Biology subjects

Stewart, R.

Publications and source records attributed to Stewart, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Molecular signatures of genetically defined jugular nociceptors and their responses to allergic airway inflammation

Abstract Rationale: Vagal sensory neurons are molecularly and functionally diverse, and they play critical roles in visceral sensation and homeostasis. The vagal system is anatomically divided into jugular and nodose ganglia, which exhibit distinct peripheral innervation patterns and physiological functions. Although recent transcriptomic studies have characterized nodose neuronal subtypes in detail, the molecular profiles of jugular sensory neurons remain poorly defined. Objectives: To define the molecular profiles of genetically identified jugular sensory neurons and determine the transcriptional responses of jugular and nodose airway afferents to allergic airway inflammation. Methods: We performed deep transcriptomic profiling of two genetically defined jugular nociceptor populations, MrgprC11 and MrgprD neurons, and compared them with airway-innervating P2ry1 nodose neurons and Phox2b nodose neurons. We further examined transcriptional changes of MrgprC11 and P2ry1 neurons following OVA-induced allergic airway inflammation. Measurements and Main Results: Transcriptomic analyses revealed distinct molecular signatures of jugular nociceptive neurons and identified subtype-specific genes across diverse functional categories. Following OVA challenge, MrgprC11 and P2ry1 neurons exhibited strikingly divergent transcriptional responses. More than 400 genes that were differentially expressed in both neuronal populations were regulated in opposite directions. GO enrichment analysis suggested that MrgprC11 jugular neurons adopted a metabolically restrained state with reduced translation and bioenergetic activities, whereas P2ry1 nodose neurons exhibited metabolically active state with enhanced translation and bioenergetic activities. Conclusions: These findings provide new insight into vagal sensory neuron diversity and reveal distinct transcriptional adaptations of jugular and nodose airway afferents during allergic inflammation.

neuroscience↗

bioRxiv2026

Microenvironmental and transcriptional determinants of the robustness of spinal cord regeneration in zebrafish

Unlike mammals, adult zebrafish can regenerate spinal cord tissue after complete transection. We report here that a substantial proportion of zebrafish receiving this injury fail to regain locomotor function, and we analyzed this critically. After stratifying large cohorts of injured animals according to functional recovery, we directly compared recovered and non-recovered individuals at matched post-injury stages. We find that permanently paralyzed animals form a tissue bridge across the lesion yet exhibit impaired axonal invasion and regrowth to downstream targets. Integrative single-nucleus profiling, spatial transcriptomics, and regulatory network analyses revealed that regenerative success is characterized by the coordinated establishment of a permissive multicellular regenerative state rather than major differences in cellular composition. Furthermore, we find that regenerative failure involves persistent non-permissive regulatory programs, including extracellular matrix remodeling, impaired adaptive T-cell activation, and reduced axon growth-associated signaling. Gene regulatory network and cellular trajectory analyses identified candidate transcriptional regulators that could not be resolved through differential expression analysis alone. Functional CRISPR F0 screening of several of these candidates identified hoxb5a as a critical determinant of regenerative outcome. Stable hoxb5a mutants exhibited impaired swimming recovery, disrupted bridge formation, and limited axon bridging after spinal cord injury. Together, our findings reveal key differences between success and failure in a model of elevated regenerative capacity, with hoxb5a acting as a regulator linking multicellular tissue remodeling to axonal regeneration and functional recovery.

developmental biology↗

bioRxiv2026

Polarized neutrons for the study of individual and collective fast dynamics in proteins

