Biology subjects

McManus, M.

Publications and source records attributed to McManus, M..

10 recordsLinked to original sources

bioRxiv2026

Hepatocyte Embryonic Ectoderm Development (Eed) Deficiency Causes Liver Injury, Fibrosis and Impacts Liver Regeneration

Regeneration depends on tightly coordinated transcriptional programs governed by a dynamic epigenetic landscape to regulate cell identity, proliferation, and tissue remodelling following injury. The liver is highly regenerative due to its ability to rapidly upregulate genes that drive the cell cycle and other genes important for hepatocyte proliferation. In the uninjured liver, many of these genes are poised for activation based on promoter occupancy of trimethylated histone 3 lysine 27 (H3K27me3) and regenerative stimulus provided by partial hepatectomy leads to evacuation of this mark, suggesting this is a key component of proregenerative gene regulation in the liver. H3K27me3 is deposited by the polycomb repressive complex 2 (PRC2). Were we deplete H3K27me3 using hepatocyte specific deletion of key component of PRC2, Embryonic Ectoderm Development (EedHepKO). This results in reduced liver size, increased hepatocyte death, proliferation, and fibrosis associated with upregulation of cell cycle and fibrogenic genes. Though these mice are less likely to survive two-thirds partial hepatectomy than WT (WT) controls, those that do survive increase liver mass faster than WTs. Importantly, genes occupied by H3K27me3 in uninjured WT livers are upregulated in EEDHepKO livers, and become further induced following PH. This demonstrates that PRC2 loss induces liver injury and dysregulates pro-regenerative gene expression, highlighting that epigenetic modulation depends requires precise targeting to achieve enhanced regenerative

pathology↗

bioRxiv2025

In vivo imaging of reactive oxygen species after myocardial ischemia-reperfusion injury: a large animal multimodal imaging and transcriptomic study

BackgroundReactive oxygen species (ROS) contribute to myocardial ischemia-reperfusion injury (IRI), but in-vivo data on the spatial myocardial distribution and systemic effects of ROS after IRI remain limited. This multimodal CMR and PET/CT study aimed to non-invasively image ROS activity in a clinically-relevant swine model of IRI using [18F]ROStrace, a fluorine-18-labeled analogue of dihydroethidium (DHE), and to investigate regional changes in ROS activity in the infarcted myocardium during the subacute post-IRI phase. MethodsIRI was induced by percutaneous occlusion of the left anterior descending artery for 90 minutes in swine (N=9). CMR and whole-body PET/CT imaging with [18F]ROStrace were performed before myocardial infarction (MI) and 3-5 days post-MI to assess ROS in non-infarct myocardium, lungs, bone marrow, spleen and skeletal muscle. Late gadolinium enhanced CMR was performed to structurally characterize infarct regions. Post-MI, in vivo [18F]ROStrace signal in infarcted myocardium was compared with remote, non-infarcted myocardium and validated via ex vivo DHE fluorescent imaging. Bulk RNA-sequencing (RNA-seq) and Gene Ontology pathway analysis were conducted on biopsies from infarct and remote myocardial tissue to identify differentially expressed genes and pathways connected to oxidative stress. ResultsDuring the subacute phase following MI, [18F]ROStrace fractional uptake rate (FUR; min-1) was significantly increased in skeletal muscle, compared to baseline (0.011{+/-}0.003 vs 0.016{+/-}0.005, p=0.04), with a trend toward increased FUR in bone marrow (0.046{+/-}0.009 vs 0.056{+/-}0.011, p=0.12) and the left ventricular free wall (0.067{+/-}0.007 vs 0.073{+/-}0.010, p=0.15). Within the myocardium, [18F]ROStrace FUR ((min-1)/(mL/min/g)) was significantly higher in infarcted compared to non-infarcted myocardium regions (0.110{+/-}0.034, vs 0.148{+/-}0.035, p=0.0005). DHE staining confirmed elevated ROS levels in the infarcted myocardium. RNA-seq identified 8,707 differentially expressed genes between infarct and remote myocardium, with downregulated pathways in the infarct associated with mitochondrial function, cellular respiration, and metabolic adaptation. ConclusionThis study demonstrated MI ROS imaging using [18F]ROStrace using a whole-body PET/CT scanner and structural assessment with CMR. Systemic and myocardial increases in ROS activity were observed post-MI, accompanied by substantial molecular alterations in infarcted tissue. These findings show potential imaging strategies to evaluate therapeutic targets that can mitigate oxidative stress after MI.

bioengineering↗

bioRxiv2025

Scalable imaging-based profiling of CRISPR perturbations with protein barcodes

Imaging-based CRISPR screens enable high-content functional genomics by capturing phenotypic changes in cells after genetic perturbation. Protein barcodes provide cost-effective, easy-to-implement, and imaging-compatible barcoding for pooled perturbations, yet their scalability has been constrained by the need for arrayed cloning, lentiviral recombination between barcodes and guides, and difficulties in decoding barcodes with high confidence. Here, we introduce poolVis and cellPool, an integrated experimental and computational platform designed to address these limitations. poolVis uses Cre-lox-mediated reconfiguration to position barcode-sgRNA pairs in proximity during viral integration, which greatly reduces barcode shuffling during pooled cloning and delivery. cellPool leverages a scalable computational workflow and the unique aspects of protein barcodes to produce unpooled image galleries from multi-terabyte scale datasets. Applying this platform to single- and double-CRISPRi profiling of cell-cycle genes and chromokinesins in the MCF10A cells uncovered established and previously unrecognized phenotypes, including nuclear morphology changes and reciprocal sign epistasis in DNA damage.

