Biology subjects

Winters, A.

Publications and source records attributed to Winters, A..

13 recordsLinked to original sources

bioRxiv2026

Tumor cell-intrinsic mtDNA instability drives cGAS-dependent temporal remodeling of the melanoma immune microenvironment

Mitochondrial DNA (mtDNA) instability is an emerging feature of cancer, yet its biological effects on the tumor microenvironment (TME) and tumor progression remain incompletely defined. Using melanocyte-specific conditional silencing of mitochondrial transcription factor A (TFAM) in an autochthonous BrafV600E/Pten-/- melanoma model, we demonstrate that tumor cell-intrinsic mtDNA instability accelerates melanoma growth and metastatic dissemination in a cGAS-dependent manner. Transcriptomic profiling of early and late tumor stages revealed that mtDNA instability drives temporal remodeling of the tumor microenvironment (TME), characterized by an early type I interferon (IFN)-rich inflammatory state that licenses immunoregulatory programs as tumors progress. Tumor-associated macrophages emerged as the principal immune cell type reshaped by tumor cell-intrinsic mtDNA stress signaling, with early enrichment of inflammatory states and a shift toward efferocytosis-associated, tissue-adaptive states. The early mtDNA instability-associated macrophage signatures mapped to human melanoma states associated with favorable survival and therapy response, whereas the late signatures mapped to states associated with poor prognosis and therapy resistance across independent cohorts. Anti-PD-1 treatment delayed progression of mtDNA-instable tumors, consistent with a therapeutic sensitivity window associated with the early IFN-rich immune state. These findings establish tumor cell-intrinsic mtDNA instability as a driver of temporal immune-stromal remodeling that promotes melanoma progression and shapes therapeutic response.

immunology↗

bioRxiv2026

Scalable probe-based single-cell transcriptional profiling for virtual cell perturbation mapping and synthetic biology phenotyping

Large-scale single-cell transcriptional phenotyping of genetic perturbations (perturb-seq) links genes to phenotypes and should enable virtual cell predictive modeling and cellular engineering. However, current perturb-seq single-cell methods are costly, information sparse and require barcodes for many applications. We developed ProPer-seq, a perturb-seq method that uses multiplexed custom DNA probe panels to measure and phenotype synthetic biology perturbations at single-cell resolution without barcodes, including multidomain proteins and sgRNAs. ProPer-seq faithfully reproduces gold-standard perturb-seq phenotypes while achieving 4-fold cost reduction and 50% increased gene detection per cell. As a scalable fixed-cell profiling method, ProPer-seq enables atlas-scale profiling for virtual-cell initiatives and demonstrates data quality suitable for training and validating predictive models. Lastly, ProPer-seqs targeted detection of modular transgenes enables library-on-library perturbation profiling of combinatorial synthetic protein design spaces. We applied this to 3,550 sgRNA x dCas9 effector combinations as well as 260 CAR x ORF combinations dynamically profiled in primary T cells, revealing principles of transcriptional control and cell state modulation by multidomain synthetic transgenes.

synthetic biology↗

bioRxiv2025

Predicting cellular responses to perturbation across diverse contexts with STATE

Cellular responses to perturbations are a cornerstone for understanding biological mechanisms and selecting drug targets. While machine learning models offer tremendous potential for predicting perturbation effects, they currently struggle to generalize to unobserved cellular contexts. Here, we introduce SO_SCPLOWTATEC_SCPLOW, a transformer model that predicts perturbation effects while accounting for cellular heterogeneity within and across experiments. SO_SCPLOWTATEC_SCPLOW predicts perturbation effects across sets of cells and is trained using gene expression data from over 100 million perturbed cells. SO_SCPLOWTATEC_SCPLOW improved discrimination of effects on large datasets by more than 30% and identified differentially expressed genes across genetic, signaling and chemical perturbations with significantly improved accuracy. Using its cell embedding trained on observational data from 167 million cells, SO_SCPLOWTATEC_SCPLOW identified strong perturbations in novel cellular contexts where no perturbations were observed during training. We further introduce Cell-Eval, a comprehensive evaluation framework that highlights SO_SCPLOWTATEC_SCPLOWs ability to detect cell type-specific perturbation responses, such as cell survival. Overall, the performance and flexibility of SO_SCPLOWTATEC_SCPLOW sets the stage for scaling the development of virtual cell models.