Neutron scattering is a powerful technique to investigate atomic structures and molecular dynamics of proteins at the nano-scale. When it comes to dynamics, incoherent and coherent scattering respectively provide information on the single and collective dynamics of nuclei. In proteins, hydrogen has the highest incoherent cross-section, and it is common practice to overlook the contribution of coherent terms stemming from all nuclei. However, the fast collective dynamics of heavier nuclei could also be studied if coherent scattering and incoherent scattering were experimentally separated. The recent advent of polarized neutron spectroscopy with sufficient flux and energy resolution has made it possible, and opens new perspectives to investigate the relative importance of coherent scattering and the information it provides on biological samples. The present study reports on the use of polarized quasi-elastic neutron scattering (QENS) and the application of a minimalistic model adapted to both individual and collective dynamics. Using a perdeuterated green fluorescent protein as a model globular protein, the study provides an interpretation of the dynamical parameters obtained with QENS, and a comparative study of the Elastic Coherent and Incoherent Scattering Factor. Based on both experiments and calculations, we discuss the relative importance of distinct and self components of coherent scattering, which is often wrongly assumed to be representative of collective dynamics only. The results highlight the current impediments rendering complicated a straightforward analysis of fast collective dynamics in hydrated protein samples.

biophysics↗

bioRxiv2026

Quantifying Evidence for Competing Biomedical Hypotheses using Large Language Models and Bayesian Analysis

Science fundamentally depends on the generation and testing of hypotheses, many of them controversial. An explosion in scientific literature has made evaluating hypotheses even within a domain a problem of scale, and risks slowing an already extensive consensus-building process. While this challenge has prompted interest in automated hypothesis evaluation tools, existing methods have not yet proven effective for comparing hypotheses. Here, we introduce KM-GPT-DCH, an algorithm that combines co-occurrence methods with large language models (LLMs) to develop a transparent and reproducible literature-based algorithm to compare controversial hypotheses using a structured scoring approach with Bayesian methods to estimate confidence. When testing the algorithm on historical controversial hypotheses previously decided, KM-GPT-DCH chooses the correct hypothesis with high confidence several years before the scientific community or public do so. We further apply the algorithm to compare twenty unresolved controversial hypothesis pairs providing guidance for future research. The method can help researchers and the public to evaluate biomedical hypotheses such as "Is it more likely that monoamine deficiency or inflammation causes depression?" It can also be used to assess and visualize historical trends in the scientific literature. A web-based implementation of the algorithm is freely available at https://skim.morgridge.org.

bioinformatics↗

bioRxiv2026

Biomimetic miR-133a inhibitor activated scaffolds optimised for spinal cord repair promote neurite outgrowth and angiogenesis via neuronal cytoskeletal remodelling

Significant challenges in spinal cord injury include the loss of neural tissue, disruption of local vasculature, and intrinsic suppression of actin mobilisation in neurons, together preventing axonal regrowth. Here, we develop an implantable biomimetic microRNA (miR) inhibitor-activated scaffold that combines optimised matrix cues with transcriptomically defined RNA-based modulation of intrinsic neuronal pathways as a platform to support neuronal cell delivery and promote neurovascular repair. First, hyaluronic acid macroporous scaffolds functionalized with collagen-IV and fibronectin supported iPSC-derived neuronal spheroid formation and neurite extension. To identify a neurotrophic target, we performed analysis of public miRNA-mRNA interaction datasets, revealing that miR-133a regulates pathways involved in neuronal actin cytoskeletal organisation. MiR-133a inhibitors were complexed with the cell-penetrating peptide RALA to form nanoparticles, demonstrated >95% scaffold loading efficiency, sustained localised release over 28 days and enhanced neurite outgrowth from motor neurons and iPSC neurons. Bulk RNA-sequencing and transcriptomic analysis of iPSC neurons within the scaffolds demonstrated coordinated upregulation of actin-remodelling, cell-matrix adhesion and metabolic pathways, indicative of a cytoskeletally adaptable neuron. When employed in an ex vivo dorsal root ganglia model, scaffold-mediated miR-133a inhibition promoted neurite extension and integration of delivered iPSC neurons with injured neural tissue. Finally, miR-133a-inhibitor-activated scaffolds upregulated neurovascular genes, increased endothelial cell migration and enhanced blood vessel formation in vivo in a chick embryo assay. These findings identify miR-133a as a neurotrophic target, elucidate the underlying mechanisms of action through transcriptomic analysis and demonstrate that biomimetic scaffold-mediated inhibition of miR-133a can enhance neuronal delivery for multifaceted spinal cord repair applications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/719922v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@13fcb26org.highwire.dtl.DTLVardef@13239c3org.highwire.dtl.DTLVardef@6e6f5eorg.highwire.dtl.DTLVardef@51ad93_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2026

