Biology subjects

Mitchell, C. A.

Publications and source records attributed to Mitchell, C. A..

9 recordsLinked to original sources

bioRxiv2026

Cell Painting and machine learning for mechanistic profiling of hepatotoxic botanicals and their constituents

BACKGROUND Botanicals are widely consumed as herbal remedies and plant-derived products, but their complex and variable chemical composition presents major challenges for safety assessment. Although hepatotoxicity has been reported for several botanicals, the constituent compounds responsible and the underlying mechanisms often remain poorly characterised. OBJECTIVES We aimed to determine whether Cell Painting could identify candidate constituent drivers of botanical bioactivity and generate hypotheses about potential mechanisms of action in primary human hepatocytes. METHODS We profiled 13 botanical extracts and 85 known constituents across multiple concentrations using Cell Painting alongside assays of metabolic activity and membrane integrity. To interpret morphological phenotypes, we trained machine-learning models using a Cell Painting reference atlas of >1,000 chemicals linked to ToxCast/Tox21 molecular and cellular bioactivity annotations. RESULTS Cell Painting detected bioactivity in 11/13 botanical extracts and 46/85 constituents, compared with 9/13 extracts and 40/85 constituents in the cell-health assays, and generally detected activity at lower concentrations. For 9/13 extracts, at least one constituent was active at a lower concentration than the corresponding botanical extract, supporting the contribution of a subset of potent constituents to mixture-level responses. Aristolochia and goldenseal were the most potent extracts across Cell Painting and cell-health assays and contained highly active constituents. Reference-atlas models generated specific mechanistic hypotheses, including cytotoxicity-, endocrine-, and genotoxicity-associated responses for several botanicals and constituents. CONCLUSION These results support Cell Painting combined with machine learning as a sensitive approach for characterising cellular responses to botanicals, prioritising candidate constituent drivers of hepatotoxicity-associated phenotypes, and generating testable hypotheses about potential mechanisms of action.

pharmacology and toxicology↗

bioRxiv2026

Identification of co-expressed gene sets for major molecular initiating events in rodent liver carcinogenesis using wild type and knock out rats

Liver tumors are the most common carcinogenic outcome observed in two-year bioassays used for regulatory risk assessment of chemicals and pharmaceuticals. Because these traditional long-term studies are resource-intensive and not feasible for most chemicals, there is a critical need to develop alternative approaches that can identify potential rodent liver carcinogens in short-term studies (e.g., [≤] 1 month). Biomarkers associated with molecular initiating events (MIEs) of early rodent liver tumorigenesis may enable the prediction of two-year outcomes from such studies. Here, we identified transcriptional changes associated with six key initiators of rat liver tumorigenesis: five transcription factors (TFs) associated with non-genotoxic modes of action (AhR, PXR, CAR, PPAR, and ER), and tumor suppressor protein p53, which is activated by diverse cellular stressors including genotoxicity. Three known chemical activators for each TF were administered orally once daily for five days at doses associated with tumorigenic responses in previous rat cancer bioassays. RNA-seq identified differentially expressed genes (DEGs) that were consistently regulated by at least two activators in wild-type but not in corresponding TF knockout animals. Gene set enrichment analysis of ranked differential expression results demonstrated enrichment of pathways reflecting established TF biology. When comparing the consensus DEGs for each TF to a library of chemically induced, liver-specific gene expression profiles, we found that in most cases the chemicals with the most similar profiles to the consensus lists were known to activate the corresponding TF. Collectively, these findings identified candidate gene sets that may have utility for monitoring the induction of carcinogenesis-associated MIEs in short-term studies.

pharmacology and toxicology↗

bioRxiv2026

A decline in skeletal muscle NOX4 abrogates adaptive homeostasis and exacerbates ageing

A decline in NFE2L2-orchestrated adaptive homeostasis and oxidative distress are thought to be key features of ageing. In contracting skeletal muscle, the ROS-producing enzyme NADPH oxidase 4 (NOX4) is a potent inducer of NFE2L2 adaptive homeostasis. Here we report that skeletal muscle NOX4 levels decline in aged mice and humans, resulting in abrogated NFE2L2 adaptive homeostasis, increased protein oxidative damage and decreased muscle function. We show that deleting NOX4 in skeletal muscle exacerbates the physiological decline associated with ageing, resulting in overt sarcopenia and frailty, characterised by physical inactivity, increased adiposity, systemic inflammation, whole-body insulin resistance and advanced liver disease in aged chow-fed mice. The systems-wide physiological decline in aged skeletal muscle NOX4-deficient mice could be corrected by activating NFE2L2 with sulforaphane and reinstating adaptive homeostatic responses otherwise induced by exercise. Our findings provide important insights into the basis for the decline in NFE2L2-orchestrated adaptive homeostasis with age and identify key mechanisms by which exercise may promote healthy ageing.

physiology↗

bioRxiv2024

The Rho effector ARHGAP18 coordinates a Hippo pathway feedback loop through YAP and Merlin to regulate the cytoskeleton and epithelial cell polarity.

