Biology subjects

Evans, S. A.

Publications and source records attributed to Evans, S. A..

10 recordsLinked to original sources

bioRxiv2026

Coevolutionary mining of prokaryotic non-coding elements with a genome language model

Microbial genomes encode compact molecular machines and diverse non-coding RNAs (ncRNAs) essential to gene regulation, pathogenesis, and many foundational biotechnologies. However, annotation remains largely protein-centric and homology-driven. Here, we introduce Minerva, a framework for coevolutionary mining that uses genome language models to predict local interactions directly from sequence as two-dimensional maps. Introducing two complementary techniques, categorical Jacobian fingerprinting and interaction heads, we demonstrate fast, accurate, alignment-free prediction of ncRNA base-pairing, monomeric protein contacts, and repetitive sequence motifs. Applied to 150 bacterial genomes, Minerva recovers known systems and predicts that 84.3% of predicted intergenic base-pairing falls outside of known annotations. In Pseudomonas, we find that the widespread TwoAYGGAY ncRNA family carries large secondary-structure extensions and is often flanked by short upstream repetitive motifs and larger downstream genomic repeats. Interpreting the coevolution maps, we find that Minerva emergently detects open-reading-frame (ORF) signatures at the DNA level despite never being trained to do so. In prophages within these genomes, we discover that Unknown Group 27 (UG27) reverse transcriptase systems encode arrays of structurally conserved yet sequence-diverse ncRNAs that template complementary DNA (cDNA) hairpin products. Together, these results establish coevolutionary mining as a scalable route to genome annotation and biological discovery across the rapidly expanding microbial universe.

bioinformatics↗

bioRxiv2026

Phage terminase recognition by the bacterial immune sensors Avs2 and Upx

Prokaryotes employ diverse defense strategies to detect and halt the progression of phage infection. Multiple defense systems sense phage proteins through direct binding, including antiviral STAND NTPases (Avs), which oligomerize upon target recognition to induce programmed cell death. The widespread Avs2 family was previously shown to detect the large terminase subunit of tailed phages, but the mechanism of terminase sensing was unknown. Here, we determine the structural basis of terminase recognition by Avs2 from Escherichia coli (EcAvs2). A cryo-EM structure at 2.3 [A] resolution reveals that EcAvs2 forms a flat, C4-symmetric tetramer in which each protomer is bound to a single terminase monomer. Terminase recognition is mediated by a large, shape complementary binding pocket in the EcAvs2 sensor domain, including specific contacts with an unexpected ATP molecule at the interface of EcAvs2 and terminase. Furthermore, we demonstrate that the defense protein Upx also recognizes diverse phage terminases, despite lacking sequence and structural homology to Avs. AlphaFold 3 models indicate that Upx binds an unfolded state of the core terminase ATPase domain, mediated by {beta}-augmentation. These findings highlight the distinct modes of terminase recognition across structurally diverse defense proteins.

molecular biology↗

bioRxiv2026

Diverse bacterial pattern recognition receptors sense the conserved phage proteome

Recognition of foreign molecules inside cells is critical for immunity across all domains of life. Proteins of the STAND NTPase superfamily1,2, including eukaryotic NOD-like receptors (NLRs), play a central role in this process3,4. In bacteria and archaea, although several STAND families sense phage proteins5-9, their functional diversity remains largely unexplored. We conducted a systematic phylogenetic analysis of prokaryotic STAND NTPases and identified at least 90 structurally distinct families associated with antiviral defense. We first show that the uncharacterized Avs7 family recognizes the major capsid protein (MCP) of tailed phages. Three cryo-EM structures of Salmonella enterica Avs7 reveal an asymmetric, butterfly-shaped tetramer that assembles stepwise via large, MCP-induced conformational changes, incorporating bacterial elongation factor Tu (EF-Tu) as a structural component that enhances defense. Using genetic screens with a library of 687 phage genes, we further show that 13 additional STAND families sense 13 conserved phage proteins, encompassing most of the core structural and replicative components of tailed phages. These include two distinct MCP-sensing families--Avs8 (PD-{lambda}-4) and Avs10 (Erebus/Hypnos/bNACHT64)--and 11 others (Avs11-21) recognizing the portal, portal adaptor, tail nozzle, head-tail connector, tail terminator, tail tube protein, tail assembly chaperone, tape measure protein, DNA polymerase, helicase/RecA-type ATPase, and single-stranded DNA annealing protein, respectively. Together, our findings highlight structurebased pattern recognition and host factor repurposing as fundamental strategies of bacterial immunity.