Gene- and domain-aware calibration increases the clinical utility of variant effect predictors

The utility of clinical genetic testing is limited because around 90% of missense variants in ClinVar remain of uncertain clinical significance. Variant effect predictors (VEPs) can score any missense variant, potentially empowering variant classification. Realizing this potential requires calibration to translate predictor scores into evidence. However, genome-wide calibration ignores predictor heterogeneity across genes, causing evidence misassignment. We developed an automated, data-adaptive framework that optimizes two complementary approaches: gene-specific calibration for genes with enough variants for calibration, and domain-aggregate calibration for other disease-associated genes, which groups variants from protein domains with similar predictor score distributions for calibration. Applied to three predictors across 2,769 genes, this framework assigned evidence to 10.6% more variants on average while generally improving evidence accuracy compared to genome-wide calibration. These calibrations and the resulting calibrated computational evidence are available through the PredictMD portal. Our framework substantially increases the clinical utility of VEPs for variant classification.

genetics↗

bioRxiv2026

A scalable approach to resolving variants of uncertain significance

Over 90% of missense variants across [~]4,000 disease-associated genes are variants of uncertain significance (VUS). Experimental variant effect measurements provide critical evidence about pathogenicity and inform disease biology, but most variants lack data and clinical translation has been limited. The Impact of Genomic Variation on Function Consortium generated experimental data for 62,215 variants across ten genes using multiplexed assays and 1,407 variants across 163 genes using arrayed assays, curated 193,139 additional community-generated variant effect measurements across 30 additional genes, and developed automated calibration methods for translating experimental data and variant effect predictions into clinical evidence. To reduce current VUS, we developed a scalable workflow using only experimental and predictive evidence, enabling reclassification of 75% of the 16,115 VUS in these genes as pathogenic or benign with <1% error. To minimize future VUS, we analyzed >90,000 unobserved variants; 62% had enough evidence to be "preclassified" as pathogenic or benign. We validated our data, evidence and classifications using All of Us and created interactive resources to enable clinical use of the calibrated data. Thus, for 40 genes, representing 1% of the clinical genome, we resolve most existing VUS and future variants, illustrating how systematic use of scalable evidence can empower genomic medicine.

genomics↗

bioRxiv2026

An integrated, scaled approach to resolve TSC2 variants of uncertain significance

Obtaining a precise genetic tuberous sclerosis diagnosis is a challenge as many missense TSC2 variants are variants of uncertain significance (VUS). VUS in TSC2 have been resolved by one-at-a-time functional assays, but these assays cannot scale to the 3,634 TSC2 missense VUS observed so far. To address this challenge, we used massively parallel sequencing to measure the steady-state abundance of almost 9,000 TSC2 missense variants and developed an mTOR pathway activity assay using genome editing and cell sorting to generate activity scores for 391 missense variants. 1,288 of 8,891 (14.49%) missense variants assayed had altered TSC2 abundance, and 69 of 391 (17.65%) missense variants assayed had altered mTOR pathway activity. Calibration and integration of these data into classification of variants identified in a clinical cohort putatively reclassified 212 of 276 (76.8%) TSC2 missense VUS. These datasets will lead to improved genetic diagnosis of tuberous sclerosis with potential positive impacts on the clinical management of patients and their families.

genetics↗

bioRxiv2025

Predicting interaction-specific protein-protein interaction perturbations by missense variants with MutPred-PPI