systems biology↗

bioRxiv2025

Genome-wide CRISPR screens identify PTGES3 as a novel AR modulator

The androgen receptor (AR) is a critical driver of prostate cancer (PCa). To study regulators of AR protein levels and oncogenic activity, we created the first live cell quantitative endogenous AR fluorescent reporters. Leveraging this novel AR reporter, we performed genome-scale CRISPRi flow cytometry sorting screens to systematically identify genes that modulate AR protein levels. We identified and validated known AR protein regulators including HOXB13 and GATA2 and also unexpected top hits including PTGES3, a poorly characterized gene in PCa. PTGES3 repression resulted in loss of AR protein, cell cycle arrest, and cell death in AR-driven PCa models. PTGES3 is not a commonly essential gene, and our data nominate it as a prime PCa therapeutic target. Clinically, analysis of PCa data demonstrate that PTGES3 expression is associated with AR-directed therapy resistance. Mechanistically, we show PTGES3 binds directly to AR, forms a protein complex with AR in the nucleus, regulates AR protein stability in vitro and in vivo and modulates AR function in the nucleus at AR target genes. PTGES3 represents a novel therapeutic target for overcoming known mechanisms of resistance to existing AR-directed therapies in PCa.

cancer biology↗

bioRxiv2025

Tahoe-100M: A Giga-Scale Single-Cell Perturbation Atlas for Context-Dependent Gene Function and Cellular Modeling

Building predictive models of the cell requires systematically mapping how perturbations reshape each cells state, function, and behavior. Here, we present Tahoe-100M, a giga-scale single-cell atlas of 100 million transcriptomic profiles measuring how each of 1,100 small-molecule perturbations impact cells across 50 cancer cell lines. Our high-throughput Mosaic platform, composed of a highly diverse and optimally balanced "cell village", reduces batch effects and enables parallel profiling of thousands of conditions at single-cell resolution at an unprecedented scale. As the largest single-cell dataset to date, Tahoe-100M enables artificial-intelligence (AI)-driven models to learn context-dependent functions, capturing fundamental principles of gene regulation and network dynamics. Although we leverage cancer models and pharmacological compounds to create this resource, Tahoe-100M is fundamentally designed as a broadly applicable perturbation atlas and supports deeper insights into cell biology across multiple tissues and contexts. By publicly releasing this atlas, we aim to accelerate the creation and development of robust AI frameworks for systems biology, ultimately improving our ability to predict and manipulate cellular behaviors across a wide range of applications.

genomics↗

bioRxiv2023

Targeting Acute Myeloid Leukemia Stem Cells Through Perturbation of Mitochondrial Calcium

We previously reported that acute myeloid leukemia stem cells (LSCs) are uniquely reliant on oxidative phosphorylation (OXPHOS) for survival. Moreover, maintenance of OXPHOS is dependent on BCL2, creating a therapeutic opportunity to target LSCs using the BCL2 inhibitor drug venetoclax. While venetoclax-based regimens have indeed shown promising clinical activity, the emergence of drug resistance is prevalent. Thus, in the present study, we investigated how mitochondrial properties may influence mechanisms that dictate venetoclax responsiveness. Our data show that utilization of mitochondrial calcium is fundamentally different between drug responsive and non-responsive LSCs. By comparison, venetoclax-resistant LSCs demonstrate a more active metabolic (i.e., OXPHOS) status with relatively high steady-state levels of calcium. Consequently, we tested genetic and pharmacological approaches to target the mitochondrial calcium uniporter, MCU. We demonstrate that inhibition of calcium uptake sharply reduces OXPHOS and leads to eradication of venetoclax-resistant LSCs. These findings demonstrate a central role for calcium signaling in the biology of LSCs and provide a therapeutic avenue for clinical management of venetoclax resistance. SignificanceWe identify increased utilization of mitochondrial calcium as distinct metabolic requirement of venetoclax-resistant LSCs and demonstrate the potential of targeting mitochondrial calcium uptake as a therapeutic strategy.

cancer biology↗

bioRxiv2023

A systematic search for RNA structural switches across the human transcriptome

RNA structural switches are key regulators of gene expression in bacteria, yet their characterization in Metazoa remains limited. Here we present SwitchSeeker, a comprehensive computational and experimental approach for systematic identification of functional RNA structural switches. We applied SwitchSeeker to the human transcriptome and identified 245 putative RNA switches. To validate our approach, we characterized a previously unknown RNA switch in the 3UTR of the RORC transcript. In vivo DMS-MaPseq, coupled with cryogenic electron microscopy, confirmed its existence as two alternative structural conformations. Furthermore, we used genome-scale CRISPR screens to identify trans factors that regulate gene expression through this RNA structural switch. We found that nonsense-mediated mRNA decay acts on this element in a conformation-specific manner. SwitchSeeker provides an unbiased, experimentally-driven method for discovering RNA structural switches that shape the eukaryotic gene expression landscape.