molecular biology↗

bioRxiv2025

Proteolytic activation of diverse antiviral defense modules in prokaryotes

Linked protease-effector modules are widespread in prokaryotic antiviral defense, yet the mechanisms of most remain poorly understood. Here we show that four of the most prevalent modules--metallo-{beta}-lactamase (MBL)-fold hydrolase, /{beta}-hydrolase, Pepco, and EACC1--form latent death effectors that are unleashed by site-specific proteolysis. Genetic, biochemical, and structural analyses reveal novel modes of effector licensing. MBL and /{beta}-hydrolase are zymogens activated by cleavage at two distinct sites, and upon proteolysis, MBL becomes a Zn2+-dependent double-stranded DNA nuclease. In contrast, Pepco and EACC1 act as pore-forming toxins via distinct mechanisms: Pepco constitutively oligomerizes into a denaturation-resistant beta barrel that is activated by cleavage after a specific isoleucine in its C-terminal tail, whereas EACC1 monomers assemble into large membrane pores following removal of an autoinhibitory domain. All four modules are fused to diverse sensors to detect a wide range of phage signals, and EACC1-DnaK chaperone fusions suggest a convergence between defense and general stress responses. These findings establish proteolysisgated activation as a dominant, modular logic for anti-phage defense and reveal parallels with eukaryotic innate immunity.

molecular biology↗

bioRxiv2025

Senescence-Associated Chromatin Rewiring Promotes Inflammation and Transposable Element Activation

Cellular senescence is a stable form of cell cycle arrest that contributes to aging and age-associated diseases through the secretion of inflammatory factors collectively known as the senescence-associated secretory phenotype (SASP). While senescence is driven by transcriptional and epigenetic changes, the contribution of higher-order genome organization remains poorly defined. Here, we present the highest-resolution Hi-C maps ([~]3 kb) to date of proliferating, quiescent, and replicative senescent (RS) human fibroblasts, enabling a comprehensive analysis of 3D genome architecture during senescence. Our analyses reveal widespread senescence-associated remodeling of chromatin architecture, including extensive compartment and subcompartment switching toward transcriptionally active states, and a dramatic increase in unique chromatin loops. These structural features correlate with local DNA hypomethylation and are largely independent of canonical CTCF binding. The altered 3D genome landscape supports expression of SASP genes, inflammation-related pathways, and neuronal gene signatures consistent with age-associated epigenetic drift. We further demonstrate that architectural changes at multiple levels, including compartments, subcompartments, and loops, facilitate the derepression of LINE-1 retrotransposons, linking 3D chromatin structure to activation of proinflammatory transposable elements. Interestingly, quiescent cells, commonly used as senescence controls, exhibited substantial overlap in inflammatory gene expression with senescent cells, raising important considerations for experimental design. Structural analysis of cell cycle genes showed distinct chromatin configurations in senescence versus quiescence, despite similar transcriptional repression. Together, our results establish a high-resolution framework for understanding how genome architecture contributes to the senescent state.

genomics↗

bioRxiv2024

Islet-antigen reactive B cells display a unique phenotype and BCR repertoire in autoantibody positive and recent-onset type 1 diabetes patients

Autoreactive B cells play an important but ill-defined role in autoimmune type 1 diabetes (T1D). To better understand their contribution, we performed single cell gene and BCR-seq analysis on pancreatic islet antigen-reactive (IAR) B cells from the peripheral blood of nondiabetic (ND), autoantibody positive prediabetic (AAB), and recent-onset T1D individuals. We found that the frequency of IAR B cells was increased in AAB and T1D. IAR B cells from these donors had altered expression of B cell signaling, pro-inflammatory, infection, and antigen processing and presentation genes. Both AAB and T1D donors demonstrated a significant increase in certain heavy and light chain V genes, and these V genes were enriched in islet-reactivity. Public clones of IAR B cells were restricted almost entirely to AAB and T1D donors. IAR B cells were clonally expanded in the autoimmune donors, particularly the AAB group. Notably, a substantial fraction of IAR B cells in AAB and T1D donors appeared to be polyreactive, which was corroborated by analysis of recombinant monoclonal antibodies. These results expand our understanding of autoreactive B cell activation during T1D and identify unique BCR repertoire changes that may serve as biomarkers for increased disease risk. One Sentence SummaryPancreatic islet antigen-reactive B cells from individuals with prediabetes and recently diagnosed with type 1 diabetes display a unique phenotype and BCR repertoire compared to non-diabetic donors.

immunology↗

bioRxiv2024

Disrupting CD38-driven T cell dysfunction restores sensitivity to cancer immunotherapy