Disruption of protein-protein interactions (PPIs) is a major mechanism of a variants deleterious effect. Computational tools are needed to assess such variants at scale, yet existing predictors rarely consider loss of specific interactions, particularly when variants perturb binding interfaces without significantly affecting protein stability. To address this problem, we present MutPred-PPI, a graph attention network that predicts interaction-specific (edgetic) effects of missense variants by operating on AlphaFold 3-based protein complex contact graphs with protein language model embeddings imposed upon nodes. We systematically evaluated our model with stringent group cross-validation as well as benchmark data recently collected within the IGVF Consortium. MutPred-PPI outperformed all baseline methods across all evaluation criteria, achieving an AUC of 0.85 on seen proteins and 0.72 on previously unseen proteins in cross-validation, demonstrating strong generalizability despite scarce training data. To demonstrate biomedical relevance, we applied MutPred-PPI to variants from ClinVar, HGMD, COSMIC, gnomAD, and two de novo neurodevelopmental disorder-linked datasets. Disease-associated variants from Clin-Var and HGMD showed strong enrichment for both quasi-null and edgetic effects, whereas population variants from gnomAD increasingly preserved interactions with higher allele frequencies. Notably, we observed a strong edgetic disruption signature in highly recurrent cancer variants from both the full COSMIC dataset and a subset of variants from oncogenes. Recurrent tumor suppressor gene variants and autism spectrum disorder-associated variants exhibited moderate quasi-null enrichment, whilst neurodevelopmental disorder-linked variants showed a weak edgetic disruption signature. These results indicate distinct PPI perturbation mechanisms across disease types and show that MutPred-PPI captures functionally relevant molecular effects of pathogenic variants.

bioinformatics↗

bioRxiv2025

Transcriptomic study of WSSV infection in Litopenaeus vannamei lymphoid organ via single nuclei RNA sequencing

BackgroundAquaculture is the fastest growing farmed food sector and is a key part of global food security. Crustacean aquaculture, while being one of the most profitable sectors, is threatened by pathogenic diseases such as white spot disease (WSD), which results in severe stock losses and threatens animal health and welfare. In this study, we used novel techniques to study the impact of white spot syndrome virus (WSSV), the causative agent of WSD, on Pacific whiteleg shrimp (Litopenaeus vannamei), and uncover additional information that may be used in creating WSSV-resistant shrimp stocks. ResultsWe successfully developed a novel nuclei isolation protocol optimized for shrimp tissues to prepare our samples for single nuclei RNA-sequencing following a pathogen challenge comprising of 32 adult whiteleg shrimp infected with WSSV either through their feed or by injection. We constructed the first penaeid shrimp lymphoid organ cell atlas to improve our understanding of the characteristics of this immune organ, constructed the UMAPs and identified marker genes that define distinct cell clusters to reveal the biological functions of different cell types within this organ. ConclusionBy comparing gene expression between the control and WSSV-infected samples we uncovered multiple genes of interest that that have the potential to be used as targets for CRISPR gene editing for WSSV-resistance in Pacific whiteleg shrimp. These genes have the potential to be valuable assets for future gene editing studies in commercially important penaeid shrimp species. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/693182v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1be071org.highwire.dtl.DTLVardef@2c3da6org.highwire.dtl.DTLVardef@835e67org.highwire.dtl.DTLVardef@aacff1_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2025

Enabling wider access to human molecular neuroscience research in pain: A simple preservation method for human dorsal root ganglion neurons in Hibernate A media

The use of human dorsal root ganglion (DRG) from organ donors opens the door for research into the molecular biology and physiology of human nociceptors; however, there are barriers to working with this tissue including logistical difficulties and limited access. We present an approach using Hibernate media to temporarily store either whole DRGs or dissociated DRG neurons prior to culturing and functional testing. Dissociation of DRGs following temporary storage (4-16hrs) in Hibernate media resulted in similar neuronal and immune cell yield as acutely dissociated DRGs. Neurons derived from DRGs stored in Hibernate media prior to dissociation exhibited similar electrophysiological properties and capsaicin responses as acutely dissociated DRG neurons. Similarly, neurons from acutely dissociated DRGs stored in Hibernate media (>24hrs) and shipped to geographically distant laboratories produced neuronal cultures displaying comparable electrophysiological properties as acutely cultured neurons. This approach overcomes insurmountable logistical burdens and increases access to freshly recovered human DRGs.

