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Hepatitis B virus (HBV) infection is a major health burden worldwide, and currently there is no cure. The persistence of HBV covalently closed circular DNA (cccDNA) is the major obstacle for antiviral trement. HBV core protein (HBc) has emerged as a promising antiviral target, as it plays important roles in critical steps of the viral life cycle. However, whether HBc could regulate HBV cccDNA transcription remains under debate. In this study, different approaches were used to address this question. In synthesized HBV cccDNA and HBVcircle transfection assays, lack of HBc showed no effect on transcription of HBV RNA as well as HBV surface antigen (HBsAg) production in a hepatoma cell line and primary human hepatocytes. Reconstitution of HBc did not alter the expression of cccDNA-derived HBV markers. Similar results were obtained from an in vivo mouse model harboring cccDNA. Chromatin immunoprecipitation (ChIP) or ChIP sequencing assays revealed transcription regulation of HBc-deficient cccDNA chromatin similar to that of wild-type cccDNA. Furthermore, treatment with capsid assembly modulators (CAMs) dramatically reduced extracellular HBV DNA but could not alter viral RNA and HBsAg. Our results demonstrate that HBc neither affects histone modifications and transcription factor binding of cccDNA nor directly influences cccDNA transcription. Although CAMs could reduce HBc binding to cccDNA, they do not suppress cccDNA transcriptional activity. Thus, therapeutics targeting capsid or HBc should not be expected to sufficiently reduce cccDNA transcription. IMPORTANCE Hepatitis B virus (HBV) core protein (HBc) has emerged as a promising antiviral target. However, whether HBc can regulate HBV covalently closed circular DNA (cccDNA) transcription remains elusive. This study illustrated that HBc has no effect on epigenetic regulation of cccDNA, and it does not participate in cccDNA transcription. Given that HBc is dispensable for cccDNA transcription, novel cccDNA-targeting therapeutics are needed for an HBV cure.

Murine hepatic cells cannot support hepatitis B virus (HBV) infection even with supplemental expression of viral receptor, human sodium taurocholate cotransporting polypeptide (hNTCP). However, the specific restricted step remains elusive. In this study, we aimed to dissect HBV infection process in murine hepatic cells.Cells expressing hNTCP were inoculated with HBV or hepatitis delta virus (HDV). HBV pregenomic RNA (pgRNA), covalently closed circular DNA (cccDNA), and different relaxed circular DNA (rcDNA) intermediates were produced in vitro. The repair process from rcDNA to cccDNA was assayed by in vitro repair experiments and in mouse with hydrodynamic injection. Southern blotting and in situ hybridization were used to detect HBV DNA. HBV, but not its satellite virus HDV, was restricted from productive infection in murine hepatic cells expressing hNTCP. Transfection of HBV pgRNA could establish HBV replication in human, but not in murine, hepatic cells. HBV replication-competent plasmid, cccDNA, and recombinant cccDNA could support HBV transcription in murine hepatic cells. Different rcDNA intermediates could be repaired to form cccDNA both in vitro and in vivo. In addition, rcDNA could be detected in the nucleus of murine hepatic cells, but cccDNA could not be formed. Interestingly, nuclease sensitivity assay showed that the protein-linked rcDNA isolated from cytoplasm was completely nuclease resistant in murine, but not in human, hepatic cells.Our results imply that the disassembly of cytoplasmic HBV nucleocapsids is restricted in murine hepatic cells. Overcoming this limitation may help to establish an HBV infection mouse model.

Different organs undergo distinct transcriptional, epigenetic and physiological alterations that guarantee their functional maturation after birth. However, the roles of epitranscriptomic machineries in these processes remain elusive. Here, we show that RNA methyltransferase enzymes Mettl3 and Mettl14 highly express in murine hepatocytes at embryonic and neonatal stages, and gradually decline during the postnatal development. Liver-specific Mettl3 deficiency causes hepatocyte hypertrophy, liver injury and growth retardation. Transcriptomic and N6-methyl-adenosine (m6A) profiling identify the neutral sphingomyelinase, Smpd3, as a target of Mettl3. Decreased decay of Smpd3 transcripts due to Mettl3 deficiency results in sphingolipid metabolism rewiring, characterized by toxic ceramide accumulation and excessive sphingomyelin hydrolysis, leading to mitochondrial damage and elevated ER stress. Pharmacological inhibition of Smpd3 ameliorates the abnormality of Mettl3-deficent liver. Our findings demonstrate that Mettl3/m6A fine-tunes sphingolipid metabolism, highlighting the pivotal role of an epitranscriptomic machinery in coordinating metabolic homeostasis and functional maturation during postnatal liver development.

