Project description:Purpose: Chronic infection with hepatitis B virus is the leading global risk factor for the development of liver cancer. A large body of research has shown the many effects an HBV infection has on cellular physiology, particularly on pathways that may be involved in the development of HBV-associated diseases. Unfortunately, a significant portion of this research has been done in systems that may not mimic what is seen in a primary hepatocyte, and is not done on a transcriptome-wide scale. Because of this, we performed an RNA-seq analysis of primary rat hepatocytes expressing HBV to determine the global changes HBV has on primary hepatocyte physiology. Methods: To do this RNA-seq analysis, triplicate samples of total RNA were collected from cultured primary rat hepatocytes infected with adenovirus expressing GFP alone (AdGFP) or GFP along with a greater than unit length copy of the HBV genome (AdHBV). Samples were collected either 24h or 48h after infection. cDNA libraries were sequenced two times using the Illumina HiSeq or Illumina NextSeq platform to generate either 1x50bp or 1x75bp reads. Reads from each sequencing run were mapped using the STAR aligner, and output BAMs were merged into a single BAM for each sample. The merged BAM was further analyzed in R using the GenomicAlignments package to quantify number of reads per transcript and DESeq2 to determine differential expression. Reads per kilobase transcript per million total reads (RPKM) was calculated by dividing reads per transcript by the transcript length and then normalizing to the total number of reads in the sample. Results: Following this pipeline, we were able to identify a number of HBV-mediated differentially expressed transcripts at 24h and 48h post-infection. Further pathway analysis of these differentially expressed transcripts identified many important cellular pathways, including those involved with cell cycle regulation and metabolism, as being differentially regulated by HBV in primary hepatocytes. mRNA profiles of HBV-expressing and non-expressing primary rat hepatocytes were generated, in triplicate, 24h and 48h post-infection using Illumina HiSeq 2500 and NextSeq 500 instruments.

Project description:Purpose: Chronic infection with hepatitis B virus is the leading global risk factor for the development of liver cancer. A large body of research has shown the many effects an HBV infection has on cellular physiology, particularly on pathways that may be involved in the development of HBV-associated diseases. Unfortunately, a significant portion of this research has been done in systems that may not mimic what is seen in a primary hepatocyte, and is not done on a transcriptome-wide scale. Because of this, we performed an RNA-seq analysis of primary rat hepatocytes either expressing HBV or not over a series of time points to determine the global changes HBV has on primary hepatocyte physiology. Methods: To do this RNA-seq analysis, triplicate samples of total RNA were collected from cultured primary rat hepatocytes (PRH) over the course of 72hr. PRH were collected immediately after isolation (0hr), or 24hr, 48hr, or 72hr after plating. In addition, PRH were infected 24hr after plating with adenovirus expressing GFP alone (AdGFP) or GFP along with a greater than unit length copy of the HBV genome (AdHBV) and collected at 48hr after plating (24hr after infection) or 72hr after plating (48hr after infection). cDNA libraries were sequenced using the Illumina NextSeq 500 platform to generate either 1x75bp reads. Reads were mapped using the STAR aligner, and output BAMs were further analyzed in R using the GenomicAlignments package, to quantify number of reads per transcript, and DESeq2, to determine differential expression. Reads per kilobase transcript per million total reads (RPKM) was calculated by dividing reads per transcript by the transcript length and then normalizing to the total number of reads in the sample. Results: Following this pipeline, we were able to identify a number of HBV-mediated differentially expressed transcripts at 48hr and 72hr. In addition, we noted considerable change to the hepatocyte transcriptome as a direct result of the isolation/plating procedure, regardless of the presence of HBV. Further pathway analysis of these differentially expressed transcripts identified many important cellular pathways, including those involved with cell cycle regulation and metabolism, as being differentially regulated by HBV in primary hepatocytes. mRNA profiles of cultured primary rat hepatocytes were generated, in triplicate, using the Illumina NextSeq 500 platform from freshly isolated cells (0hr), 24hr, 48hr, or 72hr after plating, and with or without expression of HBV 48hr or 72hr after plating.

