Biology subjects

Taylor, G. M.

Publications and source records attributed to Taylor, G. M..

12 recordsLinked to original sources

bioRxiv2025

Tissue-specific tolerance mechanisms and lymph node co-drainage converge to shape T cell immunity in the upper digestive system and regulate pancreatic cancer progression

The liver, pancreas, and duodenum share lymph nodes (LNs), providing a unique system to examine how tissue origin of self-antigens shapes T cell fate. Comparing mice expressing ovalbumin (OVA) from distinct subcellular compartments, we found that cytosolic OVA from liver or pancreas, but not gut, was immunologically ignored. High-dose hepatic secreted OVA triggered antigen-specific T cell deletion, whereas secreted pancreatic and intestinal OVA induced regulatory T (Treg) cells, revealing immunological ignorance, clonal deletion and Treg cell generation as tissue-specific tolerance mechanisms. Of these, LN co-drainage only influenced Treg cell induction, establishing gut-pancreas-liver axes: Intestinal viral infection rendered hepatocyte- and exocrine pancreas-specific T cells inflammatory; liver injury promoted pancreas- and gut-directed responses. These self-reactive T cells caused tissue destruction but enhanced pancreatic tumor control when neoantigen OVA was secreted but not cytosolic. Thus, LN co-drainage and tissue-specific tolerance mechanisms jointly shape immune homeostasis and disease susceptibility in the upper digestive system.

immunology↗

bioRxiv2025

T cell fate is dictated by different antigen presenting cells in response to dietary versus gut epithelial self-antigen

We investigated whether T cell responses and antigen-presenting cell (APC) requirements in gut-draining lymph nodes differ by antigen source, diet versus epithelium. Using mice fed ovalbumin (OVA) or expressing secreted (s), cytosolic (c), or transmembrane (tm) epithelial OVA, we compared OVA-specific T cell fates. At baseline and after reovirus infection, T cell responses were comparable across models. However, helminth infection induced Th2 cell polarization in sOVA and tmOVA but not cOVA or OVA-fed mice. BATF3 APCs were indispensable for CD4 T cell proliferation only in cOVA mice yet drove Treg cell differentiation across all epithelial OVA models. In contrast, antigen presentation by ROR{gamma}tMHC-II APCs was exclusively required for Treg cell induction by dietary OVA. These distinct APC dependencies correlated with susceptibility to pathology elicited by dietary versus epithelial self-antigens. Thus, antigen origin and presentation context are integrated to shape T cell fate, a new framework for predicting gut immune outcomes.

immunology↗

bioRxiv2025

Olfactory dysfunction in a novel model of prodromal Parkinson's disease in adult zebrafish

Olfactory dysfunction is an early clinical marker of prodromal Parkinsons disease (PD), yet the underlying mechanisms remain unclear. To explore this relationship, we developed a zebrafish model that recapitulates prodromal PD-associated olfactory impairment without affecting motor function. We used zebrafish, due to their olfactory systems similarity to mammals and their unique nervous system regenerative capacity. By injecting 6-hydroxydopamine (6-OHDA) into the dorsal telencephalic ventricle, we observed a significant loss of dopaminergic (DA) periglomerular neurons in the olfactory bulb (OB) and retrograde degeneration of olfactory sensory neurons (OSNs) in the olfactory epithelium (OE). These alterations led to impaired responses to cadaverine, an aversive odorant, while responses to alanine, an attractive odorant, remained intact. 6-OHDA triggered robust neuroinflammatory responses that was attenuated by pranlukast, an anti-inflammatory drug. By 7 days post-injection, dopaminergic synapses in the OB were remodeled, OSNs in the OE appeared recovered, and neuroinflammation subsided, leading to full recovery of olfactory responses to cadaverine. These findings highlight zebrafish remarkable neuroplasticity and suggest this novel model of prodromal PD could provide valuable insights into early PD pathology. Understanding the interplay between dopaminergic loss, neuroinflammation, and olfactory dysfunction may inform therapeutic strategies for PD patients suffering from olfactory dysfunction.

neuroscience↗

bioRxiv2023

Structure of Orthoreovirus RNA Chaperone σNS, a Component of Viral Replication Factories