systems biology↗

bioRxiv2022

A novel type of monocytic leukemia stem cell revealed by the clinical use of venetoclax-based therapy

The BCL-2 inhibitor venetoclax has recently emerged as an important component of acute myeloid leukemia (AML) therapy. Notably, use of this agent has revealed a previously unrecognized form of pathogenesis characterized by monocytic disease progression. We demonstrate that this form of disease arises from a fundamentally different type of leukemia stem cell (LSC), which we designate as monocytic LSC (m-LSC), that is developmentally and clinically distinct from the more well-described primitive LSC (p-LSC). The m-LSC is distinguished by a unique immunophenotype (CD34-, CD4+, CD11b-, CD14-, CD36-), unique transcriptional state, reliance on purine/pyrimidine metabolism, and selective sensitivity to cladribine. Critically, in some instances m-LSC and p-LSC subtypes can co-reside in the same AML patient and simultaneously contribute to overall tumor complexity. Thus, our findings demonstrate that LSC heterogeneity has direct clinical significance and highlights the need to distinguish and target m-LSCs as a means to improve clinical outcomes with venetoclax-based regimens. Statement of SignificanceThese studies identify and characterize a new type of human acute myeloid leukemia stem cell (LSC) that is responsible for monocytic disease progression in acute myeloid leukemia (AML) patients treated with venetoclax-based regimens. Our studies describe the phenotype, molecular properties, and drug sensitivities of this unique LSC subclass.

cancer biology↗

bioRxiv2022

Not just the sum of its parts: geographic variation and non-additive effects of pyrazines in the chemical defence of an aposematic moth

Chemical defences often vary within and between populations both in quantity and quality, which is puzzling if prey survival is dependent on the strength of the defence. We investigated the within-and between-population variability in chemical defence of the wood tiger moth (Arctia plantaginis). The major components of its defences, SBMP (2-sec-butyl-3-methoxypyrazine) and IBMP (2-isobutyl-3-methoxypyrazine) are volatiles that deter bird attacks. We expected the variation to reflect populations predation pressures and early-life conditions. To understand the role of the methoxypyrazines, we experimentally manipulated synthetic SBMP and IBMP and tested the birds reactions. We found a considerable variation in methoxypyrazine amounts and composition, both from wild-caught and laboratory-raised male moths. In agreement with the "cost of defence" hypothesis, the moths raised in the laboratory had a higher amount of pyrazines. We found that SBMP is more effective at higher concentrations and that IBMP is more effective only in combination with SBMP and at lower concentrations. Our results fit findings from the wild: the amount of SBMP was higher in the populations with higher predation pressure. Altogether, this suggests that, regarding pyrazine concentration, more is not always better, and highlights the importance of testing the efficacy of chemical defence and its components with relevant predators, rather than relying only on results from chemical analyses.

evolutionary biology↗

bioRxiv2021

Isolation and characterisation of quercitrin as a potent anti-sickle cell anaemia agent from Alchornea cordifolia.

Alchornea cordifolia Mull. Arg. (commonly known as Christmas Bush) has been used traditionally in Africa to treat sickle cell anaemia (a recessive disease, arising from the S haemoglobin [Hb] allele) but the active compounds are yet to be characterised. Herein we describe the use of sequential fractionation coupled with in vitro anti-sickling assays to purify the active component. Sickling was induced in HbSS genotype blood samples using sodium metabisulphite (Na2S2O5) or incubation in 100 % N2. Methanol extracts of A. cordifolia leaves and its sub-fractions showed >70 % suppression of HbSS erythrocyte sickling. Purified compound demonstrated 87.2 {+/-} 2.39 % significant anti-sickling activity and 93.1 {+/-} 2.69 % erythrocyte sickling-inhibition at 0.4 mg/mL. Nuclear magnetic resonance (NMR) spectra and high-resolution mass spectroscopy identified it as quercitrin (quercetin 3-rhamnoside). Purified quercitrin also inhibited the polymerisation of isolated HbS and stabilized sickle erythrocytes membranes. Metabolomic comparisons of blood samples using flow-infusion electrospray-high resolution mass spectrometry indicated that quercitrin could convert HbSS erythrocyte metabolomes to be similar to HbAA. Sickling was associated with changes in anti-oxidants, anaerobic bioenergy and arachidonic acid metabolism, all of which were reversed by quercitrin. The findings described could inform efforts directed to the development of an anti-sickling drug or quality control assessments of A. cordifolia preparations.

