Project description:To elucidate the possible regions of JEV genome targeted by ZAP, we performed CLIP to pull down the RNA interacting with ZAP in JEV-infected ZAP-S overexpressing cells. The enriched RNA population was subjected to NGS by use of the Ion Torrent platform.
Project description:B16F10, a murine melanoma cel line is cytolytic to JEV infection. We have identified a B16F10 cell line variant which is non-cytolytic to JEV infection. This variant cell line has two types of cells, one which is persistently infected JEV and another which is resistant to JEV infection. The JEV-resistant cells (B16F10r) were seperated from the presistently infected cells by single cell cloning. To understand the mechanism of JEV resistance microarray analysis was caried out to Identify and characterize genes differentially expressed during in uninfected/JEV-infected B16F10 and B16F10r cells.
Project description:B16F10, a murine melanoma cel line is cytolytic to JEV infection. We have identified a B16F10 cell line variant which is non-cytolytic to JEV infection. This variant cell line has two types of cells, one which is persistently infected JEV and another which is resistant to JEV infection. The JEV-resistant cells (B16F10r) were seperated from the presistently infected cells by single cell cloning. To understand the mechanism of JEV resistance microarray analysis was caried out to Identify and characterize genes differentially expressed during in uninfected/JEV-infected B16F10 and B16F10r cells. Agilent one-color experiment,Organism: Mus musculus ,Agilent Custom Mouse Whole Genome Mouse 8x60k Gene expression designed by Genotypic Technology Private Limited, Labeling kit: Agilent Quick-Amp labeling Kit (p/n5190-0442)
Project description:Crosslinking and immunoprecipitation (CLIP) is increasingly used to map transcriptome-wide binding sites of RNA-binding proteins (RBPs). We developed a method for CLIP data analysis and applied it to compare 254 nm CLIP with PAR-CLIP, which involves crosslinking of photoreactive nucleotides with 365 nm UV light. We found small differences in the accuracy of these methods in identifying binding sites of HuR, a protein that binds low-complexity sequences and Argonaute 2, which has a complex binding specificity. We show that crosslink-induced mutations lead to single-nucleotide resolution for both PAR-CLIP and CLIP. Our results confirm the expectation from original CLIP publications that RNA-binding proteins do not protect sufficiently their sites under the denaturing conditions used during the CLIP procedure, and we show that extensive digestion with sequence-specific ribonucleases strongly biases the set of recovered binding sites. We finally show that this bias can be substantially reduced by milder nuclease digestion conditions. We performed duplicate experiments for each variant of the CLIP protocol (CLIP, PAR-CLIP), each protein (HuR, Ago2), and enzymatic digestion (complete T1 digestion, mild MNase digestion). In addition, we performed a single PAR-CLIP experiment with mild T1 digestion.
Project description:B-cell chronic lymphocytic leukemia (B-CLL) is a heterogenous disease with a highly variable clinical course and analysis of ZAP-70 and CD38 expression on B-CLL cells allowed for identification of patients with good (ZAP-70-CD38-), intermediate (discordant expression of ZAP-70 and CD38) and poor (ZAP-70+CD38+) prognosis. In an attempt to identify a molecular basis that may underly this diverse clinical behaviour DNA microarray technology was employed to compare eight ZAP-70+CD38+ with eight ZAP-70-CD38- B-CLL cases. We used microarrays to detail the global programme of gene expression distinguising B-CLL from patient with good (samples 1 to 8) and poor prognosis (sample 9 to 16) and identified distinct classes of up- and down-regulated genes. Keywords: Disease progression
Project description:Crosslinking and immunoprecipitation (CLIP) is increasingly used to map transcriptome-wide binding sites of RNA-binding proteins (RBPs). We developed a method for CLIP data analysis and applied it to compare 254 nm CLIP with PAR-CLIP, which involves crosslinking of photoreactive nucleotides with 365 nm UV light. We found small differences in the accuracy of these methods in identifying binding sites of HuR, a protein that binds low-complexity sequences and Argonaute 2, which has a complex binding specificity. We show that crosslink-induced mutations lead to single-nucleotide resolution for both PAR-CLIP and CLIP. Our results confirm the expectation from original CLIP publications that RNA-binding proteins do not protect sufficiently their sites under the denaturing conditions used during the CLIP procedure, and we show that extensive digestion with sequence-specific ribonucleases strongly biases the set of recovered binding sites. We finally show that this bias can be substantially reduced by milder nuclease digestion conditions.