The reovirus {sigma}NS RNA-binding protein is required for formation of intracellular compartments during viral infection that support viral genome replication and capsid assembly. Despite its functional importance, a mechanistic understanding of {sigma}NS is lacking. We conducted structural and biochemical analyses of an R6A mutant of {sigma}NS that forms dimers instead of the higher-order oligomers formed by wildtype (WT) {sigma}NS. The crystal structure of selenomethionine-substituted {sigma}NS-R6A reveals that the mutant protein forms a stable antiparallel dimer, with each subunit having a well-folded central core and a projecting N-terminal arm. The dimers interact with each other by inserting the N-terminal arms into a hydrophobic pocket of the neighboring dimers on either side to form a helical assembly that resembles filaments of WT {sigma}NS in complex with RNA observed using cryo-EM. The interior of the crystallographic helical assembly is positively charged and of appropriate diameter to bind RNA. The helical assembly is disrupted by bile acids, which bind to the same hydrophobic pocket as the N-terminal arm, as demonstrated in the crystal structure of {sigma}NS-R6A in complex with bile acid. This finding suggests that the N-terminal arm functions in conferring context-dependent oligomeric states of {sigma}NS, which is supported by the structure of {sigma}NS lacking the N-terminal arm. We discovered that {sigma}NS displays RNA helix destabilizing and annealing activities, likely essential for presenting mRNA to the viral RNA-dependent RNA polymerase for genome replication. The RNA chaperone activity is reduced by bile acids and abolished by N-terminal arm deletion, suggesting that the activity requires formation of {sigma}NS oligomers. Our studies provide structural and mechanistic insights into the function of {sigma}NS in reovirus replication.

biochemistry↗

bioRxiv2022

A gut commensal protist protects against virus-mediated loss of oral tolerance

Loss of oral tolerance (LOT) to gluten, characterized by a T helper 1 (Th1) gluten-specific immune response, is a hallmark of celiac disease (CeD) and can be triggered by enteric viral infections. We hypothesized that certain gut microbes have the capacity to protect against virus-mediated LOT. By using our previously defined reovirus-mediated LOT CeD model, we discovered that the gut colonizing protist Tritrichomonas (T.) arnold promotes oral tolerance and protects against reovirus-mediated LOT by suppressing the reovirus-induced proinflammatory program of dietary-antigen-presenting CD103+ dendritic cells. Importantly, T. arnold did not affect antiviral host immunity, suggesting that T. arnold-mediated protection against T1L-induced LOT is not attributable to differences in antiviral host responses. Additionally, using gnotobiotic mice, we found that Tritrichomonas arnold colonization is sufficient to protect against reovirus-mediated LOT in the absence of the microbiota. Mechanistically, we show that Tritrichomonas arnold colonization restrains reovirus-induced inflammatory responses in dendritic cells and thus limit their ability to promote Th1 immune responses ex vivo. Finally, our studies using human stool samples support a role for Tritrichomonas sp. colonization in protecting against development of CeD. This study will motivate the design of effective therapies to prevent LOT to gluten in at-risk individuals and to reinstate tolerance to gluten in CeD patients. One Sentence SummaryTritrichomonas arnold protects against virus-mediated loss of oral tolerance to gluten and is underrepresented in celiac disease patients.

immunology↗

bioRxiv2021

Reovirus infection is regulated by NPC1 and endosomal cholesterol homeostasis

Cholesterol homeostasis is required for the replication of many viruses, including Ebola virus, hepatitis C virus, and human immunodeficiency virus-1. Niemann-Pick C1 (NPC1) is an endosomal-lysosomal membrane protein involved in cholesterol trafficking from late endosomes and lysosomes to the endoplasmic reticulum. We identified NPC1 in CRISPR and RNA interference screens as a putative host factor for infection by mammalian orthoreovirus (reovirus). Following internalization via clathrin-mediated endocytosis, the reovirus outer capsid is proteolytically removed, the endosomal membrane is disrupted, and the viral core is released into the cytoplasm where viral transcription, genome replication, and assembly take place. We found that reovirus infection is significantly impaired in cells lacking NPC1, but infection is restored by treatment of cells with hydroxypropyl-{beta}-cyclodextrin, which binds and solubilizes cholesterol. Absence of NPC1 did not dampen infection by infectious subvirion particles, which are reovirus disassembly intermediates that bypass the endocytic pathway for infection of target cells. NPC1 is not required for reovirus attachment to the plasma membrane, internalization into cells, or uncoating within endosomes. Instead, NPC1 is required for delivery of transcriptionally active reovirus core particles into the cytoplasm. These findings suggest that cholesterol homeostasis, ensured by NPC1 transport activity, is required for reovirus penetration into the cytoplasm, pointing to a new function for NPC1 and cholesterol homeostasis in viral infection. Author summaryGenetic screens are useful strategies to identify host factors required for viral infection. NPC1 was identified in independent CRISPR and RNA interference screens as a putative host factor required for reovirus replication. We discovered that NPC1-mediated cholesterol transport is dispensable for reovirus attachment, internalization, and disassembly but required for penetration of the viral disassembly intermediate from late endosomes into the cytoplasm. These findings pinpoint an essential function for cholesterol in the entry of reovirus and raise the possibility that cholesterol homeostasis regulates the entry of other viruses that penetrate late endosomes to initiate replication.