neuroscience↗

bioRxiv2025

SKiM-GPT: Combining Biomedical Literature-Based Discovery with Large Language Model Hypothesis Evaluation

BackgroundGenerating and testing hypotheses is a critical aspect of biomedical science. Typically, researchers generate hypotheses by carefully analyzing available information and making logical connections, which are then tested. The accelerating growth of biomedical literature makes it increasingly difficult to keep pace with connections between biological entities emerging across biomedical research. Recently developed automated means of generating hypotheses can generate many more hypotheses than can be easily tested. One such approach involves literature based discovery (LBD) systems such as Serial KinderMiner (SKiM), which surfaces putative A B C links derived from term co occurrence. However, LBD systems leave three critical gaps: (i) they find statistical associations, not biological relationships; (ii) they can produce false positive leads; and (iii) they do not assess agreement with a hypothesis in question. As a result, LBD search results often require costly manual curation to be of practical utility to the researcher. Large language models (LLMs) have the potential to automate much of this curation step, but standalone LLMs are hampered by hallucinations, lack of transparency in information sources, and the inability to reference data not included in the training corpus. ResultsWe introduce SKiM-GPT, a retrieval-augmented generation (RAG) system that combines SKiMs co-occurrence search and retrieval with frontier LLMs to evaluate user- defined hypotheses. For every chosen A-B-C SKiM hit, SKiM-GPT retrieves appropriate PubMed abstract texts, filters out irrelevant abstracts with a fine-tuned relevance model, and prompts an LLM to evaluate the users hypothesis, given the relevant abstracts. Importantly, the SKiM-GPT system is transparent and human-verifiable: it displays the retrieved abstracts, the hypothesis score, and a justification for the score grounded in the texts and written in natural language. On a benchmark consisting of 14 disease-gene-drug hypotheses, SKiM-GPT achieves strong ordinal agreement with four expert biologists (Cohens {kappa} = 0.84), demonstrating its ability to replicate expert judgment. ConclusionsSKiM-GPT is open-source (https://github.com/stewart-lab/skimgpt) and available through a web interface (https://skim.morgridge.org), enabling both wet-lab and computational researchers to systematically and efficiently evaluate biomedical hypotheses at scale.

bioinformatics↗

bioRxiv2025

Pan-cancer analysis in the real-world setting uncovers immunogenomic drivers of acquired resistance post-immunotherapy

Immune checkpoint blockade (ICB) has revolutionised cancer therapy, yet resistance -- both primary and acquired -- remains a significant obstacle, affecting the majority of patients. Here, we leveraged a large-scale, real-world clinicogenomic dataset to systematically explore the molecular underpinnings of ICB resistance in the post-progression setting. Analysing over 5,000 pan-cancer patients with clinical and pre-/post-treatment genomic and transcriptomic data, we identify distinct immunogenomic drivers of acquired vs. primary ICB resistance. Post-ICB progression, acquired resistance showed extended survival compared to primary resistance across all cancer types. The acquired resistance clinical phenotype was paralleled by a universally immune-inflamed, albeit dysfunctional, tumour microenvironment (TME) at the onset of acquired resistance, with sustained or ICB-induced inflammatory and interferon responses. We confirm previously described mechanisms of acquired resistance, including B2M loss-of-function (LoF) in non-small cell lung cancer (NSCLC), and identify novel potential mediators, including LoF of TGFBR2 in NSCLC, CYLD in head and neck cancer, and RUNX1 in triple negative breast cancer. Further supporting their involvement in resistance, these acquired ICB alterations associated with immune-escaped TMEs, characterised by active immunomodulatory oncogenic signalling, hyperproliferation and invasiveness, or altered tumour metabolism. These findings emphasise the heterogeneity of molecular drivers of acquired resistance to ICB within and across cancers, and highlight the potential for personalised therapeutic interventions post-progression to improve patient outcomes.

