Rapid and Reliable Protein-Free HBV DNA Extraction and Sensitive Branched DNA Southern Blot Assay.

HBV covalently closed circular DNA (cccDNA) plays an important role in the persistence of hepatitis B virus (HBV) infection by serving as the template for transcription of viral RNAs. To cure HBV infection, it is expected that cccDNA needs either to be eliminated or silenced. Hence, precise cccDNA quantification is essential.

Discovery of Hepatitis B Virus Surface Antigen Suppressor GS-8873.

Chronic hepatitis B (CHB) virus infection afflicts hundreds of millions of people and causes nearly one million deaths annually. The high levels of circulating viral surface antigen (HBsAg) that characterize CHB may lead to T-cell exhaustion, resulting in an impaired antiviral immune response in the host. Agents that suppress HBsAg could help invigorate immunity toward infected hepatocytes and facilitate a functional cure.

Parasympathetic neurons derived from human pluripotent stem cells model human diseases and development.

Autonomic parasympathetic neurons (parasymNs) control unconscious body responses, including "rest-and-digest." ParasymN innervation is important for organ development, and parasymN dysfunction is a hallmark of autonomic neuropathy. However, parasymN function and dysfunction in humans are vastly understudied due to the lack of a model system.

Characterization of a Novel Capsid Assembly Modulator for the Treatment of Chronic Hepatitis B Virus Infection.

The standard of care for the treatment of chronic hepatitis B (CHB) is typically lifelong treatment with nucleos(t)ide analogs (NAs), which suppress viral replication and provide long-term clinical benefits. However, infectious virus can still be detected in patients who are virally suppressed on NA therapy, which may contribute to the failure of these agents to cure most CHB patients. Accordingly, new antiviral treatment options are being developed to enhance the suppression of hepatitis B virus (HBV) replication in combination with NAs ("antiviral intensification").

Characterization of a KDM5 small molecule inhibitor with antiviral activity against hepatitis B virus.

Chronic hepatitis B (CHB) is a global health care challenge and a major cause of liver disease. To find new therapeutic avenues with a potential to functionally cure chronic Hepatitis B virus (HBV) infection, we performed a focused screen of epigenetic modifiers to identify potential inhibitors of replication or gene expression. From this work we identified isonicotinic acid inhibitors of the histone lysine demethylase 5 (KDM5) with potent anti-HBV activity.

A roadmap for serum biomarkers for hepatitis B virus: current status and future outlook.

Globally, 296 million people are infected with hepatitis B virus (HBV), and approximately one million people die annually from HBV-related causes, including liver cancer. Although there is a preventative vaccine and antiviral therapies suppressing HBV replication, there is no cure. Intensive efforts are under way to develop curative HBV therapies.

Targeted Long-Read Sequencing Reveals Comprehensive Architecture, Burden, and Transcriptional Signatures from Hepatitis B Virus-Associated Integrations and Translocations in Hepatocellular Carcinoma Cell Lines.

Hepatitis B virus (HBV) can integrate into the chromosomes of infected hepatocytes, creating potentially oncogenic lesions that can lead to hepatocellular carcinoma (HCC). However, our current understanding of integrated HBV DNA architecture, burden, and transcriptional activity is incomplete due to technical limitations. A combination of genomics approaches was used to describe HBV integrations and corresponding transcriptional signatures in three HCC cell lines: huH-1, PLC/PRF/5, and Hep3B.

Characterization of Hepatitis B Precore/Core-Related Antigens.

Current therapies rarely cure chronic hepatitis B virus (HBV) infection due to the persistence of the viral episome, the covalently closed circular DNA (cccDNA), in hepatocytes. The hepatitis B virus core-related antigen (HBcrAg), a mixture of the viral precore/core gene products, has emerged as one potential marker to monitor the levels and activities of intrahepatic cccDNA. In this study, a comprehensive characterization of precore/core gene products revealed that HBcrAg components included the classical hepatitis B virus core antigen (HBc) and e antigen (HBeAg) and, additionally, the precore-related antigen, PreC, retaining the N-terminal signal peptide.

Serum HBsAg clearance has minimal impact on CD8+ T cell responses in mouse models of HBV infection.