cell biology↗

bioRxiv2021

The human neuronal receptor NgR1 bridges reovirus capsid proteins to initiate infection

Human Nogo-66 receptor 1 (NgR1) is a receptor for mammalian orthoreoviruses (reoviruses), but the mechanism of virus-receptor engagement is unknown. NgR1 binds a variety of structurally dissimilar ligands in the adult central nervous system (CNS) to inhibit axon outgrowth. Disruption of ligand binding to NgR1 and subsequent signaling can improve neuron regrowth, making NgR1 an important therapeutic target for diverse conditions such as spinal crush injuries and Alzheimer disease. To elucidate how NgR1 mediates cell binding and entry of reovirus, we defined the affinity of interaction between virus and receptor, determined the structure of the virus-receptor complex, and identified residues in the receptor required for virus binding and infection. These studies revealed that NgR1 sequences in a central concave region of the molecule establish a bridge between two copies of the viral capsid protein, {sigma}3. This unusual binding interface produces high-avidity interactions between virus and receptor and likely primes early entry steps. NgR1 sequences engaged by reovirus also are required for NgR1 binding to ligands expressed by neurons and oligodendrocytes. These studies redefine models of reovirus cell-attachment and highlight the evolution of viruses to engage multiple receptors using distinct capsid components.

microbiology↗

bioRxiv2020

Neuron-intrinsic NF-κB Signaling Mediates Reovirus Virulence

Pathological effects of apoptosis associated with viral infections of the central nervous system are an important cause of morbidity and mortality. Reovirus is a neurotropic virus that causes apoptosis in neurons, leading to lethal encephalitis in newborn mice. Reovirus-induced encephalitis is diminished in mice with germline ablation of NF-{kappa}B subunit p50. It is not known whether the pro-apoptotic function of NF-{kappa}B is mediated by neuron-intrinsic processes, NF-{kappa}B-regulated cytokine production by inflammatory cells, or a combination of both. To determine the contribution of cell type-specific NF-{kappa}B signaling in reovirus-induced neuronal injury, we established mice that lack NF-{kappa}B p65 expression in neurons using the Cre/loxP recombination system. Following intracranial inoculation of reovirus, 50% of wild-type (WT) mice succumbed to infection, whereas more than 90% of mice lacking neural NF-{kappa}B p65 (Nsp65-/-) mice survived. While viral loads in brains of WT and Nsp65-/- were comparable, histological analysis revealed that reovirus antigen-positive areas in the brain of WT mice displayed enhanced cleaved caspase-3 immunoreactivity, a marker of apoptosis, compared with Nsp65-/- mice. These data suggest that neuron-intrinsic NF-{kappa}B-dependent factors are essential mediators of reovirus neurovirulence. RNA sequencing analysis of reovirus-infected cortices of WT and Nsp65-/- mice suggests that NF-{kappa}B activation in neurons upregulates genes involved in innate immunity, inflammation, and cell death following reovirus infection. A better understanding of the contribution of cell type-specific NF-{kappa}B-dependent signaling to viral neuropathogenesis could inform development of new therapeutics that target and protect highly vulnerable cell populations

microbiology↗

bioRxiv2020

Cytidine monophosphate N-acetylneuraminic acid synthetase and solute carrier family 35 member A1 are required for reovirus binding and infection

Engagement of cell-surface receptors by viruses is a critical determinant of viral tropism and disease. The reovirus attachment protein, {sigma}1, binds sialylated glycans and proteinaceous receptors to mediate infection, but the specific requirements on different cell types are unknown. To identify host factors required for reovirus-induced cell death, we conducted a CRISPR-knockout screen targeting over 20,000 genes in murine microglial BV2 cells. Candidate genes identified as required for reovirus to cause cell death were highly enriched for sialic acid synthesis and transport. Two of the top candidates identified, cytidine monophosphate N-acetylneuraminic acid synthetase (Cmas) and solute carrier family 35 member A1 (Slc35a1), promote sialic acid expression on the cell surface. Two reovirus strains differing in the capacity to bind sialic acid, T3SA+ and T3SA-, were used to evaluate Cmas and Slc35a1 as potential host genes required for infection. Following CRISPR-Cas9 disruption of either gene, cell-surface expression of sialic acid was diminished. These results correlated with decreased binding of strain T3SA+, which is capable of engaging sialic acid. Disruption of either gene did not alter the low-level binding of T3SA-, which does not engage sialic acid. Infectivity of T3SA+ was diminished to levels of T3SA-in cells lacking Cmas and Slc35a1 by CRISPR ablation. However, exogenous expression of Cmas and Slc35a1 into the respective null cells restored sialic acid expression and T3SA+ binding and infectivity. These results demonstrate that Cmas and Slc35a1, which mediate cell-surface expression of sialic acid, are required in murine microglial cells for efficient reovirus binding and infection. IMPORTANCEAttachment factors and receptors are important determinants of dissemination and tropism during reovirus-induced disease. In a CRISPR cell-survival screen, we discovered two genes, Cmas and Slc35a1, which encode proteins required for sialic acid expression on the cell surface, that mediate reovirus infection of microglial cells. This work elucidates host genes that render microglial cells susceptible to reovirus infection and expands current understanding of the receptors on microglial cells that are engaged by reovirus. Such knowledge may lead to new strategies to selectively target microglial cells for oncolytic applications.