cancer biology↗

bioRxiv2024

Impact of wildfire smoke and diesel exhaust on inflammatory response in aging human microglia

BackgroundAir pollution, particularly from Diesel Exhaust Particles (DEP) and Wildfire Smoke (WFS), is increasingly recognised as a significant driver of neuroinflammation linked to brain diseases. However, the role of microglia in mediating these neuroinflammatory responses remains poorly understood. This study aimed to investigate the effects of air pollution on monocyte-derived microglia-like cells (MDMi) from both young (< 40 years of age), and (older > 60 years of age) healthy individuals, focusing on immune response, cytokine secretion, nitrosative stress, and phagocytic activity. ResultsOur study demonstrated that DEP and WFS extract (WFSE) significantly upregulated expression of the oxidative stress marker, heme-oxygenase-1 (HO-1) in MDMi after 24 hr, with levels normalising by 96 hr, indicating a transient oxidative stress response. Both DEP and WFSE elicited distinct inflammatory cytokine profiles. DEP induced a rapid response, increasing TNF-, IL-6, IL-23, and IL-33 within 2 hr in young MDMi and 24 hr in aged MDMi. In contrast, WFSE triggered a delayed but sustained inflammatory response, with TNF-, IFN-{gamma}, IL-23, and IL-33 levels persisting at 96 hr in aged MDMi, highlighting an age-related vulnerability to air pollutant-induced inflammation. Both pollutants activated p38, ERK, and NF-{kappa}B pathways, with p38 activity resolving by 96 hours and ERK activation persisting, reflecting their distinct roles in cellular stress and inflammation. NF-{kappa}B p65 nuclear translocation, observed at 24 hours, highlighted its critical role in cytokine release and inflammation following exposure to DEP and WFSE. This is the first report of NF-{kappa}B activation in human microglia exposed to air pollutants. ConclusionsThese results highlight the distinct and potentially harmful effects of DEP and WFSE on immune and inflammatory responses in MDMi, particularly in ageing populations, with significant implications for brain health. DEP triggers acute oxidative stress and inflammatory responses, while WFSE induces more prolonged effects, especially in aged microglia. Both pollutants activate the MAPK and NF-{kappa}B pathways and exhibit unique cytokine profiles, underscoring their overlapping yet distinct mechanisms of action. These findings advance our understanding of air pollutant-induced neuroinflammation and its contribution to neurodegeneration, providing a foundation for developing targeted interventions to mitigate the neurotoxic effects of air pollution.

cell biology↗

bioRxiv2024

Differential Ubiquitination of Host Proteins in Response to IPNV and ISAV viruses in Atlantic salmon

