Biology subjects

Connor, S.

Publications and source records attributed to Connor, S..

8 recordsLinked to original sources

bioRxiv2026

SARS-CoV-2 Defective Viral Genomes from Distinct Genomic Regions Drive Divergent Interferon Responses

Defective viral genomes (DVGs) are naturally generated during genomic replication of many RNA viruses. When produced early in infection or supplemented at the onset of infection, DVGs can attenuate viral pathogenesis by stimulating IFN responses and antagonizing wild type (WT) virus replication, highlighting their potential as antiviral therapeutics. However, during natural infection DVGs can exert both antiviral and proviral effects depending on their generation kinetics, species, and abundance, underscoring the need to better understand their roles in viral pathogenesis. Coronaviruses (CoVs) remain a major global health threat and ubiquitously generate DVGs, yet DVGs roles during CoV infection are largely unknown. Using SARS-CoV-2 as a model, we previously identified DVGs in vitro and in patient samples and discovered two major genomic hotspots (A and B) for their generation. Here, we first showed that overall DVG abundance tended to positively correlate with COVID-19 severity, with approximately 40% of DVGs originating from a specific genomic region designated hotspot B. Analysis of a publicly available single-cell RNA-seq datasets revealed that DVGs from hotspot B, but not hotspot A, were associated with elevated IFN responses, suggesting that DVGs derived from different genomic regions vary in their ability to stimulate innate immunity. To test this directly, we constructed two representative DVGs corresponding to hotspots A and B. Both DVGs suppressed the replication of co-infecting WT virus; however, only DVG-B induced robust IFN responses, exceeding those triggered by WT virus alone. This was further confirmed in human precision-cut lung slices. Mechanistically, DVG-B-derived dsRNA exhibited a distinct subcellular distribution compared with WT virus. Complementation with the nucleocapsid (N) partially restored dsRNA organization but did not alter the IFN response. Together, our findings demonstrate that DVGs arising from distinct genomic hotspots differentially regulate IFN responses, potentially contributing to varied pathogenic outcomes during SARS-CoV-2 infection. Author summaryDefective viral genomes (DVGs) are naturally produced during RNA virus infection and can suppress viral pathogenesis by stimulating innate immune responses. However, their roles in coronavirus infection, particularly SARS-CoV-2, remain poorly understood. This study investigated the species-specific function of DVGs generated during SARS-CoV-2 infection and their impact on disease outcomes. Cohort analysis revealed that overall DVG abundance tended to positively correlate with COVID-19 severity, with approximately 40% of DVGs originating from a specific genomic region designated hotspot B. Single-cell RNA sequencing showed that DVGs from hotspot B, but not hotspot A, were associated with elevated IFN responses, suggesting that DVGs from different genomic regions vary in their immunostimulatory capacity. To directly test this, we constructed representative DVGs from both hotspots. While both suppressed wild-type virus replication, only DVG-B induced robust IFN responses both in vitro and ex vivo. Mechanistically, DVG-B produced dsRNA with distinct subcellular distribution compared to wild-type virus. Interestingly, complementation with the viral nucleocapsid protein partially restored dsRNA organization but did not alter IFN responses. These findings demonstrate that SARS-CoV-2 DVGs arising from different genomic hotspots differentially regulate innate immunity, potentially contributing to varied pathogenic outcomes during infection.

microbiology↗

bioRxiv2025

A Respiratory Syncytial Virus trailer sequence modulates viral replication and copy-back defective viral genome generation and propagation kinetics

Copy-back defective viral genomes (cbDVGs) are key inducers of antiviral responses during negative-sense RNA virus infection. Once considered byproducts of in vitro viral replication, cbDVGs have since been detected in clinical specimens and implicated in affecting infection outcomes. The molecular mechanism of cbDVG generation remains unclear, thereby hindering our ability to manipulate cbDVG production during infection for therapeutic gain. Previous work showed that respiratory syncytial virus (RSV) cbDVG re-initiation sites cluster in trailer-end hotspots R1, R2, and R3, and that a poly-U mutation in R1 selectively reduced cbDVG formation at the mutated region. Here, we reported that a 10U mutation in R2 drastically reduced cbDVGs in this region in both minigenome and recombinant virus systems. Furthermore, during high-MOI passaging of the R2-10U virus, we observed delayed detection of cbDVGs with re-initiation sites in R1-R3 (trailer cbDVGs) compared to WT, while no differences in virus titers were observed. Interestingly, we observed the rapid emergence and accumulation of a viral variant bearing a 2-ribonucleotide deletion (R2-8U) within the R2-10U mutation sequence as early as P0. Compared to R2-10U, the R2-8U virus was stable, displayed faster generation and accumulation of trailer cbDVGs, restored cbDVGs with R2 re-initiation sites, and exhibited enhanced genomic replication. Overall, our data identify a sequence in the RSV trailer whose mutation critically modulates both viral replication and the generation/propagation of trailer cbDVGs. Our data also suggest that cbDVG generation, particularly near the trailer, may be an evolutionary tradeoff for more rapid virus genomic replication.

