Project description:Genetic variations in drug metabolising enzymes play a role in how individuals respond to drugs. Pharmacogene variation data in the Ghanaian population is limited and this study looks at exploring common variations that exist in our population for commonly used drugs. In addition, the study also looks at the how variations in cytokines and HLA play a role in HBV pathogenesis. Samples were validated with PCR-RFLP for accuracy
Project description:Genetic variations in drug metabolising enzymes play a role in how individuals respond to drugs. Pharmacogene variation data in the Ghanaian population is limited and this study looks at exploring common variations that exist in our population for commonly used drugs. Samples were validated with PCR-RFLP for accuracy
Project description:We analyzed three clinical parameters with gene expression data from 122 liver tissues. Six healthy samples were used in validation. All hepatitis samples were HBV infected, which was validated by positive HBsAg or serum HBV-DNA. The samples with HCV infection or metabolic liver injury (e.g. fatty liver, chronic alcoholic hepatitis, etc.) were excluded. This dataset is part of the TransQST collection.
Project description:This study aimed to better characterize the repertoire of serum HBV RNAs during chronic HBV infection in humans, which remains understudied. Using RT-PCR, qPCR, RNA-sequencing and immuno-precipitation, we found that (i) >50% of serum samples bore different amounts of HBV replication-derived RNAs (rd-RNAs); (ii) a few samples contained RNAs transcribed from integrated HBV DNA including 5'-HBV-human-3' RNAs (integrant-derived RNAs or id-RNAs) and 5'-human-HBV-3' transcripts as a minority of serum HBV RNAs; (iii) spliced HBV RNAs were abundant in <50% of analyzed samples; (iv) most serum rd-RNAs were polyadenylated via conventional HBV polyadenylation signal; (v) pre-genomic RNA (pgRNA) was the major component of the pool of serum RNAs; (vi) area of HBV positions 1531-1739 had very high RNA reads coverage and thus should be used as a target for detecting serum HBV RNAs; (vii) vast majority of rd-RNAs and pgRNA were associated with HBV virions, but not with unenveloped capsids, exosomes, classic microvesicles or apoptotic vesicles and bodies; (viii) considerable rd-RNAs presence in the circulating immune complexes was found in a few samples; and (ix) serum rcDNA and rd-RNAs should be quantified simultaneously to evaluate HBV replication status and efficacy of anti-HBV therapy with nucleos(t)ide analogs. In summary, sera contain various HBV RNA types of different origin, which are likely secreted via different mechanisms. In addition, since we previously showed that id-RNAs were abundant or predominant HBV RNAs in many of liver and hepatocellular carcinoma tissues comparing to rd-RNAs, there is likely a mechanism favoring the egress of the replication-derived RNAs.
Project description:Human primary hepatocytes isolated from chimeric mice were infected with HBV for 7 days. The comprehensive changes of miRNA levels were determined by miRNA array. MicroRNA expression levels were compared in between control and HBV-replicating primary hepatocytes by 2K microRNA microarrays
Project description:Several classes of capsid assembly modulators (CAMs) are currently being developed for chronic hepatitis B (CHB) cure. Both class A (CAM-A) and class E (CAM-E) CAMs disrupt nucleocapsid assembly and reduce extracellular hepatitis B virus (HBV) DNA. However, only CAM-As have been shown to reduce the number of HBV-infected cells in the animal models. However, there has been limited efficacy to date of CAM-A molecules achieving this secondary mechanism of HBV-infected cell clearance in CHB clinical trials. To investigate this disconnect, we performed comparative experiments with tool compounds from each class to further explore these unique features and antiviral activity of CAM-A across HBV-infected primary human hepatocytes (PHH), as well as in two different HBV mouse models (immunodeficient mice repopulated with human hepatocytes and AAV-HBV). Mechanistic studies in HBV-infected PHH revealed that CAM-A, but not CAM-E, induced dose-dependent aggregation of HBV core protein (HBc) in the nucleus. Experiments with siRNA, resulted in identification of the ubiquitin-binding protein p62 as a factor negatively regulating the size of these aggregates. Furthermore, we found that only the CAM-A is able to induce HBc-positive cell death in vivo with the loss of HBV-infected cells positively correlated to the levels of intrahepatic HBc. Profiling of intrahepatic HBc levels across CHB patient liver biopsies demonstrated a significantly lower level of HBc per hepatocyte than either of the HBV mouse models. Taken together, these data demonstrate that CAMs of class A have a unique secondary mechanism that has potential to specifically affect viability of HBV-infected hepatocytes. At the same time, the clearance of infected hepatocytes may depend on the level of HBc expression thereby limiting the therapeutic potential for this class of molecules.
Project description:Monitor HBV mRNA reduction in response to Tazarotene and to identify global transcriptome-wide gene expression associated with Tazarotene treatment in HBV infected PHH using RNA-Seq
Project description:Cell: HBV-infected PHH cells. Methods: PHH cells were infected with 2000 genome equivalents/cell of HBV particles in the presence of 4% PEG8000. Seven days after HBV infection, ChIP-Seq analysis of cccDNA in HBV-infected PHHs was carried out. HBV-infected PHH cells were digested with micrococcal nuclease and resulting mononucleosomes purified by sucrose gradient centrifugation. Nucleosomes were enriched with anti-H3K79succ antibodies by ChIP assay and the associated DNA contained both human and HBV DNA fragments was analyzed by deep sequencing. The HBV-specific reads in ChIP-Seq pilot experiments was quantified and normalized by the reads mapped to the human genome.