Novel Approaches to Inhibition of HBsAg Expression from cccDNA and Chromosomal Integrants: A Review.

Hepatitis B virus (HBV) is a widely prevalent liver infection that can cause acute or chronic hepatitis. Although current treatment modalities are highly effective in the suppression of viral levels, they cannot eliminate the virus or achieve definitive cure. This is a consequence of the complex nature of HBV-host interactions. Major challenges to achieving sustained viral suppression include the presence of a high viral burden from the HBV DNA and hepatitis B surface antigen (HBsAg), the presence of reservoirs for HBV replication and antigen production, and the HBV-impaired innate and adaptive immune response of the host. Those therapeutic methods include cell entry inhibitors, HBsAg inhibitors, gene editing approaches, immune-targeting therapies and direct inhibitors of covalently closed circular DNA (cccDNA). Novel approaches that target these key mechanisms are now being studied in preclinical and clinical phases. In this review article, we provide a comprehensive review on mechanisms by which HBV escapes elimination from current treatments, and highlight new agents to achieve a definitive HBV cure.

Targeted Mitochondrial Delivery to Hepatocytes: A Review.

Defects in mitochondria are responsible for various genetic and acquired diseases. Mitochondrial transplantation, a method that involves introduction of healthy donor mitochondria into cells with dysfunctional mitochondria, could offer a novel approach to treat such diseases. Some studies have demonstrated the therapeutic benefit of mitochondrial transplantation and targeted delivery in vivo and in vitro within hepatocytes and the liver. This review discusses the issues regarding isolation and delivery of mitochondria to hepatocytes and the liver, and examines the existing literature in order to elucidate the utility and practicality of mitochondrial transplantation in the treatment of liver disease. Studies reviewed demonstrate that mitochondrial uptake could specifically target hepatocytes, address the challenge of non-specific localization of donor mitochondria, and provide evidence of changes in liver function following injection of mitochondria into mouse and rat disease models. While potential benefits and advantages of mitochondrial transplantation are evident, more research is needed to determine the practicality of mitochondrial transplantation for the treatment of genetic and acquired liver diseases.

Uncertainty as a critical determinant of antibiotic prescribing in patients with an asthma exacerbation: a qualitative study.

OBJECTIVE: To identify factors that influence providers' decisions to prescribe antibiotics in patients presenting to the hospital with an asthma exacerbation. METHODS: We performed semi-structured interviews with a purposive sample of providers including sixteen hospitalists, emergency room providers, or pulmonologists, and one focus group with internal medicine residents recruited from one large, urban, teaching hospital and one small, rural, community hospital. Questions were informed by the Theoretical Domains Framework to determine factors that may influence behaviors. Directed content analysis was used to code and analyze transcripts of the interviews. RESULTS: Uncertainty regarding the diagnostic (asthma vs. COPD) and the cause of exacerbation (bacterial vs. viral infection) emerged as the main driver for prescribing behavior. Provider response to uncertainty included: "watchful waiting" or immediate antibiotic prescribing. The following factors played important roles in providers' prescribing decision: 1) awareness/agreement with existing guidelines 2) confidence in their ability to apply the guidelines in challenging cases; 3) perceived risk of patient deterioration without antibiotics; 4) fear of litigation; 5) habit and clinical inertia 6) prescribing within the group 7) lack of information of antibiotic prescribing rates and 8) lack of time and/or resources. CONCLUSIONS: We identified diagnostic uncertainty as the primary determinant of antibiotic prescribing in asthma exacerbations and developed a conceptual model to explain provider responses and factors that influenced their responses. These results enhance our understanding of the factors that can contribute to low-value and wasteful practices like superfluous antibiotic prescribing and will support the development of interventions to de-implement such practices.

Identification of splice regulators of fibronectin-EIIIA and EIIIB by direct measurement of exon usage in a flow-cytometry based CRISPR screen.

The extracellular matrix protein fibronectin (FN) is alternatively spliced in a variety of inflammatory conditions, resulting in increased inclusion of alternative exons EIIIA and EIIIB. Inclusion of these exons affects fibril formation, fibrosis, and inflammation. To define upstream regulators of alternative splicing in FN, we have developed an in vitro flow-cytometry based assay, using RNA-binding probes to determine alternative exon inclusion level in aortic endothelial cells. This approach allows us to detect exon inclusion in the primary transcripts themselves, rather than in surrogate splicing reporters. We validated this assay in cells with and without FN-EIIIA and -EIIIB expression. In a small-scale CRISPR KO screen of candidate regulatory splice factors, we successfully detected known regulators of EIIIA and EIIIB splicing, and detected several novel regulators. Finally, we show the potential in this approach to broadly interrogate upstream signaling pathways in aortic endothelial cells with a genome-wide CRISPR-KO screen, implicating the TNFalpha and RIG-I-like signaling pathways and genes involved in the regulation of fibrotic responses. Thus, we provide a novel means to screen the regulation of splicing of endogenous transcripts, and predict novel pathways in the regulation of FN-EIIIA inclusion.