microbiology↗

bioRxiv2024

Transcriptomic signatures of host immune responses in aphthous ulcers, the earliest lesions of Crohn's disease, suggest that a bacterial invasive challenge, rather than global dysbiosis, is the initiating factor.

Crohns disease is a chronic, transmural inflammatory disease of the human gut. Changes in the faecal microbial composition and a reduction in species diversity (dysbiosis) are consistent features in studies of Crohns disease patients, but whether dysbiosis is a cause or consequence of inflammation remains unresolved. Genetic susceptibility also plays a role in the development of Crohns disease and has been linked to genes involved in recognition of intestinal bacteria by cells of the mononuclear phagocyte system. The earliest visible lesions in Crohns disease are aphthous ulcers, overlying Peyers patches and lymphoid follicles. To identify mechanisms underlying the earliest stages of disease initiation we compared gene expression in aphthous ulcers, Peyers patches, inflamed and endoscopically normal mucosa from patients and controls using total RNA-seq. The resulting data was subjected to network analysis to identify co-regulated gene expression signatures of cell types and processes. These results were compared to recent single cell RNA-seq analysis of intestinal macrophages in normal and diseased mucosa. The analysis of aphthous ulcers revealed signatures of epithelial stress and antimicrobial defence, plasma cell activation and immunoglobulin production, monocyte recruitment, inflammatory gene expression and induction of interferon-{gamma} and downstream target genes. These signatures were not present in the normal appearing mucosa adjacent to aphthous ulcers which were similar to healthy control mucosa. We conclude that the initial lesion in Crohns disease arises from an invasive bacterial challenge leading to intense activation of multiple host defence pathways rather than the breakdown of epithelial barrier integrity and widespread bacterial translocation.

immunology↗

bioRxiv2024

Panx1 deletion promotes synaptic reconfiguration and recruitment of cell death pathways in the Zebrafish MPTP model

Pannexin-1 channels have garnered attention for their implications in neurodevelopment, potentially having a dual role in mediating a delicate balance between cell death and survival. However, a comprehensive understanding of the underlying molecular and cellular mechanisms and Panx1s potential protective functions throughout neurodevelopment remains to be determined. Zebrafish larvae with loss of Pannexin-1a function were subjected to an acute exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Early-life changes in larvae induced by uncoupling of oxidative phosphorylation were investigated by a computational Gene Set Expression Analysis of RNA-seq data and experimental testing of light-stimulated locomotor behavior, cell death, and bioelectrical properties of local field potentials in the ascending visual pathway. A KEGG pathway analysis underscored Panx1as regulatory influence on neurodevelopment. Targeting Panx1a caused a deregulation of oxidative phosphorylation, glycolysis, reactive oxygen production, hypoxia, unfolded protein response pathways, and reduced extracellular ATP. Further, Panx1a ablation enhanced the transcriptional activation of 5 AMP-activated protein kinase (AMPK) kinase, a cellular energy sensor activated by falling energy status, largely to activate glucose and fatty acid uptake and oxidation when cellular energy is low. The activation of the AMPK pathway in Panx1a knock-out larvae correlated with the stimulation of the mammalian target of rapamycin (mTORC1) pathway that controls cellular metabolism, catabolism, immune responses, autophagy, survival, proliferation, and migration, to maintain cellular homeostasis. The differential expression of mTORC1 pathway genes associated with autophagy, and apoptosis signaling pathways. The resultant cell death was pronounced in the pallium and tectum regions. The loss of cells interrelated with a trans-synaptic a loss of synaptic neurotransmitter receptor and ion channel/transporter expression. Local field potential recordings in the optic tectum and pallium demonstrated that Panx1as involvement in modulating local neuronal networks was altered. Collectively, the results shed light on the impacts of acute MPTP treatment on locomotor behavior, transcriptomic shifts, metabolic disturbances, and the pivotal role of Panx1a in cell death. These insights enhance our comprehension of the intricate molecular and cellular mechanisms underpinning neurodevelopment, with implications for potential therapeutic strategies targeting Panx1 channels in autism and Alzheimers disease. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=172 HEIGHT=200 SRC="FIGDIR/small/590821v2_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1a8688eorg.highwire.dtl.DTLVardef@16849a3org.highwire.dtl.DTLVardef@1ba8298org.highwire.dtl.DTLVardef@1d3efff_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIA genetic model was combined with the neurotoxin MPTP to explore the roles of zebrafish Pannexin-1 channels in neurodevelopment under oxidative stress conditions. C_LIO_LIA potential beneficial impact of targeting Panx1a is superseded by synaptic plasticity loss, dysfunctional mitochondrial metabolism, and cell death pathway activation. C_LIO_LILoss of Panx1a amplifies AMPK/mTORC1 pathway activation of cell death pathways. C_LIO_LIA role of Panx1a as a regulator of energy and synaptic homeostasis was identified. C_LI