The organization of the cells cytoskeletal filaments is coordinated through a complex network of signaling cascades activated by both internal and external cues. Two major actin regulatory pathways are signal transduction through Rho family GTPases and growth and proliferation signaling through the Hippo pathway. These two pathways define the actin cytoskeleton, controlling foundational cellular attributes such as morphology and polarity and are hijacked to promote proliferation and motility in aggressive cancers. In this study, we use human epithelial cells to investigate the interplay between the Hippo and Rho Family signaling pathways, which have predominantly been characterized as independent actin regulatory mechanisms. We identify that the RhoA effector, ARHGAP18, forms a complex with the Hippo pathway transcription factor YAP to address a long-standing enigma in the field. Using super resolution STORM microscopy, we characterize single-filament-level changes in the actin cytoskeleton that arise from CRISPR/CAS9 knockout of ARHGAP18. We report that the loss of ARHGAP18 results in cytoskeletal alterations driven by both dysregulated RhoA signaling and aberrant nuclear localization of YAP. These findings indicate that the Hippo and Rho family GTPase signaling cascades are temporally and spatially coordinated in their regulation of the actin cytoskeleton.

cell biology↗

bioRxiv2023

Maternal IL-10 restricts fetal emergency myelopoiesis

Neonates, in contrast to adults, are highly susceptible to inflammation and infection. Here we investigate how late fetal liver (FL) mouse hematopoietic stem and progenitor cells (HSPC) respond to inflammation, testing the hypothesis that deficits in engagement of emergency myelopoiesis (EM) pathways limit neutrophil output and contribute to perinatal neutropenia. We show that despite similar molecular wiring as adults, fetal HSPCs have limited production of myeloid cells at steady state and fail to activate a classical EM transcriptional program. Moreover, we find that fetal HSPCs are capable of responding to EM-inducing inflammatory stimuli in vitro, but are restricted by maternal anti-inflammatory factors, primarily interleukin-10 (IL-10), from activating EM pathways in utero. Accordingly, we demonstrate that loss of maternal IL-10 restores EM activation in fetal HSPCs but at the cost of premature parturition. These results reveal the evolutionary trade-off inherent in maternal anti-inflammatory responses that maintain pregnancy but render the fetus unresponsive to EM activation signals and susceptible to infection. HIGHLIGHTSO_LIThe structure of the HSPC compartment is conserved from late fetal to adult life. C_LIO_LIFetal HSPCs have diminished steady-state myeloid cell production compared to adult. C_LIO_LIFetal HSPCs are restricted from engaging in emergency myelopoiesis by maternal IL-10. C_LIO_LIRestriction of emergency myelopoiesis may explain neutropenia in septic neonates. C_LI eTOC BLURBFetal hematopoietic stem and progenitor cells are restricted from activating emergency myelopoiesis pathways by maternal IL-10, resulting in inadequate myeloid cell production in response to inflammatory challenges and contributing to neonatal neutropenia.

developmental biology↗

bioRxiv2023

Autophagy counters inflammation-driven glycolytic impairment in aging hematopoietic stem cells

Aging of the hematopoietic system promotes various blood, immune and systemic disorders and is largely driven by hematopoietic stem cell (HSC) dysfunction (1). Autophagy is central for the benefits associated with activation of longevity signaling programs (2), and for HSC function and response to nutrient stress (3,4). With age, a subset of HSCs increases autophagy flux and preserves some regenerative capacity, while the rest fail to engage autophagy and become metabolically overactivated and dysfunctional (4). However, the signals that promote autophagy in old HSCs and the mechanisms responsible for the increased regenerative potential of autophagy-activated old HSCs remain unknown. Here, we demonstrate that autophagy activation is an adaptive survival response to chronic inflammation in the aging bone marrow (BM) niche (5). We find that inflammation impairs glucose metabolism and suppresses glycolysis in aged HSCs through Socs3-mediated impairment of AKT/FoxO-dependent signaling. In this context, we show that inflammation-mediated autophagy engagement preserves functional quiescence by enabling metabolic adaptation to glycolytic impairment. Moreover, we demonstrate that transient autophagy induction via a short-term fasting/refeeding paradigm normalizes glucose uptake and glycolytic flux and significantly improves old HSC regenerative potential. Our results identify inflammation-driven glucose hypometabolism as a key driver of HSC dysfunction with age and establish autophagy as a targetable node to reset old HSC glycolytic and regenerative capacity. One-Sentence SummaryAutophagy compensates for chronic inflammation-induced metabolic deregulation in old HSCs, and its transient modulation can reset old HSC glycolytic and regenerative capacity.