Project description:Oct4 is an essential regulator of embryonic stem (ES) cell pluripotency in vivo and in vitro, as well as a key mediator of the reprogramming of somatic cells to form induced pluriopotent stem (iPS) cells. It is not known whether activation and/or repression of specific genes by Oct4 is relevant to these functions. Here we show that fusion proteins containing the coding sequence of Oct4 or Xlpou91 (the Xenopus homologue of Oct4) fused to activating regions, but not those fused to repressing regions, behave as Oct4, suppressing differentiation and promoting maintenance of undifferentiated phenotypes in vivo and in vitro. An Oct4 activation domain fusion supported ES cell self-renewal in vitro at lower concentrations than required for Oct4 while alleviating the ordinary requirement for the cytokine LIF. At still lower levels of the fusion, LIF-dependence was restored. We conclude that the necessary and sufficient function of Oct4 in promoting pluripotency is to activate specific target genes. Two independent clones from each cell line were used as biological replications. Cy3-CTP-labelled sample targets were prepared from 2.5 ?g of total RNA using the Low RNA Input Fluorescent Linear Amplification Kit (Agilent). Cy5-CTP-labelled reference target was produced from 2.5 ?g of Stratagene Universal Mouse Reference RNA. Purified target RNA were hybridized to the NIA Mouse 44K Microarray v3.0 (whole genome 60-mer oligo arrays, Agilent Technology, design ID 015087) (Carter et al., 2005) according to the manufacturer's protocol (Two-Colour Microarray-Based Gene Expression Analysis Protocol, Version 5.0.1). Slides were scanned with an Agilent DNA Microarray Scanner (model G2505-64120) at 100% PMT for the Cy3 channel and 10% PMT for the Cy5 channel with a scan resolution of 5µm. Data was analyzed using NIA Array Analysis (Sharov et al., 2005) using standard statistical conditions (FDR<0.05, 2-fold expression levels change) to unveil genes with changes in expression levels between the samples and controls.

Project description:Aims/hypothesis: Duct cells isolated from adult human pancreas can be reprogrammed to express islet beta cell genes by adenoviral transduction of the developmental transcription factor neurogenin3 (Ngn3). In this study we aimed to fully characterize the extent of this reprogramming and intended to improve it. Methods: The extent of the Ngn3-mediated duct-to-endocrine cell reprogramming was measured employing genome wide mRNA profiling. By modulation of the Delta-Notch signaling or addition of pancreatic endocrine transcription factors Myt1, MafA and Pdx1 we intended to improve the reprogramming. Results: Ngn3 stimulates duct cells to express a focused set of genes that are abundant in islet endocrine cells and/or neural tissues. This neuro-endocrine shift, however, covers a minor fraction (5%) of the estimated genome-wide transcriptome difference between duct and islet endocrine cells. Interestingly, transduction of exogenous Ngn3 activates endogenous Ngn3 suggesting auto-activation of this gene. Furthermore, pancreatic endocrine reprogramming of human duct cells can be moderately enhanced by inhibition of Delta-Notch signaling as well as by co-expressing the transcription factor Myt1, but not MafA and Pdx1. Conclusions/interpretation: The results provide further insight into the plasticity of adult human duct cells and suggest measurable routes to enhance Ngn3-mediated in vitro reprogramming protocols for regenerative beta cell therapy in diabetes. We used Affymetrix HG133A and HG133B to get a comprehensive view on the reprogramming potential in vitro of human pancreatic duct cell cultures (n=3-4) at 3 and 14/20 days after ectopic adenoviral expression of murine neurogenin 3 as compared to GFP-expressing control vectors. The microarray analysis was performed on 3 independent samples that each contained RNA extracted from a pool of 3 independent donor pancreata. The total number of non-selected donor organs is 9. Transcripts were considered as differentially regulated by Ngn3 when 1.5 fold (LCB, unpaired P < 0.05) up- or down-regulated in AdGFP-Ngn3 versus AdGFP controls, at 3 and/or 14 dpi. Transcripts that showed differential expression between day 3 and day 14 in AdGFP-Ngn3 duct cells but not in AdGFP control cells, were also considered Ngn3-regulated

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Hepatitis B virus-mediated alterations to the primary hepatocyte transcriptome

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