systems biology↗

bioRxiv2025

How the characteristics of a virtual environment affects the perception of travel distance through it

Although simulated self-motion through virtual environments has been widely used to investigate perceptual odometry, the characteristics of the virtual environments used, and the reported results have varied greatly. Here, we systematically vary the characteristics of the environment through which observers are moved in order to explore the effect of (1) the structure of an environment including the presence and texture of a ground surface, (2) the naturalism and scale of an environment, (3) colour, and (4) the density of a starfield and how it might affect perceived travel distance. In all four experiments, participants were visually moved forwards through a virtual environment and perceived travel distance was estimated by either (1) stopping at the location of a previously seen target (the Move-To-Target Task) or (2) adjusting the position of a target to indicate a previously travelled distance (the Adjust-Target Task). Data were analyzed in terms of gain (perceived travel distance/actual travel distance). Results show no significant differences that depended on the structure of an environment or on the presence or absence of a ground surface (Experiment 1), or on the naturalism of the environment (Experiment 2), or on whether the environment was in colour or in black and white (Experiment 3). However, there was a small effect of the texture of the ground surface and of the scale of the environment. In Experiment 4, we show that there may be a very low ceiling effect in the density of a starfield needed to accurately estimate travel distance. Together these experiments have implications for the design of real and virtual environments where perceived motion is important and will enable us to further predict our perception of moving through an environment.

neuroscience↗

bioRxiv2025

Multiplexed Perturbation Enables Scalable Pooled Screens

CRISPR-based genetic perturbation screens have revolutionized the ability to link genes to cellular phenotypes with unprecedented precision and scale. However, conventional pooled CRISPR screens require large cell numbers to achieve adequate sgRNA representation, posing technical and financial challenges. Here, we investigate the impact of co-delivery of multiple guide RNAs via high multiplicity of infection (MOI) in pooled CRISPR interference (CRISPRi) screens as a strategy to enhance screening efficiency while reducing cell numbers. We systematically evaluate screen performance across varying MOIs, assessing the effects of multiplexing on knockdown efficiency, sgRNA representation, and potential interference of multiple sgRNA phenotypes. Our data demonstrate that sgRNA multiplexing (MOI 2.5-10) can maintain screen performance while enabling significant reductions in cell number requirements. We further apply these optimized conditions to conduct a genome-wide CRISPR screen for regulators of the intracellular adhesion molecule ICAM-1, successfully identifying novel candidates using as few as half a million cells. This study provides a framework for adopting multiplexed sgRNA strategies to streamline CRISPR screening applications in resource-limited settings.

molecular biology↗

bioRxiv2024

Acute infectious mononucleosis generates persistent, functional EBNA-1 antibodies with high cross-reactivity to alpha crystalline beta

Epstein-Barr Virus (EBV) infects over 95% of the worlds population and is the most common cause of infectious mononucleosis (IM). Epidemiologic studies have linked EBV with certain cancers or autoimmune conditions, including multiple sclerosis (MS). Recent studies suggest that molecular mimicry between EBV proteins, particularly EBV nuclear antigen 1 (EBNA-1), and self-proteins is a plausible mechanism through which EBV infection may contribute to the development of autoimmune disorders. We used a systems immunology approach to investigate the magnitude, specificity, and functional properties of EBNA-1 specific antibodies in a cohort of 97 young adults with IM from presentation through 1-year post-primary infection compared to a control cohort of EBV-seropositive individuals. Levels of EBNA-1 specific IgG1 and IgG3 binding antibodies increased over the course of infection. EBNA-1 antibodies capable of mediating antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent complement deposition (ADCD) were detected at or after 6 months. Binding and ADCP- and ADCD-leveraged antibodies primarily targeted a region of EBNA-1 known to elicit cross-reactive antibodies to several self-peptides in individuals with MS. Significantly higher binding and ADCD-active antibodies targeting EBNA-1 were observed in individuals with at least one HLA-DRB1*15:01 allele, a known genetic risk factor for MS; Importantly, high levels of antibodies capable of binding alpha crystalline beta (CRYAB) and mediating complement deposition were detected at 6 months and 1-year following IM; CRYAB antibodies were resistant to denaturing forces, indicating an affinity matured response. Blocking experiments confirmed that CRYAB antibodies were cross-reactive with EBNA-1. Altogether, these results demonstrate that high levels of functional antibodies targeting EBNA-1 are generated in early EBV infection, some of which are cross-reactive with CRYAB. Further investigation is warranted to determine how these antibody responses may contribute to the subsequent development of MS.

