DUOX2 regulates secreted factors in virus-infected respiratory epithelial cells that contribute to neutrophil attraction and activation.

The first line of defense against respiratory viruses relies on the antiviral and proinflammatory cytokine response initiated in infected respiratory epithelial cells. The cytokine response not only restricts virus replication and spreading, but also orchestrates the subsequent immune response. The epithelial Dual Oxidase 2 (DUOX2) has recently emerged as a regulator of the interferon antiviral response. Here, we investigated the role of DUOX2 in the inflammatory cytokine response using a model of A549 cells deficient in DUOX2 generated using Crispr-Cas9 and infected by Sendai virus. We found that the absence of DUOX2 selectively reduced the induction of a restricted panel of 14 cytokines and chemokines secreted in response to Sendai virus by 20 to 89%. The secreted factors produced by epithelial cells upon virus infection promoted the migration, adhesion, and degranulation of primary human neutrophils, in part through the DUOX2-dependent secretion of TNF and chemokines. In contrast, DUOX2 expression did not impact neutrophil viability or NETosis, thereby highlighting a selective impact of DUOX2 in neutrophil functions. Overall, this study unveils previously unrecognized roles of epithelial DUOX2 in the epithelial-immune cells crosstalk during respiratory virus infection.

TNF receptor-associated factor 6 interacts with ALS-linked misfolded superoxide dismutase 1 and promotes aggregation.

Amyotrophic lateral sclerosis (ALS) is a fatal disease, characterized by the selective loss of motor neurons leading to paralysis. Mutations in the gene encoding superoxide dismutase 1 (SOD1) are the second most common cause of familial ALS, and considerable evidence suggests that these mutations result in an increase in toxicity due to protein misfolding. We previously demonstrated in the SOD1G93A rat model that misfolded SOD1 exists as distinct conformers and forms deposits on mitochondrial subpopulations. Here, using SOD1G93A rats and conformation-restricted antibodies specific for misfolded SOD1 (B8H10 and AMF7-63), we identified the interactomes of the mitochondrial pools of misfolded SOD1. This strategy identified binding proteins that uniquely interacted with either AMF7-63 or B8H10-reactive SOD1 conformers as well as a high proportion of interactors common to both conformers. Of this latter set, we identified the E3 ubiquitin ligase TNF receptor-associated factor 6 (TRAF6) as a SOD1 interactor, and we determined that exposure of the SOD1 functional loops facilitates this interaction. Of note, this conformational change was not universally fulfilled by all SOD1 variants and differentiated TRAF6 interacting from TRAF6 noninteracting SOD1 variants. Functionally, TRAF6 stimulated polyubiquitination and aggregation of the interacting SOD1 variants. TRAF6 E3 ubiquitin ligase activity was required for the former but was dispensable for the latter, indicating that TRAF6-mediated polyubiquitination and aggregation of the SOD1 variants are independent events. We propose that the interaction between misfolded SOD1 and TRAF6 may be relevant to the etiology of ALS.

Structural basis of STAT2 recognition by IRF9 reveals molecular insights into ISGF3 function.

Cytokine signaling through the JAK/STAT pathway controls multiple cellular responses including growth, survival, differentiation, and pathogen resistance. An expansion in the gene regulatory repertoire controlled by JAK/STAT signaling occurs through the interaction of STATs with IRF transcription factors to form ISGF3, a complex that contains STAT1, STAT2, and IRF9 and regulates expression of IFN-stimulated genes. ISGF3 function depends on selective interaction between IRF9, through its IRF-association domain (IAD), with the coiled-coil domain (CCD) of STAT2. Here, we report the crystal structures of the IRF9-IAD alone and in a complex with STAT2-CCD. Despite similarity in the overall structure among respective paralogs, the surface features of the IRF9-IAD and STAT2-CCD have diverged to enable specific interaction between these family members. We derive a model for the ISGF3 complex bound to an ISRE DNA element and demonstrate that the observed interface between STAT2 and IRF9 is required for ISGF3 function in cells.

Oxidation-state-dependent photochemistry of sulfur-bridged anthracenes.

What's up sulfur? The photochemical reactivity, including a mechanistic study of sulfur-bridged anthracenes is reported. The oxidation state of the bridging sulfur (SOn ) dictates the excited-state behavior of these molecules.

Pinpointing cysteine oxidation sites by high-resolution proteomics reveals a mechanism of redox-dependent inhibition of human STING.

Protein function is regulated by posttranslational modifications (PTMs), among which reversible oxidation of cysteine residues has emerged as a key regulatory mechanism of cellular responses. Given the redox regulation of virus-host interactions, the identification of oxidized cysteine sites in cells is essential to understand the underlying mechanisms involved. Here, we present a proteome-wide identification of reversibly oxidized cysteine sites in oxidant-treated cells using a maleimide-based bioswitch method coupled to mass spectrometry analysis. We identified 2720 unique oxidized cysteine sites within 1473 proteins with distinct abundances, locations, and functions. Oxidized cysteine sites were found in numerous signaling pathways, many relevant to virus-host interactions. We focused on the oxidation of STING, the central adaptor of the innate immune type I interferon pathway, which is stimulated in response to the detection of cytosolic DNA by cGAS. We demonstrated the reversible oxidation of Cys148 and Cys206 of STING in cells. Molecular analyses led us to establish a model in which Cys148 oxidation is constitutive, whereas Cys206 oxidation is inducible by oxidative stress or by the natural ligand of STING, 2'3'-cGAMP. Our data suggest that the oxidation of Cys206 prevented hyperactivation of STING by causing a conformational change associated with the formation of inactive polymers containing intermolecular disulfide bonds. This finding should aid the design of therapies targeting STING that are relevant to autoinflammatory disorders, immunotherapies, and vaccines.