Hepatitis B virus (HBV) infection is prevalent throughout the world, with approximately 300 million people chronically infected. HBV infection can cause acute and chronic liver hepatitis. Long-term chronic infection can increase the incidence of liver cirrhosis and hepatocellular carcinoma (HCC). At present, there are two types of approved drugs for chronic HBV infection: nucleos(t)ide analogs (NAs) and interferon α (IFNα), currently pegylated IFNα (PegIFNα). Although these drugs can inhibit viral replication and reduce the risk of liver cirrhosis and HCC, the limitations are obvious: HBV surface antigen (HBsAg) and covalently closed circular DNA (cccDNA) pool cannot be efficiently eliminated. To overcome these limitations, new drugs are required urgently to be designed and developed. In recent years, with the deepening of research on HBV, drugs targeting different targets of viral complex replication mechanisms and interactions with the host immune system have been developed. Drugs targeting viral complex replication mechanisms include HBV entry inhibitors, cccDNA formation and transcription inhibitors, gene editing targeting HBV DNA, HBV specific siRNAs, capsid inhibitors, polymerase inhibitors and HBV secretion inhibitors. Entry inhibitors have been used successfully in treating viral infections. Myrcludex B, a synthetic N-myristoylated lipopeptide derived from HBV preS1 protein, competes with the virus for binding to the solute carrier family 10 member 1 (SLC10A1 or NTCP). This agent prevents HBV and Hepatitis D virus (HDV) infection and in cells and animal models. cccDNA is the transcriptional template of HBV RNAs and is an ideal target for clearing HBV. DNA ligase inhibitors, which can inhibit cccDNA formation and are being developed as anticancer agents, may be tested for treatment of chronic HBV infection, if the side effects are tolerable. The genome editing tool, clustered regularly interspaced short palindromic repeats associated nuclease 9 (CRISPR/Cas9), has many innovative applications in different fields because of its potential for gene therapy. These have provided a clear proof of concept that this approach has the potential to treat or even cure patients with chronic HBV infection. RNA interference technology can be used to manipulate virus gene expression and may be developed for HBV treatment. HBV nucleocapsid formation and pgRNA packaging are critical steps of viral life cycle that may be targeted by antiviral agents. Inside the viral capsid, pgRNA is reverse transcribed into minus-strand DNA by the viral polymerase, with its terminal protein domain acting as a primer for initiation of reverse transcription. HBV polymerase is also a good drug target. Drugs targeting the host immune system include that stimulating the innate immune system, like IFNα, Toll-like receptor (TLR) agonists and stimulating cytoplasmic nucleic acid sensors, and that activating an adaptive immune response, like checkpoint inhibitors, T-cell redirection and therapeutic vaccines. IFN-based therapies are considered as immunomodulatory strategies. TLRs are expressed either at the cell surface or on endosome membranes, where they recognize molecular patterns associated with invading pathogens. Stimulation of TLR1, TLR2, TLR3, and TLR4 on hepatocytes has direct antiviral effects against HBV. The RNA helicases DExD/H-box helicase 58 (DDX58, also called RIG-I) and IFN induced with helicase C domain 1 (IFIH1, also called MDA5) are RNA sensors in the cytoplasm that induce production of cytokines by activating IRF3 and NF-kB. Activation of RIG-I induces an antiviral response against HBV. PD-1 is the inhibitory receptor expressed by most HBV specific T cells, so agents are in development to disrupt the interaction between PD-1 and its ligand, PD-L1. A therapeutic vaccine against HBV needs to overcome mechanisms of HBV-specific immune tolerance. Because natural resolution of HBV infection is accompanied by lifelong T-cell- and antibody-mediated immunity, these vaccines should induce HBV-specific B- and T-cell immune responses. Here, we review recent advances in development of directing-acting antiviral (DAA), host-targeting agents and immunomodulatory therapies. Some of which have entered clinical trials. We also discuss strategies for unbiased high-throughput screens to identify compounds that inhibit HBV and for repurposing existing drugs.