Viral diseases remain a major barrier to the sustainable production of farmed fish, primarily attributable to the absence of effective prevention and treatment options. Understanding host-pathogen interactions can guide the development of vaccines, antiviral therapies, or gene editing strategies. Ubiquitination is a key cell signalling molecule, known to regulate many aspects of immune functions but currently understudied in fish. This study leverages ubiquitin-enriched mass spectroscopy complemented with RNA sequencing to characterize the role of ubiquitination in response to infection. A challenge experiment was conducted by infecting Atlantic salmon head kidney (SHK-1) cells with Infectious salmon anaemia virus (ISAV) and Infectious pancreatic necrosis virus (IPNV). At 24- and 48 hours post-infection dramatic changes were observed in the global ubiquitination state of host proteins. Many post-translational modifying proteins increased in abundance upon ISAV infection, whilst IPNV infection resulted in a reduction in abundance of many of these proteins. Transcriptomics showed a delay in the activation of the antiviral response to ISAV infection, with major upregulation of genes associated with immune pathways only at 48h. On the contrary, IPNV infection resulted in upregulation of classic innate immune response genes at both timepoints. Clear activation of Rig-like receptor pathways is demonstrated in both infections, in addition to upregulation of both conserved and novel antiviral TRIM E3 ubiquitin ligate genes. Network analysis identified clusters of immune genes and putatively regulatory proteins showing differential ubiquitination upon viral infection. This study highlights the importance of post-translational control of the host innate immune response to viruses in Atlantic salmon. Clear differences in ubiquitination between two viruses indicate either virus-specific post-translational regulation or viral antagonism of the immune response. Additionally, the ubiquitination of various proteins was linked to the regulation of innate immune pathways, suggesting a direct role of ubiquitination in the regulation of antiviral responses. AUTHOR SUMMARYUbiquitination is a main cellular regulatory mechanism in all animal species, controlling the activity of many proteins within the cell. The role of ubiquitination in the regulation of immunity is well-established, and several direct interactions with viruses have been described, either as part of a strategy of the host to fight the infection or as a viral mechanism to evade the host immune response. In aquaculture, viral diseases currently represent the most important threat to the sustainability of the industry. However, very little is known about the interplay between ubiquitination and viral pathogens in fish species. In our study, we have assessed the ubiquitination response of Atlantic salmon to two production-relevant viruses, Infectious salmon anaemia virus (ISAV) and Infectious pancreatic necrosis virus (IPNV). We found remarkable differences in the ubiquitination profiles between the two infections, suggesting a key role of ubiquitination on the early immune response. We also discovered an association between the ubiquitination of certain regulatory proteins and the activation of antiviral pathways. This information could help develop new strategies to tackle viral diseases in aquaculture, such as the development of more effective vaccines or antiviral therapies, or inform gene editing efforts to generate disease resistant fish.

molecular biology↗

bioRxiv2024

Passage of Zika virus in Rag1-deficient mice selects for unique envelope glycosylation motif mutants that show enhanced replication

N-linked glycosylation of flavivirus envelope proteins is widely viewed as a requirement for optimal folding, processing and/or transit of envelope proteins, and the assembling virons, through the endoplasmic reticulum (ER) and Golgi. Herein we show that serial passage of ZIKVNatal in Rag1-/- mice generated two unique envelope glycan-deficient mutants, ZIKV-V153D and ZIKV-N154D, that, surprisingly, produced titers [~]1 to 2.6 logs higher than the glycosylated parental ZIKVNatal in Vero E6 cells and human brain organoids. RNA-Seq of infected organoids suggested that this increased replication fitness was associated with upregulation of the unfolded protein response (UPR). Cell death, cellular viral RNA and viral protein levels were not significantly affected, arguing that these glycan mutants enjoyed faster ER/Golgi folding, processing, assembly, transit, and virion egress, assisted by an upregulated UPR. Thus, ZIKV envelope N-linked glycosylation is not essential for promoting envelope folding, assembly and transit through the ER/Golgi, as aspartic acid (D) substitutions in the glycosylation motif achieve this with significantly greater efficiency. V153D and N154D mutants have not been employed in flavivirus envelope glycosylation studies. Instead, mutants such as N154A have been used, which may impart unfavorable properties that have a greater impact than the loss of the glycan. ZIKV-V153D and -N154D may avoid this by preserving the surface negative charge provided by the glycan moiety in the parental ZIKVNatal. In Ifnar-/- mice ZIKV-V153D and -N154D showed faster viremia onsets, but reduced viremic periods, than the parental ZIKVNatal, consistent with the contention that these glycans have evolved to delay neutralizing antibody activity. IMPORTANCEStudies seeking to understand the role(s) of N-linked glycosylation of flavivirus envelope proteins often introduce amino acid substitutions that disrupt the glycosylation motif, which in ZIKV has the sequence 153VNDT156. Unfortunately, such substitutions, for instance N154A, may themselves impart unfavorable properties on envelope that have a greater impact than the loss of the glycan moiety. Herein we describe two unique glycosylation motif mutants, ZIKV-V153D and -N154D that were positively selected during passage of ZIKVNatal in Rag1-/- mice. These N154 glycan-deficient viruses produced viral titers up to [~]400 fold higher than the parental ZIKVNatal in Vero cells and in human brain organoids. Such glycans are thus clearly not a requirement for optimal folding and trafficking of ZIKV envelope through the endoplasmic reticulum/Golgi. These results provide new insights into the molecular mechanisms underpinning viral fitness in vitro and in vivo, and also have implications for virus-like-particle vaccine design and production.

