Biology subjects

Cohen, N. A.

Publications and source records attributed to Cohen, N. A..

13 recordsLinked to original sources

bioRxiv2026

Evaluating Spike Antigenicity across Endemic Human Coronavirus Models using Flow Virometry

While SARS-CoV-2 research has advanced rapidly since COVID-19, endemic human coronaviruses (HCoVs) remain comparatively understudied. Tools to phenotype spike (S), the primary antigenic target on coronaviruses, at the single-virion level could improve vaccine design by capturing variation in epitope availability and spike abundance. Here, we establish a calibrated flow virometry (FV) platform to quantify S antigenicity on native endemic (HCoV-229E, HCoV-OC43) and epidemic (SARS-CoV-2) coronaviruses directly in cell culture supernatants. FV revealed cell line-dependent differences in S antigenicity, including receptor-induced changes in epitope accessibility. Comparison of virion-associated S with recombinant stabilized S by ELISA and biolayer interferometry showed consistent binding for HCoV-OC43, MERS-CoV, and SARS-CoV-2, but differences for HCoV-229E, with FV resolving heterogeneity not captured by bulk assays. Finally, FV showed that HCoV-229E from patient-derived air-liquid interface cultures exhibited reduced antibody binding and distinct S antigenicity compared to cell line-derived virions. Together, these findings establish FV as a platform for single-virion analysis of HCoV antigenicity.

microbiology↗

bioRxiv2025

Clade C MERS-CoV camel strains vary in protease utilizationduring viral entry

MERS-CoV is a lethal pathogen with pandemic potential. Clade A and B MERS-CoV viruses have caused outbreaks in the Middle East since 2012 when they initially spilled over from camels to humans. Clade C viruses, however, are only found in camels across Africa and the spillover potential of these viruses seems to be lower than for clade A/B strains but remains to be fully understood. Here, we report that clade C spikes are less well-cleaved at the S1/S2 boundary than clade A or B viral spikes and that most clade C spikes induce reduced syncytium formation. Additionally, we demonstrate that several East African clade C strains are less able to utilize the TMPRSS2-mediated pathway for viral entry in both cell lines and primary nasal epithelial cultures. We map the molecular basis of this reduced TMPRSS2 usage to the N-terminal domain (NTD) and subdomain 2 (SD2) of East African clade C MERS-CoV. We suggest that reduced usage of the TMPRSS2-mediated entry pathway may underlie the reduced replication of East African clade C strains in humans, while the reduced replication of West African strains remains to be further investigated. Overall, we suggest that altered protease usage may contribute to differential tropism of East African clade C strains and indicate geographically distinct selection pressures on spike between MERS-CoV strains circulating in camels. Significance StatementClade A/B MERS-CoV outbreaks have caused 957 deaths since the first spillover in 2012; meanwhile, Clade C MERS-CoV strains have been found in camels across Africa but have not been reported to cause outbreaks. Investigating why these viruses do not successfully transmit to humans will be key to understanding the pandemic potential of the African MERS-CoV camel reservoir. Our study indicates that clade C viruses exhibit less spike cleavage and that East African clade C isolates are less able to utilize the TMPRSS2-mediated pathway during viral entry of both human cell lines and primary nasal cells. Differences in viral entry pathways could alter cellular and organ tropism and inform our understanding of the pandemic potential of these viruses.

microbiology↗

bioRxiv2025

Human olfactory organoids as an in vitro model of the olfactory epithelium

Abstract/Key PointsO_LIA novel human olfactory epithelial organoid model has been generated from primary cell culture. C_LIO_LIHuman olfactory organoids exhibit immunostaining and gene expression for olfactory cells. C_LIO_LIOlfactory organoids mobilize Ca2+ in response to odorants, a surrogate for neuronal function. C_LI

cell biology↗

bioRxiv2023

Interferon signaling in the nasal epithelium distinguishes among lethal and common cold respiratory viruses and is critical for viral clearance

All respiratory viruses establish primary infections in the nasal epithelium, where efficient innate immune induction may prevent dissemination to the lower airway and thus minimize pathogenesis. Human coronaviruses (HCoVs) cause a range of pathologies, but the host and viral determinants of disease during common cold versus lethal HCoV infections are poorly understood. We model the initial site of infection using primary nasal epithelial cells cultured at air-liquid interface (ALI). HCoV-229E, HCoV-NL63 and human rhinovirus-16 are common cold-associated viruses that exhibit unique features in this model: early induction of antiviral interferon (IFN) signaling, IFN-mediated viral clearance, and preferential replication at nasal airway temperature (33{degrees}C) which confers muted host IFN responses. In contrast, lethal SARS-CoV-2 and MERS-CoV encode antagonist proteins that prevent IFN-mediated clearance in nasal cultures. Our study identifies features shared among common cold-associated viruses, highlighting nasal innate immune responses as predictive of infection outcomes and nasally-directed IFNs as potential therapeutics.