BACKGROUND & AIMS: Our understanding about the interaction of the Hepatitis B virus (HBV) with its host cells is still quite limited. Spliceosome associated factor 1 (SART1) has recently been found to restrict Hepatitis C Virus. We aim to dissect its role in HBV infection that remains a big threat to global health. METHODS: SART1 was knocked down by RNA interference and over-expressed by lentiviral or adeno-associated virus (AAV) vectors in HBV infected cell cultures and in vivo in HBV replicating mice evaluating HBV replication markers. Luciferase reporter assays were used to determine viral or host factor promoter activities, and chromatin immunoprecipitation (ChIP) to investigate protein-DNA interactions. RESULTS: In HBV infected cell cultures, downregulation of SART1 did not affect HBV cccDNA but resulted in markedly enhancing HBV RNA, antigen expression and progeny virus production. On the other hand, HBV transcription and replication were significantly inhibited by overexpression of SART1. Similar results were observed in AAV-HBV infected mice persistently replicating HBV. Inhibition of Janus kinases showed no effect SART1-mediated inhibition of HBV replication. HBV promoter assays revealed that SART1 reduced HBV core promoter activity. By screening known HBV transcription factors, we found that SART1 specifically suppressed the expression of hepatocyte nuclear factor 4α (HNF4α). Luciferase reporter and ChIP assays demonstrated a direct downregulation of HNF4α expression by association of SART1 with HNF4α proximal P1 promoter element. CONCLUSIONS: We identify SART1 as a novel host factor suppressing HBV cccDNA transcription. Besides its effect on interferon stimulated genes, SART1 exerts an anti-HBV activity by suppressing HNF4α expression that is essential for transcription of HBV cccDNA. LAY SUMMARY: Hepatitis B virus (HBV) infects hepatocytes and establishes a covalently closed circular DNA (cccDNA), which remains a major obstacle for antiviral treatments. Spliceosome associated factor 1 (SART1) has been described to inhibit hepatitis C virus infection through direct regulation of interferon stimulated genes. In this study, by using various HBV models, we demonstrate that SART1 restrains HBV cccDNA transcription through suppression of HBV key transcription factor HNF4α.

Background. Researchers have demonstrated that GSDMB is highly expressed in cancer tissues and located in amplicons, genomic regions that are often amplified during cancer development. We evaluated the role of GSDMB in bladder cancer using publicly available data.Methods. The relationship between clinical correlation features and GSDMB were analyzed with the Wilcoxon rank sum test (2 groups), Kruskal-Wallis test (multiple groups) and logistic regression. Clinicopathologic characteristics associated with overall survival in TCGA patients using Cox regression and the Kaplan-Meier method. Gene Set Enrichment Analysis (GSEA) was performed using TCGA data set. Results. GSDMB was differentially expressed between the tumor group and the normal group and was highly expressed in the tumor group. Reduced GSDMB expression in BLCA as significantly associated with grade (OR = 4.55, High Grade vs. Low Grade), clinical stage (OR = 2.34, Ⅱvs. Ⅳ), status (OR = 0.51 survival vs. death), all p-values Conclusions. High GSDMB mRNA expression is an independent risk factor for excellent prognosis in bladder cancer.

Asymptomatic transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a potential problem for pandemic control through public health strategies. Our results demonstrate that asymptomatic COVID-19 patients have better outcomes than symptomatic patients. This may have been due to more active cellular immune responses and normal liver function. Since asymptomatic patients have no clinical symptoms which can easily prevent timely diagnosis and treatment, they may cause a greater risk of virus transmission than symptomatic patients, which poses a major challenge to infection control. Evidence suggests that nonpharmaceutical public health interventions, like social distancing and face mask ordinances, play important roles in the control of COVID-19. Looking forward, it may be necessary to proceed cautiously while reopening businesses in areas of epidemicity to prevent potential waves of COVID-19 in the future., Since the outbreak of coronavirus disease 2019 (COVID-19) in Wuhan, China, it has rapidly spread around the world. Persons with asymptomatic disease exhibit viral shedding, resulting in transmission, which presents disease control challenges. However, the clinical characteristics of these asymptomatic individuals remain elusive. We collected samples of 25 asymptomatic and 27 symptomatic COVID-19 patients. Viral titers of throat swabs were determined by quantitative reverse transcription-PCR (qRT-PCR). COVID-19 IgG and IgM were examined. Complete blood counts were determined, and serum biochemistry panels were performed. Cytokines, including gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin 2 (IL-2), IL-4, IL-6, and IL-10 were evaluated. T cell, B cell, and NK cell counts were measured using flow cytometry. Although similar viral loads were detected, asymptomatic patients had significantly faster virus turnover than symptomatic patients. Additionally, asymptomatic patients had higher counts of lymphocytes, T cells, B cells, and NK cells. While liver damage was observed in symptomatic patients, as indicated by elevated liver enzymes and decreased liver-synthesized proteins in the blood, asymptomatic patients showed normal liver measurements. Lactate dehydrogenase, a COVID-19 risk factor, was significantly lower in asymptomatic patients. These results suggest that asymptomatic COVID-19 patients had normal clinical indicators and faster viral clearance than symptomatic patients. Lymphocytes may play a role in their asymptomatic phenotype. Since asymptomatic patients may be a greater risk of virus transmission than symptomatic patients, public health interventions and a broader range of testing may be necessary for the control of COVID-19. IMPORTANCE Asymptomatic transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a potential problem for pandemic control through public health strategies. Our results demonstrate that asymptomatic COVID-19 patients have better outcomes than symptomatic patients. This may have been due to more active cellular immune responses and normal liver function. Since asymptomatic patients have no clinical symptoms which can easily prevent timely diagnosis and treatment, they may cause a greater risk of virus transmission than symptomatic patients, which poses a major challenge to infection control. Evidence suggests that nonpharmaceutical public health interventions, like social distancing and face mask ordinances, play important roles in the control of COVID-19. Looking forward, it may be necessary to proceed cautiously while reopening businesses in areas of epidemicity to prevent potential waves of COVID-19 in the future.