neuroscience↗

bioRxiv2022

Lung tumor-infiltrating Treg have divergent transcriptional profiles and function linked to checkpoint blockade response

Regulatory T cells (Treg) are conventionally viewed to suppress endogenous and therapyinduced anti-tumor immunity; however, their role in modulating responses to immune checkpoint blockade (ICB) is unclear. In this study, we integrated single-cell RNAseq/TCRseq of >73,000 tumor-infiltrating Treg (TIL-Treg) from anti-PD-1-treated and treatment naive non-small cell lung cancers (NSCLC) with single cell analysis of tumor-associated antigen (TAA)-specific Treg derived from a murine tumor model. We identified 10 subsets of human TIL-Treg, most of which have high concordance with murine TIL-Treg subsets. Notably, one subset selectively expresses high levels of OX40 and GITR, whose engangement by cognate ligand mediated proliferative programs and NF-kB activation, as well as multiple genes involved in Treg suppression, in particular LAG3. Functionally, the OX40hiGITRhi subset in the most highly suppressive ex vivo and Treg expression of OX40, GITR and LAG3, correlated with resistance to PD-1 blockade. Surprisingly, in the murine tumor model, we found that virtually all TIL-Treg expressing T cell receptors that are specific for TAA fully develop a distinct Th1-like signature over a two-week period after entry into the tumor, down-regulating FoxP3 and up-regulating expression of TBX21 (Tbet), IFN{gamma} and certain pro-inflammatory granzymes. Application of a gene score from the murine TAA-specific Th1-like Treg subset to the human single-cell dataset revealed a highly analogous subcluster that was enriched in anti-PD-1 responding tumors. These findings demonstrate that TIL-Treg partition into multiple distinct transcriptionally-defined subsets with potentially opposing effects on ICB-induced anti-tumor immunity and suggest that TAA-specific TIL-Treg may positively contribute to anti-tumor responses. One-Sentence SummaryWe define 10 subsets of lung cancer-infiltrating regulatory T cells, one of which is highly suppressive and enriched in anti-PD-1 non-responders and the other is Th1-like and is enriched in PD-1 responders.

immunology↗

bioRxiv2021

An extremes of phenotype approach confirms significant genetic heterogeneity in patients with ulcerative colitis.