microbiology↗

bioRxiv2024

TMEM106B-mediated SARS-CoV-2 infection allows for robust ACE2-independent infection in vitro but not in vivo

Angiotensin converting enzyme 2 (ACE2) serves as the primary entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, ACE2-independent entry has been observed in vitro for SARS-CoV-2 strains containing the E484D amino acid substitution in the spike protein. In this study, we conducted a whole genome CRISPR-Cas9 knockout screen using a SARS-CoV-2 strain containing the spike-E484D substitution (SARS-CoV-2MA1) to identify the ACE2-independent entry mechanisms. Our findings revealed that SARS-CoV-2MA1 infection in HEK293T cells relied on heparan sulfate and endocytic pathways, with TMEM106B emerging as the most significant contributor. While SARS-CoV-2MA1 productively infected human brain organoids and K18-hACE2 mouse brains, it did not infect C57BL/6J or Ifnar-/- mouse brains. This suggests that ACE2-independent entry via TMEM106B, which is a protein that is predominantly expressed in the brain, did not overtly increase the risk of SARS-CoV-2 neuroinvasiveness in wild-type mice. Importantly, SARS-CoV-2MA1 did not replicate in Ace2-/- mouse respiratory tracts. Overall, this suggests that robust ACE2-independent infection by SARS-CoV-2E484D is likely a phenomenon specific to in vitro conditions, with no apparent clinical implications.

microbiology↗

bioRxiv2023

Macrophages undergo functionally significant reprograming of nucleotide metabolism upon classical activation

During an immune response, macrophages specifically rewire their metabolism to support functional changes. Using a multi-omics approach, we identified nucleotide metabolism as one of the most significantly rewired pathways across the metabolic network in classically activated macrophages. Further isotopic tracing studies revealed the substantial changes in nucleotide de novo synthesis, degradation, and salvage fluxes in stimulated macrophages, as well as the key reactions where metabolic regulation occurs: 1) de novo synthesis of purine nucleotides is shut down and particularly blocked at the last step of IMP synthesis catalyzed by ATIC; 2) de novo synthesis of pyrimidines is maintained up to UMP, but further synthesis of CTP (catalyzed by CTPS) and dTMP (catalyzed by TYMS) is greatly reduced; 3) Nucleotide degradation to nitrogenous bases is increased, but further oxidation of purine bases (catalyzed by XOR) is inhibited, causing a great accumulation of nucleosides and bases; and 4) cells switch to salvaging the nucleosides and bases as the primary means to maintain purine nucleotides. Mechanistically, we found these changes are driven by a combination of transcriptional regulation and enzyme inhibition. Nitric oxide (NO) was identified as a major regulator, driving the strong inhibition of ATIC and XOR, and the transcriptional downregulation of Tyms. To understand the functional impact of the activation-induced switch from purine de novo synthesis to salvage, we knocked out the purine salvage enzyme Hprt. Hprt knockout significantly alters functional gene expression in activated macrophages, suppresses macrophage migration, and increases pyroptosis. Furthermore, knocking out Hprt or Xor increases the proliferation of the intracellular parasite Toxoplasma gondii in macrophages. Together, these results comprehensively uncovered the dynamic rewiring of nucleotide metabolism in classically activated macrophages, elucidated the key regulatory mechanisms, and identified the functional significance of such rewiring.

cell biology↗