Detection of cell-cell interactions via photocatalytic cell tagging.

The growing appreciation of immune cell-cell interactions within disease environments has led to extensive efforts to develop immunotherapies. However, characterizing complex cell-cell interfaces in high resolution remains challenging. Thus, technologies leveraging therapeutic-based modalities to profile intercellular environments offer opportunities to study cell-cell interactions with molecular-level insight. We introduce photocatalytic cell tagging (PhoTag) for interrogating cell-cell interactions using single-domain antibodies (VHHs) conjugated to photoactivatable flavin-based cofactors. Following irradiation with visible light, the flavin photocatalyst generates phenoxy radical tags for targeted labeling. Using this technology, we demonstrate selective synaptic labeling across the PD-1/PD-L1 axis in antigen-presenting cell-T cell systems. In combination with multiomics single-cell sequencing, we monitored interactions between peripheral blood mononuclear cells and Raji PD-L1 B cells, revealing differences in transient interactions with specific T cell subtypes. The utility of PhoTag in capturing cell-cell interactions will enable detailed profiling of intercellular communication across different biological systems.

Structurally Conserved Domains between Flavivirus and Alphavirus Fusion Glycoproteins Contribute to Replication and Infectious-Virion Production.

Alphaviruses and flaviviruses have class II fusion glycoproteins that are essential for virion assembly and infectivity. Importantly, the tip of domain II is structurally conserved between the alphavirus and flavivirus fusion proteins, yet whether these structural similarities between virus families translate to functional similarities is unclear. Using in vivo evolution of Zika virus (ZIKV), we identified several novel emerging variants, including an envelope glycoprotein variant in ß-strand c (V114M) of domain II. We have previously shown that the analogous ß-strand c and the ij loop, located in the tip of domain II of the alphavirus E1 glycoprotein, are important for infectivity. This led us to hypothesize that flavivirus E ß-strand c also contributes to flavivirus infection. We generated this ZIKV glycoprotein variant and found that while it had little impact on infection in mosquitoes, it reduced replication in human cells and mice and increased virus sensitivity to ammonium chloride, as seen for alphaviruses. In light of these results and given our alphavirus ij loop studies, we mutated a conserved alanine at the tip of the flavivirus ij loop to valine to test its effect on ZIKV infectivity. Interestingly, this mutation inhibited infectious virion production of ZIKV and yellow fever virus, but not West Nile virus. Together, these studies show that shared domains of the alphavirus and flavivirus class II fusion glycoproteins harbor structurally analogous residues that are functionally important and contribute to virus infection in vivo.IMPORTANCE Arboviruses are a significant global public health threat, yet there are no antivirals targeting these viruses. This problem is in part due to our lack of knowledge of the molecular mechanisms involved in the arbovirus life cycle. In particular, virus entry and assembly are essential processes in the virus life cycle and steps that can be targeted for the development of antiviral therapies. Therefore, understanding common, fundamental mechanisms used by different arboviruses for entry and assembly is essential. In this study, we show that flavivirus and alphavirus residues located in structurally conserved and analogous regions of the class II fusion proteins contribute to common mechanisms of entry, dissemination, and infectious-virion production. These studies highlight how class II fusion proteins function and provide novel targets for development of antivirals.

Design of a Multiuse Photoreactor To Enable Visible-Light Photocatalytic Chemical Transformations and Labeling in Live Cells.

Despite the growing use of visible-light photochemistry in both chemistry and biology, no general low-heat photoreactor for use across these different disciplines exists. Herein, we describe the design and use of a standardized photoreactor for visible-light-driven activation and photocatalytic chemical transformations. Using this single benchtop photoreactor, we performed photoredox reactions across multiple visible light wavelengths, a high-throughput photocatalytic cross-coupling reaction, and in vitro labeling of proteins and live cells. Given the success of this reactor in all tested applications, we envision that this multi-use photoreactor will be widely used in biology, chemical biology, and medicinal chemistry settings.

Autoimmune anti-DNA and anti-phosphatidylserine antibodies predict development of severe COVID-19.

High levels of autoimmune antibodies are observed in COVID-19 patients but their specific contribution to disease severity and clinical manifestations remains poorly understood. We performed a retrospective study of 115 COVID-19 hospitalized patients with different degrees of severity to analyze the generation of autoimmune antibodies to common antigens: a lysate of erythrocytes, the lipid phosphatidylserine (PS) and DNA. High levels of IgG autoantibodies against erythrocyte lysates were observed in a large percentage (up to 36%) of patients. Anti-DNA and anti-PS antibodies determined upon hospital admission correlated strongly with later development of severe disease, showing a positive predictive value of 85.7% and 92.8%, respectively. Patients with positive values for at least one of the two autoantibodies accounted for 24% of total severe cases. Statistical analysis identified strong correlations between anti-DNA antibodies and markers of cell injury, coagulation, neutrophil levels and erythrocyte size. Anti-DNA and anti-PS autoantibodies may play an important role in the pathogenesis of COVID-19 and could be developed as predictive biomarkers for disease severity and specific clinical manifestations.