microbiology↗

bioRxiv2023

SARS-CoV-2 nsp15 endoribonuclease antagonizes dsRNA-induced antiviral signaling

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 has caused millions of deaths since emerging in 2019. Innate immune antagonism by lethal CoVs such as SARS-CoV-2 is crucial for optimal replication and pathogenesis. The conserved nonstructural protein 15 (nsp15) endoribonuclease (EndoU) limits activation of double-stranded (ds)RNA-induced pathways, including interferon (IFN) signaling, protein kinase R (PKR), and oligoadenylate synthetase/ribonuclease L (OAS/RNase L) during diverse CoV infections including murine coronavirus and Middle East respiratory syndrome (MERS)-CoV. To determine how nsp15 functions during SARS-CoV-2 infection, we constructed a mutant recombinant SARS-CoV-2 (nsp15mut) expressing a catalytically inactive nsp15. Infection with SARS-CoV-2 nsp15 mut led to increased activation of the IFN signaling and PKR pathways in lung-derived epithelial cell lines and primary nasal epithelial air-liquid interface (ALI) cultures as well as significant attenuation of replication in ALI cultures compared to wild-type (WT) virus. This replication defect was rescued when IFN signaling was inhibited with the Janus activated kinase (JAK) inhibitor ruxolitinib. Finally, to assess nsp15 function in the context of minimal (MERS-CoV) or moderate (SARS-CoV-2) innate immune induction, we compared infections with SARS-CoV-2 nsp15mut and previously described MERS-CoV nsp15 mutants. Inactivation of nsp15 had a more dramatic impact on MERS-CoV replication than SARS-CoV-2 in both Calu3 cells and nasal ALI cultures suggesting that SARS-CoV-2 can better tolerate innate immune responses. Taken together, SARS-CoV-2 nsp15 is a potent inhibitor of dsRNA-induced innate immune response and its antagonism of IFN signaling is necessary for optimal viral replication in primary nasal ALI culture. SIGNIFICANCESevere acute respiratory syndrome coronavirus (SARS-CoV)-2 causes a spectrum of respiratory disease ranging from asymptomatic infections to severe pneumonia and death. Innate immune responses during SARS-CoV-2 infection have been associated with clinical disease severity, with robust early interferon responses in the nasal epithelium reported to be protective. Thus, elucidating mechanisms through which SARS-CoV-2 induces and antagonizes host innate immune responses is crucial to understanding viral pathogenesis. CoVs encode various innate immune antagonists, including the conserved nonstructural protein 15 (nsp15) which contains an endoribonuclease (EndoU) domain. We demonstrate that SARS-CoV-2 EndoU is a crucial interferon antagonist, by providing further evidence for the role of the conserved CoV nsp15 in antagonizing innate immune activation, thereby optimizing CoV replication.

microbiology↗

bioRxiv2023

Comparison of SARS-CoV-2 variants in primary human nasal cultures indicates Delta as most cytopathic and Omicron as fastest replicating

The SARS-CoV-2 pandemic was marked with emerging viral variants, some of which were designated as variants of concern (VOCs) due to selection and rapid circulation in the human population. Here we elucidate functional features of each VOC linked to variations in replication rate. Patient-derived primary nasal cultures grown at air-liquid-interface (ALI) were used to model upper-respiratory infection and human lung epithelial cell lines used to model lower-respiratory infection. All VOCs replicated to higher titers than the ancestral virus, suggesting a selection for replication efficiency. In primary nasal cultures, Omicron replicated to the highest titers at early time points, followed by Delta, paralleling comparative studies of population sampling. All SARS-CoV-2 viruses entered the cell primarily via a transmembrane serine protease 2 (TMPRSS2)-dependent pathway, and Omicron was more likely to use an endosomal route of entry. All VOCs activated and overcame dsRNA-induced cellular responses including interferon (IFN) signaling, oligoadenylate ribonuclease L degradation and protein kinase R activation. Among the VOCs, Omicron infection induced expression of the most IFN and IFN stimulated genes. Infections in nasal cultures resulted in cellular damage, including a compromise of cell-barrier integrity and loss of nasal cilia and ciliary beating function, especially during Delta infection. Overall, Omicron was optimized for replication in the upper-respiratory system and least-favorable in the lower-respiratory cell line; and Delta was the most cytopathic for both upper and lower respiratory cells. Our findings highlight the functional differences among VOCs at the cellular level and imply distinct mechanisms of pathogenesis in infected individuals. ImportanceComparative analysis of infections by SARS-CoV-2 ancestral virus and variants of concern including Alpha, Beta, Delta, and Omicron, indicated that variants were selected for efficiency in replication. In infections of patient-derived primary nasal cultures grown at air-liquid-interface to model upper-respiratory infection, Omicron reached highest titers at early time points, a finding that was confirmed by parallel population sampling studies. While all infections overcame dsRNA-mediated host responses, infections with Omicron induced the strongest interferon and interferon stimulated gene response. In both primary nasal cultures and lower-respiratory cell line infections by Delta were most damaging to the cells as indicated by syncytia formation, loss of cell barrier integrity and nasal ciliary function.