molecular biology↗

bioRxiv2020

Combinatorial metabolic engineering platform enabling stable overproduction of lycopene from carbon dioxide by cyanobacteria

Cyanobacteria are simple, efficient, genetically-tractable photosynthetic microorganisms representing ideal biocatalysts for CO2 capture and conversion, in principle. In practice, genetic instability and low productivity are key, linked problems in engineered cyanobacteria. We took a massively parallel approach, generating and characterising libraries of synthetic promoters and RBSs for the cyanobacterium Synechocystis, and assembling a sparse combinatorial library of millions of metabolic pathway-encoding construct variants. Laboratory evolution suppressed variants causing metabolic burden in Synechocystis, leading to expected genetic instability. Surprisingly however, in a single combinatorial round without iterative optimisation, 80% of variants chosen at random overproduced the valuable terpenoid lycopene from atmospheric CO2 over many generations, apparently overcoming the trade-off between stability and productivity. This first large-scale parallel metabolic engineering of cyanobacteria provides a new platform for development of genetically stable cyanobacterial biocatalysts for sustainable light-driven production of valuable products directly from CO2, avoiding fossil carbon or competition with food production.

synthetic biology↗

bioRxiv2019

Transcriptional terminators allow leak-free chromosomal integration of genetic constructs in cyanobacteria

Cyanobacteria are promising candidates for sustainable bioproduction of chemicals from sunlight and carbon dioxide. However, the genetics and metabolism of cyanobacteria are less well understood than model heterotrophic organisms, and the suite of well characterised cyanobacterial genetic tools and parts is less mature and complete. Transcriptional terminators use specific RNA structures to halt transcription and are routinely used in both natural and recombinant contexts to achieve independent control of gene expression and insulate genes and operons from one another. Insulating gene expression can be particularly important when heterologous/synthetic genetic constructs are inserted at genomic locations where transcriptional read-through from chromosomal promoters occurs, resulting in poor control of expression of the introduced genes. To date, few terminators have been described and characterised in cyanobacteria. In this work, nineteen heterologous, synthetic or putative native Rho-independent (intrinsic) terminators were tested in the model freshwater cyanobacterium, Synechocystis sp. PCC 6803, from which eleven strong terminators were identified. A subset of these strong terminators was then used to successfully insulate a chromosomally-integrated rhamnose-inducible rhaBAD expression system from hypothesised read-through from a neighbouring chromosomal promoter, resulting in greatly improved inducible control. The addition of validated strong terminators to the cyanobacterial toolkit will allow improved independent control of introduced genes.

synthetic biology↗

bioRxiv2018

Start-Stop Assembly: a functionally scarless DNA assembly system optimised for metabolic engineering.

DNA assembly allows individual DNA constructs or designed mixtures to be assembled quickly and reliably. Most methods are either: (i) Modular, easily scalable and suitable for combinatorial assembly, but leave undesirable scar sequences; or (ii) bespoke (non-modular), scarless but less suitable for construction of combinatorial libraries. Both have limitations for metabolic engineering. To overcome this trade-off we devised Start-Stop Assembly, a multi-part, modular DNA assembly method which is both functionally scarless and suitable for combinatorial assembly. Crucially, 3 bp overhangs corresponding to start and stop codons are used to assemble coding sequences into expression units, avoiding scars at sensitive coding sequence boundaries. Building on this concept, a complete DNA assembly framework was designed and implemented, allowing assembly of up to 15 genes from up to 60 parts (or mixtures); monocistronic, operon-based or hybrid configurations; and a new streamlined assembly hierarchy minimising the number of vectors. Only one destination vector is required per organism, reflecting our optimisation of the system for metabolic engineering in diverse organisms. Metabolic engineering using Start-Stop Assembly was demonstrated by combinatorial assembly of carotenoid pathways in E. coli resulting in a wide range of carotenoid production and colony size phenotypes indicating the intended exploration of design space.\n\nGRAPHICAL ABSTRACT\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=153 SRC=\"FIGDIR/small/361626_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (45K):\norg.highwire.dtl.DTLVardef@1cdd32eorg.highwire.dtl.DTLVardef@1e394cforg.highwire.dtl.DTLVardef@3ec8f5org.highwire.dtl.DTLVardef@1fb72e7_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