Enabling high-throughput enzyme discovery and engineering with a low-cost, robot-assisted pipeline.

As genomic databases expand and artificial intelligence tools advance, there is a growing demand for efficient characterization of large numbers of proteins. To this end, here we describe a generalizable pipeline for high-throughput protein purification using small-scale expression in E. coli and an affordable liquid-handling robot.

Simultaneous enhancement of multiple functional properties using evolution-informed protein design.

A major challenge in protein design is to augment existing functional proteins with multiple property enhancements. Altering several properties likely necessitates numerous primary sequence changes, and novel methods are needed to accurately predict combinations of mutations that maintain or enhance function. Models of sequence co-variation (e.

Simultaneous enhancement of multiple functional properties using evolution-informed protein design.

Designing optimized proteins is important for a range of practical applications. Protein design is a rapidly developing field that would benefit from approaches that enable many changes in the amino acid primary sequence, rather than a small number of mutations, while maintaining structure and enhancing function. Homologous protein sequences contain extensive information about various protein properties and activities that have emerged over billions of years of evolution.

Programmable antivirals targeting critical conserved viral RNA secondary structures from influenza A virus and SARS-CoV-2.

Influenza A virus's (IAV's) frequent genetic changes challenge vaccine strategies and engender resistance to current drugs. We sought to identify conserved and essential RNA secondary structures within IAV's genome that are predicted to have greater constraints on mutation in response to therapeutic targeting. We identified and genetically validated an RNA structure (packaging stem-loop 2 (PSL2)) that mediates in vitro packaging and in vivo disease and is conserved across all known IAV isolates.

Signatures of immune dysfunction in HIV and HCV infection share features with chronic inflammation in aging and persist after viral reduction or elimination.

Chronic inflammation is thought to be a major cause of morbidity and mortality in aging, but whether similar mechanisms underlie dysfunction in infection-associated chronic inflammation is unclear. Here, we profiled the immune proteome, and cellular composition and signaling states in a cohort of aging individuals versus a set of HIV patients on long-term antiretroviral therapy therapy or hepatitis C virus (HCV) patients before and after sofosbuvir treatment. We found shared alterations in aging-associated and infection-associated chronic inflammation including T cell memory inflation, up-regulation of intracellular signaling pathways of inflammation, and diminished sensitivity to cytokines in lymphocytes and myeloid cells.

Upregulation of CD47 Is a Host Checkpoint Response to Pathogen Recognition.

It is well understood that the adaptive immune response to infectious agents includes a modulating suppressive component as well as an activating component. We now show that the very early innate response also has an immunosuppressive component. Infected cells upregulate the CD47 "don't eat me" signal, which slows the phagocytic uptake of dying and viable cells as well as downstream antigen-presenting cell (APC) functions.

A functional subset of CD8 T cells during chronic exhaustion is defined by SIRPα expression.

Prolonged exposure of CD8 T cells to antigenic stimulation, as in chronic viral infections, leads to a state of diminished function termed exhaustion. We now demonstrate that even during exhaustion there is a subset of functional CD8 T cells defined by surface expression of SIRPα, a protein not previously reported on lymphocytes. On SIRPα CD8 T cells, expression of co-inhibitory receptors is counterbalanced by expression of co-stimulatory receptors and it is only SIRPα cells that actively proliferate, transcribe IFNγ and show cytolytic activity.

The balance of type 1 and type 2 immune responses in the contexts of hepatitis B infection and hepatitis D infection.

Background And Aim: Hepatitis delta virus (HDV) infection is the most rapidly progressive chronic viral hepatitis. Little is understood about the immune responses to HDV. This study aims to characterize the systemic immune environments of hepatitis B virus (HBV) and HDV patients at various disease stages.

Hepatitis D Viremia Among Injection Drug Users in San Francisco.

People who inject drugs (PWID) are commonly exposed to hepatitis B virus (HBV) and hepatitis D virus (HDV). We evaluated the prevalence of HDV viremia among hepatitis B surface antigen (HBsAg)-positive PWID (n = 73) using a new quantitative microarray antibody capture (Q-MAC) assay, HDV western blot, and HDV RNA. HDV Q-MAC performed well in this cohort: anti-HDV, 100% sensitivity and specificity; HDV viremia, 61.

Future Therapy for Hepatitis B Virus: Role of Immunomodulators.

Although currently available therapies for chronic hepatitis B virus infection can suppress viremia and provide long-term benefits for patients, they do not lead to a functional cure for most patients. Advances in our understanding of the virus-host interaction and the recent remarkable success of immunotherapy in cancer offer new and promising strategies for developing immune modulators that may become important components of a total therapeutic approach to hepatitis B, some of which are now in clinical development. Among the immunomodulatory agents currently being investigated to combat chronic HBV are toll-like receptor agonists, immune checkpoint inhibitors, therapeutic vaccines, and engineered T cells.

A novel quantitative microarray antibody capture assay identifies an extremely high hepatitis delta virus prevalence among hepatitis B virus-infected mongolians.

Unlabelled: Hepatitis delta virus (HDV) causes the most severe form of human viral hepatitis. HDV requires a hepatitis B virus (HBV) coinfection to provide HDV with HBV surface antigen envelope proteins. The net effect of HDV is to make the underlying HBV disease worse, including higher rates of hepatocellular carcinoma.

Phosphatidylinositol 4,5-bisphosphate is an HCV NS5A ligand and mediates replication of the viral genome.

Background & Aims: Phosphoinositides (PIs) bind and regulate localization of proteins via a variety of structural motifs. PI 4,5-bisphosphate (PI[4,5]P2) interacts with and modulates the function of several proteins involved in intracellular vesicular membrane trafficking. We investigated interactions between PI(4,5)P2 and hepatitis C virus (HCV) nonstructural protein 5A (NS5A) and effects on the viral life cycle.