Biology subjects

Brumwell, A.

Publications and source records attributed to Brumwell, A..

7 recordsLinked to original sources

bioRxiv2026

Single-cell proteomics resolves major cell populations and protein-defined chondrocyte states in the adult human osteoarthritic joint

Objective Single-cell RNA sequencing has transformed our understanding of cellular heterogeneity within osteoarthritic (OA) joint tissues, yet transcript abundance provides only an indirect measure of cellular phenotype and function. Recent advances in mass spectrometry now enable proteins to be measured directly at single-cell resolution. Here, we applied single-cell proteomics (SCP) to primary human OA cartilage and synovium to determine whether cellular heterogeneity within the diseased joint can be resolved directly at the proteome level. Design Cartilage and synovium were obtained from patients undergoing total knee arthroplasty for OA. Low-input proteomic profiling was first performed to establish proteome depth from limited cell numbers and validate biologically relevant clustering. SCP was then applied to individual cells, followed by unsupervised clustering, differential protein abundance analysis and iterative biological annotation. Results Low-input proteomics identified 7,800 proteins from 1,000 cells while retaining tissue- and cell-associated biological differences. Across the SCP dataset, ~2,300 unique proteins were detected, resolving chondrocyte, synovial fibroblast and immune-cell populations that closely paralleled those previously defined by single-cell transcriptomics. Higher-resolution analysis distinguished lining and sublining fibroblasts, discrete immune populations and multiple protein-defined chondrocyte states. Conclusions SCP enables direct characterisation of cellular heterogeneity within the human OA joint at the level of the proteome. By independently recovering established cellular populations while revealing protein-defined chondrocyte states, our findings establish SCP as a powerful complement to single-cell transcriptomics and open a new route to investigating functional cellular states and disease mechanisms that cannot be inferred from RNA abundance alone.

genetics↗

bioRxiv2025

Origins and diversity of Exmoor ponies: A mitogenome framework for assessing maternal lineage diversification in endangered pony breeds

In response to growing concerns over human-driven biodiversity loss, rewilding efforts increasingly employ semi-wild herbivores to restore ecosystems. The Exmoor pony is often cited as an ancient breed retaining primitive traits suited for such projects, yet its genetic history remains underexplored. We combined studbook records, mitochondrial DNA (mtDNA) D-loop, and whole mitogenome sequences to evaluate maternal lineage diversity for improved management recommendations and the phylogenetic placement of Exmoor ponies among modern and ancient breeds. We also assessed whether data from multiple breeds already available based on D-loop sequences could be organised into a useful framework for resolving the complex history of modern horse breeds. We identified eight mtDNA haplotypes among 88 Exmoor pony samples, representing all extant maternal lines. While phylogenetic trees based on D-loop sequences lacked resolution, clustering published sequences to a mitogenome-derived haplogroup framework allowed assessment of the distribution and relationships of lineages across diverse horse populations, including ancient DNA samples and Przelwalskis horses. The most basal lineages of Exmoors were traced back to the Pleistocene, supporting their ancient origin and status as a primitive population, with a possible ancestral role in the development of some modern breeds. The founder lineages were found to be polyphyletic, with more derived haplogroups representing subsequent mixture with other domestic horse breeds. Although nuclear genome sequencing is needed to fully assess levels of admixture, we identified new haplotypes, along with rare or basal haplotypes that could be prioritised for conservation in rewilding studies. Our results highlight the potential for broad testing of hypotheses about origins and relationships among horse breeds using short regions of DNA mapped onto a more robust phylogenetic framework resolved through comparison of whole mitogenomes.

evolutionary biology↗

bioRxiv2023

Autophagy-dependent alternative splicing event produces a more stable ribosomal protein S24 isoform that aids in hypoxic cell survival

Overlapping or convergent stress-activated molecular pathways exist to coordinate cell fate in response to stimuli such as hypoxia, oxidative stress, DNA damage, and unfolded proteins. Cells can remodel the splicing and translation machineries to mount a specialized gene expression response to certain stresses. Here, we show that hypoxic human cells in 2D and 3D culture models increase the relative abundance by 1.7- to 2.6-fold and 4.7- to 11.5-fold, respectively, of a longer mRNA variant of ribosomal protein S24 (RPS24L) compared to a shorter mRNA variant (RPS24S) by favoring the inclusion of a 22 bp cassette exon. Mechanistically, RPS24L and RPS24S are induced and repressed, respectively, by distinct parallel pathways in hypoxia: RPS24L is induced in an autophagy-dependent manner, while RPS24S is reduced by mTORC1 repression and in a HIF-dependent manner. RPS24L is a more stable mRNA in hypoxia and produces a more stable protein isoform compared to RPS24S. Cells overexpressing RPS24L display improved survival and growth in hypoxia relative to control cells and cells overexpressing RPS24S, which display impaired survival. Previous work from our group showed a correlation between RPS24L levels and tumor hypoxia in prostate cancer. These data highlight RPS24L as a stress-induced alternative splicing event that favors hypoxic cell survival, which could be exploited by cancer cells in the tumor microenvironment.

