were cultivated in the LB medium until the exponential phase. Four biological replicas were pooled in pairs to get two replicas for RNA extraction, library preparation and sequencing. Tumor samples were collected from mice infected with Wt Salmonella and SF102. Bacterial RNA was extracted from single tumors. The tumor samples were split into two technical replicas for sequencing library preparation.
Project description:The goal is to identify estrogen-dependent gene expression patterns using RNAseq for 3 different ERα forms in human osteoblasts. We analyzed 3 different forms of estrogen receptor alpha (ERα), 1) Wild-type, 2) NERKI (DNA-binding mutant) and 3) NOER (Nuclear Only ERα, not membrane). Each was infected into hFOB (human osteoblast) cells and treated with either vehicle or E2 (10nM) (n=3 for each group/treatment). RNAseq was then performed.
Project description:Acetylation of lysine residues is conserved across organisms and plays important roles in various cellular functions. Maintaining intracellular pH homeostasis is crucial for the survival of enteric bacteria in acidic gastric tract. However, it remains unkown whether bacteria can utilize reversible protein acetylation system to adapt to acidic environment. Here we demonstrate that the protein acetylation/deacetylation is critical for Salmonella Typhimurium to survive in acidic environment. We first used RNA-seq to analyze the transcriptome patterns under acid stress, and found that the transcriptional levels of genes involved in NAD+/NADH metabolism were significantly changed, leading to the increase of intracellular NAD+/NADH ratio. Moreover, acid stress down-regulated the transcriptional level of pat (encoding an acetyltranseferase) and genes encoding adenylate cyclase and cAMP-regulatory protein (CRP) which regulates pat positively. Acid signal also affects TCA cycle to promote the consumption of Ac-CoA, which reduced the donor of acetylation. Lowered acetylation level is not only bacterial’s response to acid stress, but also positively regulates the survival rate of S. Typhimurium. The deletion mutant of pat had more stable intracellular pH, which paralleled with higher survival rate after acid treatment compared with the wild type strain and deletion mutant of cobB. Our data suggest that bacteria can down-regulate protein acetylation level to prevent intracellular pH further falling in acid stress, and this work may provide a new perspective to understand the bacterial acid resistance mechanism. To use RNA-seq to analyze the transcriptome patterns under acid stress
Project description:Salmonella causes a range of diseases in different hosts, including enterocolitis and systemic infection. Lysine acetylation regulates many eukaryotic cellular processes, but its function in prokaryotes is largely unknown. Reversible acetylation in Salmonella Typhimurium depends on acetyltransferase Pat and nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase CobB. Here, we used cell and animal models to evaluate the virulence of pat and cobB deletion mutants in S. Typhimurium, and found that pat is essential for bacterial intestinal colonization, systemic infection and host inflammation response. Next, to understand the underlying mechanism, genome-wide transcriptome was analyzed. RNA-seq data showed the expression of Salmonella pathogenicity islands 1 (SPI-1) is partially dependent on pat. In addition, we found that HilD is a substrate of Pat, which is essential for maintaining HilD protein level. Taken together, these results suggested that protein acetylation system regulates SPI-1 expression by controlling HilD in a post-translational manner to mediate S. Typhimurium virulence. To use RNA-seq to analyze the transcriptome patterns of pat or cobB mutation in Salmonella Typhimurium 14028s.
Project description:The aim of the study is to identify AR target gens in LNCaP cells 5 samples corresponding to LSD1 K114me2 or CHD1 genome-binding in DHT-treated and control LNCaP cells. 6 samples corresponding to LSD1 K114me2 or CHD1 genome-binding in LNCaP cells knocked-down for LSD1 or CHD1 and treated with DHT.
Project description:A recent study by Castelo-Branco, P., et al. Methylation of the TERT promoter and risk stratification of childhood brain tumours: an integrative genomic and molecular study. Lancet Oncol 2013;14:534-542 found upstream of the transcription start site (UTSS) hypermethylation of TERT is associated with tumor progression and poor prognosis in paediatric brain tumours. They interpreted that the UTSS region of telomerase reverse transcriptase (TERT) gene is a potentially accessible biomarker for various cancers. This study, we aimed to explore the role of TERT in hepatocellular carcinoma (HCC) and to investigate whether the UTSS region of TERT promoter shows the same methylation pattern in HCC. We analyzed a methylation assay of TERT including the UTSS region in 125 paired HCC samples using Mass Array EpiTyper (Sequenom, SanDiego, CA, USA). To obtain the relationship between TERT promoter methylation status and TERT expression level, we analysed a validation group of 12 paired HCC samples and acquired the FPKM value of TERT gene.
Project description:Comparison of small RNA fractions derived from piRNA clusters and transposon sequences in control and Su(var)3-7 null mutant. Small RNA profile of 3-day old control and Su(var)3-7 mutant ovaries were generated by high-throughput sequencing on Illumina HiSeq 2000
Project description:Bacterial transcription is controlled by many transcription factors and promoters. Currently many promoters are mapped only in in vitro conditions. We use an intra-macrophage model to map the promoters and their expression in vivo. Transcriptome analysis of S.Typhimurium 4/74 within macrophages using RNA-seq in biological replicates. dRNA-seq also performed in biological replicates.
Project description:New tools for studying bacterial transcripts at the single nucleotide level offer an unparalleled opportunity to understand the bacterial transcriptome. For the model pathogen Salmonella enterica serovar Typhimurium, it is necessary to identify the regulatory inputs for all RNA transcripts, including small RNAs (sRNAs) and coding genes. Here, we use RNA-seq to define the transcriptomes of mutants lacking 18 global regulatory systems that, among other functions, modulate the expression of the SPI1 and SPI2 Type Three secretion systems in S. Typhimurium strain 4/74. We directly compared the roles of the major regulators of transcription, and reported the effects of the regulatory mutations on expression of sRNAs. We also use this method to describe the impact of the RNA chaperone Hfq upon the steady state levels of 280 sRNA transcripts. Transcriptome analysis of S. Typhimurium 4/74 using RNA isolated wild-type and mutants grown under infection-relevant conditions.
Project description:Background: West Nile virus is an emerging infection of biodefense concern and there are no available treatments or vaccines. Here we used a high-throughput method based on a novel gene expression analysis, RNA-Seq, to give a global picture of differential gene expression by primary human macrophages of 10 healthy donors infected in vitro with WNV. Results: From a total of 50 million reads per sample, we employed a Bayesian hierarchical mixture model to identify 4,026 transcripts that were differentially expressed after infection. Both predicted and novel gene changes were detected, as were gene isoforms, and while many of the genes were expressed by all donors, some were unique. Knock-down of genes not previously known to be associated with WNV resistance identified their critical role in control of viral infection. Conclusions: Our study distinguishes both common gene pathways as well as novel cellular responses. Such analysis will be valuable for translational studies of susceptible and resistant individuals -- and for targeting therapeutics -- in multiple biological settings. Differential gene expression by primary human macrophages of 10 healthy donors infected in vitro with WNV were generated by RNA-Seq.