immunology↗

bioRxiv2023

Genome-wide screens identify SEL1L as an intracellular rheostat controlling collagen turnover

Accumulating evidence has implicated impaired extracellular matrix (ECM) clearance as a key factor in fibrotic disease. Despite decades of research elucidating the effectors of ECM clearance, relatively little is understood regarding the upstream regulation of this process. Collagen is the most abundant constituent of normal and fibrotic ECM in mammalian tissues. Its catabolism occurs through extracellular proteolysis and cell-mediated uptake of collagen fragments for intracellular degradation. Given the paucity of information regarding the regulation of this latter process, we executed unbiased genome-wide screens to understand the molecular underpinnings of cell-mediated collagen clearance. Using this approach, we discovered a previously unappreciated mechanism through which collagen biosynthesis is sensed by cells internally and directly regulates clearance of extracellular collagen. The sensing mechanism is dependent on endoplasmic reticulum-resident protein SEL1L and occurs via a noncanonical function of SEL1L. This pathway functions as a homeostatic negative feedback loop that limits collagen accumulation in tissues. In human fibrotic lung disease, the induction of this collagen clearance pathway by collagen synthesis is impaired, thereby contributing to the pathological accumulation of collagen in lung tissue. Thus cell-autonomous, rheostatic collagen clearance is a previously unidentified pathway of tissue homeostasis.

cell biology↗

bioRxiv2021

Nucleolar-based Dux repression is essential for 2-cell stage exit

Upon fertilisation, the mammalian embryo must switch from dependence on maternal transcripts to transcribing its own genome, and in mice involves the transient upregulation of MERVL transposons and MERVL-driven genes at the 2-cell stage. The mechanisms and requirement for MERVL and 2-cell (2C) gene upregulation are poorly understood. Moreover, this MERVL-driven transcriptional program must be rapidly shut off to allow 2C exit and developmental progression. Here, we report that robust ribosomal RNA (rRNA) synthesis and nucleolar maturation are essential for exit from the 2C state. 2C-like cells and 2C embryos show similar immature nucleoli with altered structure and reduced rRNA output. We reveal that nucleolar disruption via blocking Pol I activity or preventing nucleolar phase separation enhances conversion to a 2C-like state in embryonic stem cells (ESCs) by detachment of the MERVL activator Dux from the nucleolar surface. In embryos, nucleolar disruption prevents proper Dux silencing and leads to 2-4 cell arrest. Our findings reveal an intriguing link between rRNA synthesis, nucleolar maturation and gene repression during early development.

developmental biology↗

bioRxiv2021

Prevalent mouse phenotypes in the unexplored druggable genome

Among the estimated ~23,000 protein encoding human genes, the class of druggable genes- defined by their ability to bind drug-like compounds- represents an enticing collection of targets for clinical intervention. Yet many if not most of these genes remain poorly understood and understudied. Here we evaluate three major classes of druggable genes (GPCRs, ion channels, and kinases) and found that a third of these remain largely ignored yet display significant mouse phenotypes upon genetic ablation. We show that both well-studied and understudied druggable genes share a similar number and spectrum of phenotypes. Moreover, many of the mouse phenotypes arising from the ablation of both well-studied and understudied druggable genes show similarities with symptoms in rare human diseases. Collectively these data diminish the notion that most poorly studied genes may not be especially important and highlight therapeutic opportunities and potential disease models among poorly characterized druggable genes.

genetics↗

bioRxiv2019

High throughput CRISPR screening identifies genes involved in macrophage viability and inflammatory pathways

AbstractMacrophages are critical cells of the innate immune system involved in the recognition and destruction of invading microbes in addition to the resolution of inflammation and maintenance of homeostasis. Understanding the genes involved in all aspects of macrophage biology is essential to gaining new insights into immune system dysregulation during diseases that range from autoinflammatory to cancer. Here we utilize high throughput clustered regularly interspaced short palindromic repeats (CRISPR) screening to generate a resource that identifies genes required for macrophage viability and function. First, we employ a pooled based CRISPR/Cas nuclease active screening approach to identify essential genes required for macrophage viability by targeting genes within coding exons. In addition, we also target 3UTRs to gain insights into new cis-regulatory regions that control expression of these essential genes. Second, using our recently generated NF-{kappa}B reporter macrophage line, we perform a fluorescence-activated cell sorting (FACS)-based high-throughput genetic screen to identify regulators of inflammation. We identify a number of novel positive and negative regulators of the NF-{kappa}B pathway as well as unraveling complexities of the TNF signaling cascade showing it can function in an autocrine manner to negatively regulate the pathway. Utilizing a single complex library design we are capable of interrogating various aspects of macrophage biology, generating a resource for future studies.\n\nAuthor SummaryExcess inflammation is associated with a variety of autoimmune diseases and cancers. Macrophages are important mediators of this inflammatory response. Defining the genes involved in their viability and effector function is needed to completely understand these two important aspects of macrophage biology. Here we screened over 21,000 genes and generated a resource guide of genes required for macrophage viability as well as novel positive and negative regulators of NF-{kappa}B signaling.

immunology↗