cell biology↗

bioRxiv2021

Stromal inflammation is a targetable driver of hematopoietic aging

Hematopoietic aging is marked by a loss of regenerative capacity and skewed differentiation from hematopoietic stem cells (HSC) leading to dysfunctional blood production. Signals from the bone marrow (BM) niche dynamically tailor hematopoiesis, but the effect of aging on the niche microenvironment and the contribution of the aging niche to blood aging still remains unclear. Here, we characterize the inflammatory milieu in the aged marrow cavity that drives both stromal and hematopoietic remodeling. We find decreased numbers and functionality of osteogenic mesenchymal stromal cells (MSC) at the endosteum and expansion of pro-inflammatory perisinusoidal MSCs with deterioration of sinusoidal endothelium in the central marrow, which together create a degraded and inflamed old niche. Molecular mapping at single cell resolution confirms disruption of cell identities and enrichment of inflammatory response genes in niche populations. Niche inflammation, in turn, drives chronic activation of emergency myelopoiesis pathways in old HSCs and multipotent progenitors (MPP), which promotes myeloid differentiation at the expense of lymphoid and erythroid commitment and hinders hematopoietic regeneration. Remarkably, niche deterioration, HSC dysfunction and defective hematopoietic regeneration, can be improved by blocking inflammatory IL-1 signaling. Our results demonstrate that targeting niche inflammation is a tractable strategy to restore blood production during aging.

cell biology↗

bioRxiv2020

Deletion of INPP5E in the murine retina impairs axoneme formation and prevents photoreceptor disc morphogenesis

INPP5E (pharbin) is a ubiquitously-expressed, farnesylated phosphatidylinositol polyphosphate 5-phosphatase which modulates the phosphoinositide composition of membranes. INPP5E resides in primary cilia, and mutations or loss of INPP5E are associated with ciliary dysfunction. INPP5E missense mutations of the phosphatase catalytic domain cause Joubert syndrome in humans, a syndromic ciliopathy affecting multiple tissues including brain, liver, kidney and retina. We show that, differing from other primary cilia, INPP5E is present in the wildtype photoreceptor inner segment and absent in the outer segment--a modified primary cilium dedicated to phototransduction. We generated Inpp5eF/F;Six3Cre (in short, retInpp5e-/-) mice which exhibit a rapidly progressing rod-cone degeneration nearly completed by postnatal day 21 (P21) in the central retina. Mutant cone outer segments contain vesicles instead of discs as early as P8. While P10 mutant outer segments contain phototransduction and structural proteins, they do not form axonemes and fail to elaborate disc membranes. Connecting cilia of retInpp5e-/- rods appear normal, although IFT-B/A particles accumulate at their distal ends suggesting disrupted intraflagellar transport. These results show that ablation of INPP5E does not impair the secretory pathway responsible for delivery of outer segment-specific proteins, but blocks axonemal extension and prevents disc morphogenesis.

neuroscience↗

bioRxiv2020

Structural analysis of the PTEN:P-Rex2 signalling node reveals how cancer-associated mutations coordinate to hyperactivate Rac1

The PTEN:P-Rex2 complex is one of the most commonly mutated signaling nodes in metastatic cancer. Assembly of the PTEN:P-Rex2 complex inhibits the activity of both proteins, and its dysregulation can drive PI3K-AKT signaling and cell proliferation. Here, using extensive crosslinking mass spectrometry and functional studies, we provide crucial mechanistic insights into PTEN:P-Rex2 complex assembly and co-inhibition. PTEN is anchored to P-Rex2 by interactions between the PTEN PDZ-BM tail and the second PDZ domain of P-Rex2. This interaction bridges PTEN across the P-Rex2 surface, occluding PTEN membrane-binding and PI(3,4,5)P3 hydrolysis. Conversely, PTEN both allosterically promotes an autoinhibited P-Rex2 conformation and occludes G{beta}{gamma} binding and GPCR activation. These insights allow us to define a new gain-of-function class of cancer mutations within the PTEN:P-Rex2 interface that uncouples PTEN inhibition of Rac1 signaling. These findings provide a mechanistic framework to understand the dysregulation of the PTEN:P-Rex2 signaling node in metastatic cancer.

biochemistry↗