microbiology↗

bioRxiv2023

Neuron-dependent tuft cell expansion initiates sinonasal allergic Type 2 inflammation

Withdrawal StatementThe authors have withdrawn this manuscript owing to inaccuracies in the calculation of tuft cell numbers and errors in the selection of immunofluorescence images used to support our claims. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

immunology↗

bioRxiv2022

Taste receptor T1R3 in nasal cilia detects Staphylococcus aureus D-amino acids to enhance apical glucose uptake

Bitter (T2R) and sweet/umami (T1R) taste receptors serve chemosensory roles throughout the body. In airway cilia, T2Rs detect bacterial metabolites to stimulate bactericidal nitric oxide. T1Rs in solitary chemosensory cells detect glucose in airway surface liquid (ASL) and bacterial D-stereoisomer amino acids to regulate antimicrobial peptides. Using differentiated air-liquid interface cultures of primary nasal cells, we show that the T1R3 receptor is also expressed in human and mouse nasal cell cilia. D-amino acids produced at low mM concentrations by Staphylococcus aureus activate T1R3 to decrease ASL glucose and increase apical glucose uptake by increasing GLUT2 and GLUT10 expression through a {beta}-arrestin pathway. Our data suggests that T1R3 localized to cilia functions as an immune detector for D-amino acids to reduce ASL glucose and potentially limit bacterial growth. Given that glucose protected S. aureus against bactericidal NO produced during T2R activation, the reduced ASL glucose with T1R3 activation may also sensitize bacteria to other innate defenses. HIGHLIGHTSO_LINasal motile cilia express the T1R3 subunit of the sweet taste receptor C_LIO_LIS. aureus D-amino acids activate cilia T1R3 to enhance mucosal glucose uptake C_LIO_LIReduced airway surface liquid glucose likely reduces S. aureus growth C_LIO_LIReduced airway glucose also sensitizes S. aureus to epithelial NO production C_LI

physiology↗

bioRxiv2022

Infection of primary nasal epithelial cells differentiates among lethal and seasonal human coronaviruses

The nasal epithelium is the initial entry portal and primary barrier to infection by all human coronaviruses (HCoVs). We utilize primary nasal epithelial cells grown at air-liquid interface, which recapitulate the heterogeneous cellular population as well as mucociliary clearance functions of the in vivo nasal epithelium, to compare lethal (SARS-CoV-2 and MERS-CoV) and seasonal (HCoV-NL63 and HCoV-229E) HCoVs. All four HCoVs replicate productively in nasal cultures but diverge significantly in terms of cytotoxicity induced following infection, as the seasonal HCoVs as well as SARS-CoV-2 cause cellular cytotoxicity as well as epithelial barrier disruption, while MERS-CoV does not. Treatment of nasal cultures with type 2 cytokine IL-13 to mimic asthmatic airways differentially impacts HCoV replication, enhancing MERS-CoV replication but reducing that of SARS-CoV-2 and HCoV-NL63. This study highlights diversity among HCoVs during infection of the nasal epithelium, which is likely to influence downstream infection outcomes such as disease severity and transmissibility.

microbiology↗

bioRxiv2022

Injury-induced pulmonary tuft cells are heterogenous, arise independent of key Type 2 cytokines, and are dispensable for dysplastic repair

While the lung bears significant regenerative capacity, severe viral pneumonia can chronically impair lung function by triggering dysplastic remodeling. The connection between these enduring changes and chronic disease remains poorly understood. We recently described the emergence of tuft cells within Krt5+ dysplastic regions after influenza injury. Using bulk and single cell transcriptomics, we characterized and delineated multiple distinct tuft cell populations that arise following influenza clearance. Distinct from intestinal tuft cells which rely on Type 2 immune signals for their expansion, neither IL-25 nor IL-4R signaling are required to drive tuft cell development in dysplastic/injured lungs. Furthermore, tuft cells were also observed upon bleomycin injury, suggesting that their development may be a general response to severe lung injury. While intestinal tuft cells promote growth and differentiation of surrounding epithelial cells, in the lungs of tuft cell deficient mice, Krt5+ dysplasia still occurs, goblet cell production is unchanged, and there remains no appreciable contribution of Krt5+ cells into more regionally appropriate alveolar Type 2 cells. Together, these findings highlight unexpected differences in signals necessary for lung tuft cell amplification and establish a framework for future elucidation of tuft cell functions in pulmonary health and disease.