Antibody-mediated clearance of hepatitis B surface antigen (HBsAg) from the circulation of chronically infected patients (i.e., seroconversion) is usually associated with increased HBV-specific T cell responsiveness.

Discovery of GS-9688 (Selgantolimod) as a Potent and Selective Oral Toll-Like Receptor 8 Agonist for the Treatment of Chronic Hepatitis B.

Toll-like receptor 8 (TLR8) recognizes pathogen-derived single-stranded RNA fragments to trigger innate and adaptive immune responses. Chronic hepatitis B (CHB) is associated with a dysfunctional immune response, and therefore a selective TLR8 agonist may be an effective treatment option. Structure-based optimization of a dual TLR7/8 agonist led to the identification of the selective TLR8 clinical candidate ()-2-((2-amino-7-fluoropyrido[3,2-]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (GS-9688, ()-).

Toll-Like Receptor 8 Agonist GS-9688 Induces Sustained Efficacy in the Woodchuck Model of Chronic Hepatitis B.

Background And Aims: GS-9688 (selgantolimod) is an oral selective small molecule agonist of toll-like receptor 8 in clinical development for the treatment of chronic hepatitis B. In this study, we evaluated the antiviral efficacy of GS-9688 in woodchucks chronically infected with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to hepatitis B virus.

Approach And Results: WHV-infected woodchucks received eight weekly oral doses of vehicle, 1 mg/kg GS-9688, or 3 mg/kg GS-9688.

Woolly Monkey-HBV Infection in Squirrel Monkeys as a Surrogate Nonhuman Primate Model of HBV Infection.

Development of curative therapies for chronic hepatitis B virus (HBV) infection will likely require new animal models. Here, we evaluate HBV infection in squirrel monkeys based on the high-sequence homology of the HBV receptor, Na+/taurocholate co-transporting peptide (NTCP), between humans and squirrel monkeys. HBV PreS1 peptide was examined for binding human and squirrel monkey NTCP.

Microwave measurement of giant unilamellar vesicles in aqueous solution.

A microwave technique is demonstrated to measure floating giant unilamellar vesicle (GUV) membranes in a 25 μm wide and 18.8 μm high microfluidic channel. The measurement is conducted at 2.

Toll-like receptor 7 agonist GS-9620 induces prolonged inhibition of HBV via a type I interferon-dependent mechanism.

Background & Aims: GS-9620, an oral agonist of toll-like receptor 7 (TLR7), is in clinical development for the treatment of chronic hepatitis B (CHB). GS-9620 was previously shown to induce prolonged suppression of serum viral DNA and antigens in the woodchuck and chimpanzee models of CHB. Herein, we investigated the molecular mechanisms that contribute to the antiviral response to GS-9620 using in vitro models of hepatitis B virus (HBV) infection.

Anti-HBV response to toll-like receptor 7 agonist GS-9620 is associated with intrahepatic aggregates of T cells and B cells.

Background & Aims: GS-9620, an oral agonist of toll-like receptor 7, is in clinical development for the treatment of chronic hepatitis B (CHB). GS-9620 was previously shown to induce prolonged suppression of serum viral DNA and antigens in the chimpanzee and woodchuck models of CHB. Herein, we investigated the immunomodulatory mechanisms underlying these antiviral effects.

Development of a digital droplet PCR assay to measure HBV DNA in patients receiving long-term TDF treatment.

The COBAS TaqMan assay has a lower limit of quantification (LLOQ) of 169 HBV copies/mL and a lower limit of detection (LLOD) of 58 copies/mL. HBV DNA below the TaqMan LLOQ is classified as target not detected (TND) (<58 copies/mL) or target detected (TD) (between 58 and 169 copies/mL). Here we have developed a more sensitive digital droplet PCR (ddPCR) assay to evaluate the impact of long-term tenofovir disoproxil fumarate (TDF) treatment in patients that did or did not achieve HBsAg seroconversion.

Capsid Phosphorylation State and Hepadnavirus Virion Secretion.