pharmacology and toxicology↗

bioRxiv2020

Modeling consensus and distance to delivering indoor residual spray (IRS) implementation strategies to control malaria transmission

BackgroundIndoor residual spraying (IRS) is an effective method to control malaria-transmitting Anopheles mosquitoes and often complements insecticide-treated mosquito nets, the predominant malaria vector control intervention. With insufficient funds to cover every household, malaria control programs must balance the malaria risk to a particular human community against the financial cost of spraying that community. This study creates a framework for modeling the distance to households for targeting IRS implementation, and applies it to potential risk prioritization strategies in four provinces (Luapula, Muchinga, Eastern, and Northern) in Zambia. MethodsWe used optimal network models to assess the travel distance of routes between operations bases and human communities identified through remote sensing. We compared network travel distances to Euclidean distances, to demonstrate the importance of accounting for road route costs. We then compared the distance to reaching communities for different risk prioritization strategies assuming sufficient funds to spray 50% of households, using four underlying malarial risk maps: a) predicted Plasmodium falciparum parasite rate in 2-10 year olds (PfPR), or b) predicted probability of the presence of each of three main malaria transmitting anopheline vectors (An. arabiensis, An. funestus, An. gambiae) ResultsThe estimated one-way network route distance to reach communities to deliver IRS ranged from 0.05 - 115.69 Km. Euclidean distance over and under-estimated these routes by -101.21 - 41.79 Km per trip, as compared to the network route method. There was little overlap between risk map prioritization strategies, both at a district-by-district scale, and across all four provinces. At both scales, agreement for inclusion or exclusion from IRS across all four prioritization strategies occurred in less than 10% of houses. The distances to reaching prioritized communities were either lower, or not statistically different from non-prioritized communities, at both scales of strategy. ConclusionVariation in distance to targeted communities differed depending on risk prioritization strategy used, and higher risk prioritization did not necessarily translate into greater distances in reaching a human community. These findings from Zambia suggest that areas with higher malaria burden may not necessarily be more remote than areas with lower malaria burden.

bioinformatics↗

bioRxiv2020

SCITO-seq: single-cell combinatorial indexed cytometry sequencing

The development of DNA-barcoded antibodies to tag cell-surface molecules has enabled the use of droplet-based single cell sequencing (dsc-seq) to profile the surface proteomes of cells. Compared to flow and mass cytometry, the major limitation of current dsc-seq-based workflows is the high cost associated with profiling each cell, thus precluding its use in applications where millions of cells are required. Here, we introduce SCITO-seq, a new workflow that combines combinatorial indexing and commercially available dsc-seq to enable cost-effective cell surface proteomic sequencing of greater than 105 cells per microfluidic reaction. We demonstrate SCITO-seqs feasibility and scalability by profiling mixed species cell lines and mixed human T and B lymphocytes. To further demonstrate its applicability, we show comparable cellular composition estimates in peripheral blood mononuclear cells obtained with SCITO-seq and mass cytometry. SCITO-seq can be extended to include simultaneous profiling of additional modalities such as transcripts and accessible chromatin or tracking of experimental perturbations such as genome edits or extracellular stimuli.

genomics↗

bioRxiv2019

Subtle, seasonal and potentially important: Are phytotoxic foliage leachates overlooked in plant invasion studies?

Invasive species pose a threat to global biodiversity. Recently there has been a growth in research into the chemical aspect of invasions, with some plants reported to introduce compounds to the soil which impair the growth of competing native plants. One such species is Rhododendron ponticum, which has become a damaging invasive shrub in Britain. Here we investigate whether water-soluble compounds introduced from R. ponticum leaves in Ceredigion, Wales, suppressed seed germination, and whether this varied temporally. Leachates from leaf material collected monthly (March to July) were analysed for their chemical composition by liquid chromatography-mass spectrometry and Lactuca sotiva bioassays. In total, 19 compounds were identified, displaying both qualitative and quantitative seasonal variation. Total concentrations peaked in June, however L sativa germination inhibition was only found in leachates from April and May (15% and 17% respectively). The study provides evidence that phytotoxic chemical introduction from R. ponticum leaves is seasonal and most bioactive at a time of year when the seeds of other species germinate. We propose this is one of several mechanisms including shading, lowering of soil pH and the deposition of thick litter, that this species exploits when in competition with other species. We argue that for studies on the phytotoxicity of invasive plants, there is a need to consider temporal variation in the rate and quality of bioactive chemicals produced and released, local precipitation rates, persistence of chemicals in the soil plus synergistic interactions between individual chemicals.

ecology↗