Clinical Evaluation of In-House-Produced 3D-Printed Nasopharyngeal Swabs for COVID-19 Testing.

3D-printed alternatives to standard flocked swabs were rapidly developed to provide a response to the unprecedented and sudden need for an exponentially growing amount of diagnostic tools to fight the COVID-19 pandemic. In light of the anticipated shortage, a hospital-based 3D-printing platform was implemented in our institution for the production of swabs for nasopharyngeal and oropharyngeal sampling based on the freely available, open-source design provided to the community by University of South Florida's Health Radiology and Northwell Health System teams as a replacement for locally used commercial swabs. Validation of our 3D-printed swabs was performed with a head-to-head diagnostic accuracy study of the 3D-printed "Northwell model" with the cobas PCR Media® swab sample kit. We observed an excellent concordance (total agreement 96.8%, Kappa 0.936) in results obtained with the 3D-printed and flocked swabs, indicating that the in-house 3D-printed swab could be used reliably in the context of a shortage of flocked swabs. To our knowledge, this is the first study to report on autonomous hospital-based production and clinical validation of 3D-printed swabs.

Variable oxidation state sulfur-bridged bithiazole ligands tune the electronic properties of ruthenium(ii) and copper(i) complexes.

The synthesis of homoleptic and heteroleptic ruthenium(ii) and copper(i) complexes containing sulfur-bridged bithiazole ligands of varying oxidation states are reported. The complexes have been characterized using 1D and 2D NMR spectroscopy, X-ray single crystal diffraction, electrochemistry, UV-vis absorbance and fluorescence spectroscopy. The stability, photophysical and electrochemical properties were found to be dependent on the oxidation state of the non-coordinating sulfur. The ruthenium and copper species were found to be non-emissive in solution at room temperature, though all displayed weak emission when doped in a PMMA matrix, which increased in intensity on cooling to 77 K. The electrochemical HOMO-LUMO gap was found to be dependent on the oxidation state of the sulfur in the bridging ligand in all complexes, illustrating an additional handle for tuning electrochemical properties.

The Combination of IFN ß and TNF Induces an Antiviral and Immunoregulatory Program via Non-Canonical Pathways Involving STAT2 and IRF9.

Interferon (IFN) ß and Tumor Necrosis Factor (TNF) are key players in immunity against viruses. Compelling evidence has shown that the antiviral and inflammatory transcriptional response induced by IFNß is reprogrammed by crosstalk with TNF. IFNß mainly induces interferon-stimulated genes by the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway involving the canonical ISGF3 transcriptional complex, composed of STAT1, STAT2, and IRF9. The signaling pathways engaged downstream of the combination of IFNß and TNF remain elusive, but previous observations suggested the existence of a response independent of STAT1. Here, using genome-wide transcriptional analysis by RNASeq, we observed a broad antiviral and immunoregulatory response initiated in the absence of STAT1 upon IFNß and TNF costimulation. Additional stratification of this transcriptional response revealed that STAT2 and IRF9 mediate the expression of a wide spectrum of genes. While a subset of genes was regulated by the concerted action of STAT2 and IRF9, other gene sets were independently regulated by STAT2 or IRF9. Collectively, our data supports a model in which STAT2 and IRF9 act through non-canonical parallel pathways to regulate distinct pool of antiviral and immunoregulatory genes in conditions with elevated levels of both IFNß and TNF.

DUSP1 regulates apoptosis and cell migration, but not the JIP1-protected cytokine response, during Respiratory Syncytial Virus and Sendai Virus infection.

The host antiviral response involves the induction of interferons and proinflammatory cytokines, but also the activation of cell death pathways, including apoptosis, to limit viral replication and spreading. This host defense is strictly regulated to eliminate the infection while limiting tissue damage that is associated with virus pathogenesis. Post-translational modifications, most notably phosphorylation, are key regulators of the antiviral defense implying an important role of protein phosphatases. Here, we investigated the role of the dual-specificity phosphatase 1 (DUSP1) in the host defense against human respiratory syncytial virus (RSV), a pathogenic virus of the Pneumoviridae family, and Sendai virus (SeV), a model virus being developed as a vector for anti-RSV vaccine. We found that DUSP1 is upregulated before being subjected to proteasomal degradation. DUSP1 does not inhibit the antiviral response, but negatively regulates virus-induced JNK/p38 MAPK phosphorylation. Interaction with the JNK-interacting protein 1 scaffold protein prevents dephosphorylation of JNK by DUSP1, likely explaining that AP-1 activation and downstream cytokine production are protected from DUSP1 inhibition. Importantly, DUSP1 promotes SeV-induced apoptosis and suppresses cell migration in RSV-infected cells. Collectively, our data unveils a previously unrecognized selective role of DUSP1 in the regulation of tissue damage and repair during infections by RSV and SeV.