The C-terminal domain (CTD) of hepadnavirus core protein is involved in multiple steps of viral replication. In particular, the CTD is initially phosphorylated at multiple sites to facilitate viral RNA packaging into immature nucleocapsids (NCs) and the early stage of viral DNA synthesis. For the avian hepadnavirus duck hepatitis B virus (DHBV), CTD is dephosphorylated subsequently to facilitate the late stage of viral DNA synthesis and to stabilize NCs containing mature viral DNA.

Antiviral response and resistance analysis of treatment-naïve HCV infected patients receiving multiple doses of the NS3 protease inhibitor GS-9256.

The NS3 protease inhibitor (PI) GS-9256 has demonstrated antiviral activity in a monotherapy study and in combination with other DAAs for treatment of chronic hepatitis C virus (HCV) infection. The resistance profile of GS-9256 was investigated in a phase 1 monotherapy study of patients with HCV genotype (GT) 1 infection. No PI resistance associated substitutions (RASs) at positions 36, 155, 156, 168 and 170 were observed at baseline by population sequencing (15% cutoff) in the 54 patients enrolled in the study, however the PI RAS Q80K were detected in 41% of patients at baseline.

Preclinical characterization of the novel HCV NS3 protease inhibitor GS-9256.

Background: GS-9256 is an inhibitor of HCV NS3 protease with a macrocyclic structure and novel phosphinic acid pharmacophore.

Methods: Key preclinical properties of GS-9256 including in vitro antiviral activity, cross-resistance and pharmacokinetic properties were investigated in non-human species.

Results: In genotype (GT) 1b Huh-luc cells with a replicon encoding luciferase, GS-9256 had a mean 50% effective concentration (EC) value of 20.

The Smc5/6 Complex Restricts HBV when Localized to ND10 without Inducing an Innate Immune Response and Is Counteracted by the HBV X Protein Shortly after Infection.

The structural maintenance of chromosome 5/6 complex (Smc5/6) is a restriction factor that represses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing HBV X protein (HBx), which targets Smc5/6 for degradation. However, the mechanism by which Smc5/6 suppresses HBV transcription and how HBx is initially expressed is not known.

Coprescribing naloxone for patients on chronic opioid therapy: Les-sons learned from a patient-safety initiative in primary care training sites.

Objective: To describe the development and implementation of a resident-led effort to increase coprescription of naloxone in a primary care setting.

Design: An exploratory, prospective pilot project to increase coprescription rates of naloxone.

Setting: Four primary care offices in western Connecticut serving as medical home training sites for primary care residents.

Cell-Free Hepatitis B Virus Capsid Assembly Dependent on the Core Protein C-Terminal Domain and Regulated by Phosphorylation.

Unlabelled: Multiple subunits of the hepatitis B virus (HBV) core protein (HBc) assemble into an icosahedral capsid that packages the viral pregenomic RNA (pgRNA). The N-terminal domain (NTD) of HBc is sufficient for capsid assembly, in the absence of pgRNA or any other viral or host factors, under conditions of high HBc and/or salt concentrations. The C-terminal domain (CTD) is deemed dispensable for capsid assembly although it is essential for pgRNA packaging.

In Vitro Antiviral Activity and Resistance Profile Characterization of the Hepatitis C Virus NS5A Inhibitor Ledipasvir.

Ledipasvir (LDV; GS-5885), a component of Harvoni (a fixed-dose combination of LDV with sofosbuvir [SOF]), is approved to treat chronic hepatitis C virus (HCV) infection. Here, we report key preclinical antiviral properties of LDV, including in vitro potency, in vitro resistance profile, and activity in combination with other anti-HCV agents. LDV has picomolar antiviral activity against genotype 1a and genotype 1b replicons with 50% effective concentration (EC50) values of 0.

Subwavelength grating based metal-oxide nano-hair structures for optical vortex generation.

An all-dielectric, subwavelength grated based metal-oxide nano-hair structure for optical vortex beam generation has been presented in the paper. The nano-hair structure fabricated with alternating layers of alumina/hafnia on a fused silica substrate has a high diffraction efficiency of ~90% around the design wavelength, λ(o) = 1.55 μm and is insensitive to the polarization of the incident optical beam.