Ulcerative colitis (UC) is a major form of inflammatory bowel disease with increasing global incidence. There is significant phenotypic heterogeneity defined by a range of clinical variables including age of onset and disease extent. Clinical outcomes range from long-term remission on minimal therapy to surgical resection. Close to 70% of UC risk can be attributed to genetics and understanding the genetic mechanisms contributing to this risk and disease heterogeneity is vital for understanding disease pathogenesis and improving patient outcomes through targeted screening and therapies. This study aims to characterise the genetic heterogeneity of UC by identifying genomic risk variants specific to mild and/or severe forms of UC, exploring variations in the effect size of known risk variants and assessing the clinical value of a genetic risk score (GRS). We conducted genome-wide association (GWA) analyses in 287 patients with mild UC, 311 patients with severe UC and 583 age- and gender-matched controls. Odds ratios (OR) for mild vs control, severe vs control and combined mild and severe UC vs control were calculated. Using the combined UC data, two independent loci in the HLA region reached genome-wide significance. An additional genome-wide significant signal on chromosome 1 was identified in severe cases only. OR for known risk loci varied between mild and severe patients and were similar to previously published results. Effect estimates from the most recent UC GWA meta-analysis were used to calculate a GRS for each individual. A higher mean GRS was observed in both mild and severe UC cases compared to controls however, there was no difference between the mean GRS for mild and severe UC. Heterogeneity in effect sizes of UC associated variants between mild and severe disease burden suggests the presence of genetically distinct signatures. While large consortium data are needed to identify genome-wide significant variants, additional risk loci may be identified by targeted recruitment of individuals with a history of severe disease. Author SummaryUlcerative colitis (UC) is a chronic and often debilitating form of inflammatory bowel disease affecting approximately 0.3% of the population in industrialized economies. The disease displays significant clinical heterogeneity including age at presentation, disease severity, and the propensity to develop disease-related complications. Several previous studies have demonstrated the heritability of UC, identifying over 30 loci specific to the disease. The majority of these loci have small to modest effect sizes other than those within the Human Leucocyte Antigen (HLA) region on chromosome 6. Using stringent clinical criteria for defining mild and severe forms of UC in an extremes of phenotype approach, we undertook a genome wide association study in a dataset of 1222 participants to investigate genetic heterogeneity in this disease. We demonstrated substantial differences in genetic associations in severe UC as compared to mild UC. While over 2,000 SNPs achieved genome-wide significance in the severe UC analysis, none reached significance for mild UC. These results were reflected in significant differences in odds ratios. We identified Complement Factor B (CFB) as a potential susceptibility gene for severe UC in the Caucasian population with additional tissue gene expression demonstrating a positive correlation with disease severity.

genetics↗

bioRxiv2019

Sub-nucleosomal organization in urine cell-free DNA

Cell-free DNA (cfDNA) in urine is a promising analyte for noninvasive diagnostics. However, urine cfDNA is highly fragmented and whether characteristics of these fragments reflect underlying genomic architecture is unknown. Here, we perform comprehensive characterization of fragmentation patterns in urine cfDNA. We show modal size and genome-wide distribution of urine cfDNA fragments are consistent with transient protection from degradation by stable intermediates of nucleosome disassembly. Genome-wide nucleosome occupancy and fragment sizes in urine cfDNA are informative of cell of origin and renal epithelial cells are amongst the highest contributors in urine. Compared to a nucleosome occupancy map based on control urine samples, we observe a higher fraction of fragments with aberrant ends in cancer patients, distinguishing cancer samples with an area under the curve of 0.89. Our results demonstrate sub-nucleosomal organization in urine cfDNA and are proof-of-principle that genome-wide fragmentation analysis of urine cfDNA can enable cancer diagnostics.

genomics↗

bioRxiv2017

Recurrent loss of heterozygosity correlates with clinical outcome in pancreatic neuroendocrine cancer

Pancreatic neuroendocrine tumors (pNETs) are uncommon cancers arising from pancreatic islet cells. Analysis of gene mutation, copy number and RNA expression of 57 sporadic pNETs showed that pNET genomes are dominated by aneuploidy. Remarkably, ~25% of pNETs had genomes characterized by recurrent loss of heterozygosity (LoH) of the same 10 chromosomes, accompanied by bi-allelic MEN1 inactivation, and these cases had generally poor clinical outcome. Another ~25% of all pNETs had chromosome 11 LoH and bi-allelic MEN1 inactivation, lacking the recurrent LoH pattern - these had universally good clinical outcome. Some level of aneuploidy was common, and overall ~80% of pNETs had LoH of [≥]1 chromosome. This aneuploidy led to changes in RNA expression at the level of whole chromosomes and allowed pathogenic germline variants (e.g. ATM) to be expressed unopposed, inactivating downstream tumor suppressor pathways. Some pNETs appear to utilize VHL gene methylation or mutation to activate pseudo-hypoxia. Contrary to expectation neither tumor morphology within well-differentiated pNETs nor single gene mutation had significant associations with clinical outcome, nor did expression of RNAs reflecting the activity of immune, differentiation, proliferative or tumor suppressor pathways. MEN1 was the only statistically significant recurrently mutated driver gene in pNETs. Only one pNET had clearly oncogenic and actionable SNVs (in PTEN and FLCN) confirmed by corroborating RNA expression changes. The two distinct patterns of aneuploidy described here, associated with markedly poor and good clinical outcome respectively, define a novel oncogenic mechanism and the first route to genomic precision oncology for this tumor type.

cancer biology↗