A central problem in cancer immunotherapy with immune checkpoint blockade (ICB) is the development of resistance, which affects 50% of patients with metastatic melanoma1,2. T cell exhaustion, resulting from chronic antigen exposure in the tumour microenvironment, is a major driver of ICB resistance3. Here, we show that CD38, an ecto-enzyme involved in nicotinamide adenine dinucleotide (NAD+) catabolism, is highly expressed in exhausted CD8+ T cells in melanoma and is associated with ICB resistance. Tumour-derived CD38hiCD8+ T cells are dysfunctional, characterised by impaired proliferative capacity, effector function, and dysregulated mitochondrial bioenergetics. Genetic and pharmacological blockade of CD38 in murine and patient-derived organotypic tumour models (MDOTS/PDOTS) enhanced tumour immunity and overcame ICB resistance. Mechanistically, disrupting CD38 activity in T cells restored cellular NAD+ pools, improved mitochondrial function, increased proliferation, augmented effector function, and restored ICB sensitivity. Taken together, these data demonstrate a role for the CD38-NAD+ axis in promoting T cell exhaustion and ICB resistance, and establish the efficacy of CD38 directed therapeutic strategies to overcome ICB resistance using clinically relevant, patient-derived 3D tumour models.

cancer biology↗

bioRxiv2021

Single Cell Transcriptomics Reveals Global Markers of Transcriptional Diversity Across Different Forms of CellularSenescence

Cellular Senescence is a state of irreversible cell cycle arrest, and the accumulation of senescent cells contributes to age- related organismal decline. The detrimental effects of cellular senescence are due to the senescence associated secretory phenotype (SASP), an array of signaling molecules and growth factors secreted by senescent cells that contribute to the sterile inflammation associated with aging tissues. Recent studies, both in vivo and in vitro, have highlighted the heterogeneous nature of the senescence phenotype. In particular, single cell transcriptomics has revealed that Oncogene Induced Senescence (OIS) is characterized by the presence of subpopulations of cells expressing different SASP profiles. We have generated a comprehensive dataset via single-cell transcriptional profiling of genetically homogenous clonal cell lines from different forms of senescence, including OIS, Replicative Senescence (RS), and DNA Damage Induced Senescence (DDIS). We identified subpopulations of cells that are common to all three major forms of senescence and show that the expression profiles of these subpopulations are driven by markers formerly identified in individual forms of senescence. These common signatures are characterized by chromatin modifiers, inflammation, extracellular matrix remodeling, and Ribosomal protein expression. The expression patterns of these subpopulations recapitulate primary and secondary senescence, a phenomenon where a preexisting (primary) senescent cell induces senescence in a neighboring (secondary) cell through cell-to-cell contact. Since it is still unclear what type of senescence occurs in-vivo with age, it is important to know that the formation of primary and secondary populations is common to multiple types of senescence since this mechanism could help explain how senescent cells accumulate in aged organisms. Finally, we show that these subpopulations show differential susceptibility to the senolytic agent Navitoclax, suggesting that senolytic agents targeting the apoptotic pathways may be clearing only a subset of senescent cells based on their inflammatory profiles in-vivo.

genomics↗

bioRxiv2021

Single cell analysis of the aging hypothalamus

Alterations in metabolism, sleep patterns, body composition, and hormone status are all key features of aging. The hypothalamus is a well-conserved brain region that controls these homeostatic and survival-related behaviors. Despite the importance of this brain region in healthy aging, little is known about the intrinsic features of hypothalamic aging. Here, we utilize single nuclei RNA-sequencing to assess the transcriptomes of 40,064 hypothalamic nuclei from young and aged female mice. We identify cell type-specific signatures of aging in neurons, astrocytes, and microglia, as well as among the diverse collection of neuronal subtypes in this region. We uncover key changes in cell types critical for metabolic regulation and body composition, as well as in an area of the hypothalamus linked to cognition. In addition, our analysis reveals an unexpected female-specific feature of hypothalamic aging. Specifically, we discover that the master regulator of X-inactivation, Xist, is elevated with age, particularly in hypothalamic neurons. Moreover, using machine learning, we show that levels of X-chromosome genes, and Xist itself, are the best predictors of cellular age. Together, this study identifies critical cell-specific changes of the aging hypothalamus in mammals, and uncovers a novel marker of neuronal aging in females.

neuroscience↗

bioRxiv2021

LINE-1 Retrotransposon expression in cancerous, epithelial and neuronal cells revealed by 5'-single cell RNA-Seq

LINE-1 retrotransposons are sequences capable of copying themselves to new genomic loci via an RNA intermediate. New studies implicate LINE-1 in a range of diseases, especially in the context of aging, but without an accurate understanding of where and when LINE-1 is expressed, a full accounting of its role in health and disease is not possible. We therefore developed a method - 5 scL1seq - that makes use of a widely available library preparation method (10x Genomics 5 single cell RNA-seq) to measure LINE-1 expression in tens of thousands of single cells. We recapitulated the known pattern of LINE-1 expression in tumors - present in cancer cells, absent from immune cells - and identified hitherto undescribed LINE-1 expression in human epithelial cells and mouse hippocampal neurons. In both cases, we saw a modest increase with age, supporting recent research connecting LINE-1 to age related diseases.

bioinformatics↗