Bacterial species of Bacillus, Lactobacillus, and Bifidobacterium in the intestinal tract have been used as probiotics. Selections for probiotic candidates by the culture-based approaches are time-consuming and labor-consuming. The aim of this study was to develop a new method based on sequencing strategies to select the probiotic Bacillus, Lactobacillus, and Bifidobacterium. The Illumina-based sequencing strategies with different specific primers for Bacillus, Clostridium, and Bifidobacterium were applied to analyze diversity of the genera in goat feces. The average number of different Bacillus, Clostridium, and Bifidobacterium OTUs (operational taxonomic units) at the 97% similarity level ranged from 1922 to 63172. The coverage index values of Bacillus, Clostridium, and Bifidobacterium calculated from the bacterial OTUs were 0.89, 0.99, and 1.00, respectively. The most genera of Bacillus (37.9%), Clostridium (53%), and Bifidobacterium (99%) were detected in goat feces by the Illumina-based sequencing with the specific primers of the genera, respectively. Higher phylogenetic resolutions of the genera in goat feces were successfully established. The results suggest that the selection for probiotic Bacillus, Clostridium, and Bifidobacterium based on the Illumina sequencing with their specific primers is reliable and feasible, and the core Bacillus, Clostridium, and Bifidobacterium species of healthy goats possess the potentials as probiotic microbial consortia.

Metal-resistant endophytic fungi from roots improved phytoremediation efficacy of host plants; however, the effects of endophytic fungi from plant aerial parts on host plants are unknown. The aim of this study was to develop a feasible method to screen fungal endophytes from stems and roots of Brassica napus and to investigate effects of the endophytic fungi on growth and phytoremediation efficiency of the plant. Endophytic Fusarium sp. CBRF44, Penicillium sp. CBRF65, and Alternaria sp. CBSF68 with different traits were isolated from roots and stems of rapes grown in a metal-contaminated soil. Fusarium sp. CBRF44 (resistant to 5 mM Cd and 15 mM Pb, isolated from roots) and Alternaria sp. CBSF68 (resistant to 1 mM Cd and 10 mM Pb, isolated from stems) could produce indole-3-acetic acid (IAA) and siderophore; Penicillium sp. CBRF65 (tolerate 2 mM Cd and 20 mM Pb, isolated from roots) could not produce IAA and siderophore but showed the highest phosphate-solubilizing activities. Fusarium sp. CBRF44 and Penicillium sp. CBRF65 significantly increased the rape biomass and promoted the extraction efficacy of Pb and Cd, while Alternaria sp. CBSF68 did not show similar results. Penicillium sp. CBRF65 and Fusarium sp. CBRF44 could be frequently recovered from inoculated rape roots, while Alternaria sp. CBSF68 was scarcely recovered. The results indicate that the colonizing capacity of endophytic fungi in roots is important to improve phytoremediation efficacy of host plants.

Burgers’ equation $${w_t} = \in {w_{xx}} - w{w_x}$$ (1.1) was first introduced by J.M. Burgers about fifty years ago when he studied fluid flow, particularly, modeling turbulence. However, it was later found that the equation is a very useful mathematical model for such diverse physical problems as shock flows, traffic flow, acoustic transmission in fogs, etc., [8]. The dissipation may arise from viscosity, heat conduction, chemical reaction, mass diffusion, thermal radiation, etc. In a typical application, i.e., fluid dynamics, ω is the velocity of the wave and є is the viscosity of the compressible flow. However, one should understand that the parameters may have different physical meanings in different applications.