cell biology↗

bioRxiv2018

Species-level bacterial community profiling of the healthy sinonasal microbiome using Pacific Biosciences sequencing of full-length 16S rRNA genes

BackgroundPan-bacterial 16S rRNA microbiome surveys performed with massively parallel DNA sequencing technologies have transformed community microbiological studies. Current 16S profiling methods, however, fail to provide sufficient taxonomic resolution and accuracy to adequately perform species-level associative studies for specific conditions. This is due to the amplification and sequencing of only short 16S rRNA gene regions, typically providing for only family- or genus-level taxonomy. Moreover, sequencing errors often inflate the number of taxa present. Pacific Biosciences (PacBios) long-read technology in particular suffers from high error rates per base. Herein we present a microbiome analysis pipeline that takes advantage of PacBio circular consensus sequencing (CCS) technology to sequence and error correct full-length bacterial 16S rRNA genes, which provides high-fidelity species-level microbiome data\n\nResultsAnalysis of a mock community with 20 bacterial species demonstrated 100% specificity and sensitivity. Examination of a 250-plus species mock community demonstrated correct species-level classification of >90% of taxa and relative abundances were accurately captured. The majority of the remaining taxa were demonstrated to be multiply, incorrectly, or incompletely classified. Using this methodology, we examined the microgeographic variation present among the microbiomes of six sinonasal sites, by both swab and biopsy, from the anterior nasal cavity to the sphenoid sinus from 12 subjects undergoing trans-sphenoidal hypophysectomy. We found greater variation among subjects than among sites within a subject, although significant within-individual differences were also observed. Propiniobacterium acnes (recently renamed Cutibacterium acnes [1]) was the predominant species throughout, but was found at distinct relative abundances by site.\n\nConclusionsOur microbial composition analysis pipeline for single-molecule real-time 16S rRNA gene sequencing (MCSMRT, https://github.com/jpearl01/mcsmrt) overcomes deficits of standard marker gene based microbiome analyses by using CCS of entire 16S rRNA genes to provide increased taxonomic and phylogenetic resolution. Extensions of this approach to other marker genes could help refine taxonomic assignments of microbial species and improve reference databases, as well as strengthen the specificity of associations between microbial communities and dysbiotic states.

bioinformatics↗

bioRxiv2018

Cytokine profile in human olfactory cleft mucus and associated changes in olfactory function

Multiple factors, including physical changes of the nasal mucosa and epithelium and exposure to air-borne environmental agents, appear to contribute to age-related olfactory loss. However, the molecular aspects of aging-associated olfactory loss in humans are not well understood. Although inflammation can be a significant underlying cause for olfactory impairment, whether aging increases the levels of inflammatory cytokines in the human olfactory mucosa and whether any inflammatory markers are associated with age-related olfactory loss remain unclear. Using a noninvasive method for collecting human olfactory mucus, we characterized and compared inflammatory cytokines, chemokines, and some growth factors, in the mucus collected from the olfactory cleft or the anterior nasal cavity from 12 healthy, young (18-40 years old) and 12 elderly (60-85 years old) individuals. We also hoped to identify candidate molecular biomarkers associated with age-associated olfactory loss in humans. Olfactory thresholds were obtained for two odorants and individual mucus samples were analyzed using multiplex assays for the levels of 30 cytokines. Results indicated elevated levels of certain inflammatory cytokines (IL-12, MCP-1) in olfactory mucus of the elderly, and high levels of some inflammatory factors (MCP-1, IL-8, IL-13 and VEGF) were associated with reduced olfactory sensitivity, suggesting that inflammation may play a role in olfactory decline associated with aging.

neuroscience↗

bioRxiv2018

Tissue dependent expression of bitter receptor TAS2R38 mRNA

TAS2R38 is a human bitter receptor gene with a common but inactive allele and people homozygous for the inactive form cannot perceive certain bitter compounds. The frequency of the inactive and active form of this receptor is nearly equal in many human populations and heterozygotes (with one copy of the active form and another copy of the inactive form) have the most common diplotype. However, heterozygotes differ markedly in their perception of bitterness, in part perhaps because of differences in TAS2R38 mRNA expression. Other tissues including the nasal sinuses express this receptor, where it contributes to pathogen defense. We asked whether heterozygotes with high TAS2R38 mRNA expression in taste tissue were also likely to express more TAS2R38 mRNA in sinonasal tissue. To that end, we measured TAS2R38 bitter taste receptor mRNA by qPCR in biopsied tissue, and learned that expression amount of one is not related to the other. We confirmed the general independence of expression in other tissue expressing TAS2R38 mRNA using autopsy data from the GTEx project. Taste tissue TAS2R38 mRNA expression among heterozygotes is unlikely to predict expression in other tissues, perhaps reflecting tissue-dependent function and hence regulation of this protein.

molecular biology↗