molecular biology↗

bioRxiv2021

IRE1α drives lung epithelial progenitor dysfunction to establish a niche for pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a disease of progressive interstitial fibrosis, which leads to severe debilitation, respiratory failure, and death. In IPF, environmental exposures interact with genetic risk factors to engender critical patho-etiological events in lung epithelial cells, including endoplasmic reticulum (ER) stress and TGF{beta} signaling, but the interactions between these disparate pathways are not well understood. We previously showed that kinase inhibitors of the IRE1 bifunctional kinase/RNase--a central mediator of the unfolded protein response (UPR) to ER stress--protected mice from bleomycin-induced pulmonary fibrosis. Here we show that a nanomolar-potent, mono-selective kinase inhibitor of IRE1 (KIRA8) decreases ER-stress induced TGF{beta} signaling and the senescence-associated secretory phenotype (SASP) in the lung epithelium after bleomycin exposure. A recently-described subset of "damage-associated transient progenitors" (DATPs) display IRE1-regulated pathological gene signatures that are quelled by KIRA8, in vivo. After injury, these cells uniquely express integrin v{beta}6, a key activator of TGF{beta} in pulmonary fibrosis. KIRA8 inhibition of IRE1 decreases both DATP number and Itgb6 expression in remaining cells, with a decrease in local collagen accumulation. Single-cell RNA sequencing from IPF lungs revealed an analogous Itgb6+ cell population that may also be regulated by IRE1. These findings suggest that lung epithelial progenitor cells sit at the center of the fibrotic niche, and IRE1 signaling locks them into a dysfunctional state that establishes and perpetuates pathological fibrosis.

cell biology↗

bioRxiv2020

Human alveolar Type 2 epithelium transdifferentiates into metaplastic KRT5+ basal cells during alveolar repair

Understanding differential lineage potential of orthologous stem cells across species can shed light on human disease. Here, utilizing 3D organoids, single cell RNAseq, and xenotransplants, we demonstrate that human alveolar type 2 cells (hAEC2s), unlike murine AEC2s, are multipotent and able to transdifferentiate into KRT5+ basal cells when co-cultured with primary fibroblasts in 3D organoids. Trajectory analyses and immunophenotyping of epithelial progenitors in idiopathic pulmonary fibrosis (IPF) indicate that hAEC2s transdifferentiate into metaplastic basal cells through alveolar-basal intermediate (ABI) cells that we also identify in hAEC2-derived organoids. Modulating hAEC2-intrinsic and niche factors dysregulated in IPF can attenuate metaplastic basal cell transdifferentiation and preserve hAEC2 identity. Finally, hAEC2s transplanted into fibrotic immune-deficient murine lungs engraft as either hAEC2s or differentiated KRT5+ basal cells. Our study indicates that hAEC2s-loss and expansion of alveolar metaplastic basal cells in IPF are causally connected, which would not have been revealed utilizing murine AEC2s as a model. HighlightsO_LIHuman AEC2s transdifferentiate into KRT5+ basal cells when accompanied by primary adult human lung mesenchyme in 3D organoid culture. C_LIO_LIAlterations of hAEC2-intrinsic and niche factors dysregulated in IPF can modify metaplastic hAEC2 transdifferentiation. C_LIO_LIhAEC2s engraft into fibrotic lungs of immune-deficient mice and transdifferentiate into metaplastic basal cells. C_LIO_LITranscriptional trajectory analysis suggests that hAEC2s in IPF gives rise to metaplastic basal cells via alveolar-basal intermediate cells. C_LI

cell biology↗

bioRxiv2019

Translational control of breast cancer plasticity

Plasticity of neoplasia, whereby cancer cells attain stem-cell-like properties, is required for disease progression and represents a major therapeutic challenge. We report that in breast cancer cells NANOG, SNAIL and NODAL transcripts manifest multiple isoforms characterized by different 5 Untranslated Regions (5UTRs), whereby translation of a subset of these isoforms is stimulated under hypoxia. This leads to accumulation of corresponding proteins which induce plasticity and \"fate-switching\" toward stem-cell like phenotypes. Surprisingly, we observed that mTOR inhibitors and chemotherapeutics induce translational activation of a subset of NANOG, SNAIL and NODAL mRNA isoforms akin to hypoxia, engendering stem cell-like phenotypes. Strikingly, these effects can be overcome with drugs that antagonize translational reprogramming caused by eIF2 phosphorylation (e.g. ISRIB). Collectively, our findings unravel a hitherto unappreciated mechanism of induction of plasticity of breast cancer cells, and provide a molecular basis for therapeutic strategies aimed at overcoming drug resistance and abrogating metastasis.

cancer biology↗

bioRxiv2018

Small molecule inhibition of IRE1α kinase/RNase has anti-fibrotic effects in the lung

Endoplasmic reticulum stress (ER stress) has been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a disease of progressive fibrosis and respiratory failure. ER stress activates a signaling pathway called the unfolded protein response (UPR) that either restores homeostasis or promotes apoptosis. The bifunctional kinase/RNase IRE1 is a UPR sensor that promotes apoptosis if ER stress remains high (i.e., a \"terminal\" UPR). Using multiple small molecule inhibitors against IRE1, we show that ER stress-induced apoptosis of murine alveolar epithelial cells can be mitigated in vitro. In vivo, we show that bleomycin exposure to murine lungs causes early ER stress to activate IRE1 and the terminal UPR prior to development of pulmonary fibrosis. Small-molecule IRE1 kinase-inhibiting RNase attenuators (KIRAs) that we developed were used to evaluate the importance of IRE1 activation in bleomycin-induced pulmonary fibrosis. One such KIRA--KIRA7--provided systemically to mice at the time of bleomycin exposure decreases terminal UPR signaling and prevents lung fibrosis. Administration of KIRA7 14 days after bleomycin exposure even promoted the reversal of established fibrosis. Finally, we show that KIRA8, a nanomolar-potent, monoselective KIRA compound derived from a completely different scaffold than KIRA7, likewise promoted reversal of established fibrosis. These results demonstrate that IRE1 may be a promising target in pulmonary fibrosis and that kinase inhibitors of IRE1 may eventually be developed into efficacious anti-fibrotic drugs.

molecular biology↗