Project description:B-cell chronic lymphocytic leukemia (B-CLL) is a heterogenous disease with a highly variable clinical course and analysis of ZAP-70 and CD38 expression on B-CLL cells allowed for identification of patients with good (ZAP-70-CD38-), intermediate (discordant expression of ZAP-70 and CD38) and poor (ZAP-70+CD38+) prognosis. In an attempt to identify a molecular basis that may underly this diverse clinical behaviour DNA microarray technology was employed to compare eight ZAP-70+CD38+ with eight ZAP-70-CD38- B-CLL cases. We used microarrays to detail the global programme of gene expression distinguising B-CLL from patient with good (samples 1 to 8) and poor prognosis (sample 9 to 16) and identified distinct classes of up- and down-regulated genes. Experiment Overall Design: To compare the transcriptosomes of good prognosis CLL cases (ZAP-70-CD38-) to poor prognosis cases (ZAP-70+CD38+), we purified CD19+ cells from peripheral blood samples by immunomagnetic isolation using MidiMacs, resulting in >95% purity of leukemic cells as detected by FACS analysis of CD19+CD5+ cells. The leukemic cells were freshly purified from untreated patients and RNA was directly isolated from fresh cells without further ex vivo treatment of the cells. Eight immunomagnetically purified peripheral blood derived ZAP-70+CD38+ CLL cases were compared with eight ZAP-70-CD38- B-CLL cases.
Project description:Infection by neurotropic virus Japanese Encephalitis Virus (JEV) is characterized by profound neuronal cell death and neuroinflammation. Long non-coding RNAs (lncRNAs) are critical regulatory players in diverse biological processes, including viral pathogenesis. We use whole transcriptomic sequencing to identify a lncRNA JINR1 (JEV-induced non-coding RNA 1) induced upon JEV infection in neuronal cells. Transcription factor NF-κB triggers JINR-1 expression during JEV infection. Loss of JINR-1 impairs virus replication and reduces JEV-induced neuronal cell death and expression of genes involved in ER stress and neuroinflammation. Mechanistically, JINR1 inhibits the expression of mir-216-5p, which inhibits host factors GRP78 and c-JUN and the viral genome. In line with its role as a pro-viral host factor, JINR1 interacts with RBM10 and NF-κB to promote the expression of genes involved in ER stress and neuroinflammation. Our results suggest a role for JINR-1 in promoting JEV-induced cell death and neuroinflammation.
Project description:Crosslinking and immunoprecipitation (CLIP) is increasingly used to map transcriptome-wide binding sites of RNA-binding proteins (RBPs). We developed a method for CLIP data analysis and applied it to compare 254 nm CLIP with PAR-CLIP, which involves crosslinking of photoreactive nucleotides with 365 nm UV light. We found small differences in the accuracy of these methods in identifying binding sites of HuR, a protein that binds low-complexity sequences and Argonaute 2, which has a complex binding specificity. We show that crosslink-induced mutations lead to single-nucleotide resolution for both PAR-CLIP and CLIP. Our results confirm the expectation from original CLIP publications that RNA-binding proteins do not protect sufficiently their sites under the denaturing conditions used during the CLIP procedure, and we show that extensive digestion with sequence-specific ribonucleases strongly biases the set of recovered binding sites. We finally show that this bias can be substantially reduced by milder nuclease digestion conditions.
Project description:Flaviviruses, particularly Japanese encephalitis virus (JEV) and West Nile virus (WNV), are important causes of virus-induced central nervous system (CNS) disease in humans. We used microarray analysis to identify cellular genes that are differentially regulated following infection of the brain with JEV (P3) or WNV (New York 99). Gene expression data for these flaviviruses was compared to that induced following infection of the brain with reovirus (Type 3 Dearing), an unrelated neurotropic virus. Although several studies have described gene expression changes following virus infection of the brain, this report is the first to directly compare large-scale gene expression data from different viruses. We found that a large number of genes were up-regulated in common to infections with all 3 viruses (fold change > 2, P < 0.001), including genes associated with interferon signaling, the immune system, inflammation and cell death/survival signaling. In addition, genes associated with glutamate signaling were down-regulated in common to infections with all 3 viruses (fold change > 2, P < 0.001). These genes may serve broad spectrum therapeutic targets for virus-induced CNS disease. A distinct set of genes were up-regulated following flavivirus-infection, but not following infection with reovirus. These genes were associated with tRNA charging and may serve as therapeutic targets for flavivirus-induce CNS disease. Gene expression in the brain following WNV or JEV infection. WNV- or JEV-infected (N=3) vs. mock-infected (N=3) mouse brain.