Biology subjects

Wilkins, M.

Publications and source records attributed to Wilkins, M..

11 recordsLinked to original sources

bioRxiv2026

Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

Genome-wide association studies have identified rare and common mutations associated with increased risk of pulmonary arterial hypertension (PAH), but the mechanism by which impaired SOX17 expression increases PAH risk is not known. Notably, SOX17 plays a critical role in endothelial identity during development by suppressing RUNX1 through binding to its promoter and directing stem and progenitor cells toward an endothelial rather than a hematopoietic cell fate. RUNX1 functions as a key regulator of myeloid differentiation, aberrant angiogenesis and adverse cardiac remodeling. Previously, we found that RUNX1 inhibition reverses pulmonary hypertension (PH) in multiple animal models. Here, we hypothesize that impaired expression of SOX17 in PAH leads to endothelial cell (EC) dysfunction by failing to suppress RUNX1. METHODSHuman pulmonary artery endothelial cells (HPAECs) with stable SOX17 CRISPR/Cas9 knockout or RUNX1 overexpression were generated and examined for endothelial and hematopoietic gene expression, proliferation, migration, apoptosis, and angiogenesis. Immortalized lymphoblastoid cell lines (LCLs) from PAH patients with SOX17 mutations and healthy controls were reprogrammed into induced pluripotent stem cells (iPSCs) and differentiated into ECs. The effect of RUNX1 inhibition on Sugen/hypoxia-PH was examined in rats, SOX17 enhancer knockout (SOX17enhKO) mice, and Cdh5-CreERT2;Runx1(flox/flox);SOX17enhKO triple transgenic mice. SOX17 and RUNX1 expression were analyzed in peripheral blood samples from PAH patients (n=359). RESULTSHPAECs with SOX17 deletion or RUNX1 overexpression exhibited decreased expression of EC markers, enhanced proliferation and migration, defective angiogenesis, and decreased apoptosis. RUNX1 siRNA knockdown or RUNX1 inhibition by Ro5-3335 partially restored the endothelial properties in SOX17 KO HPAECs. ECs differentiated from SOX17 mutant PAH patient iPSCs exhibited upregulated RUNX1 expression and loss of endothelial identity, which was also partially restored by RUNX1 siRNA or Ro5-3335. In addition, SOX17enhKO mice had increased RUNX1 expression and susceptibility to Sugen/hypoxia-induced PH (SuHx-PH). Treatment with RUNX1 inhibitors or inducible endothelial-specific deletion of RUNX1 rescued SuHx-PH susceptibility in SOX17enhKO mice. RUNX1 inhibitors Ro5-3335 and Ro24-7429 also reversed SuHx-PH in wild-type rats. In addition, plasma RUNX1 expression was higher in PAH patients lacking detectable SOX17 expression than in patients with detectable SOX17 expression. CONCLUSIONSImpaired SOX17 expression increases the risk of PAH through insufficient suppression of RUNX1, leading to pulmonary endothelial dysfunction. RUNX1 inhibition mitigates PH associated with SOX17 deficiency and may represent a novel therapeutic strategy for PAH, especially those with rare or common SOX17 mutations.

cell biology↗

bioRxiv2026

Dengue viruses serotypes 2 and 4 exhibit distinct infection kinetics and modulation of anti-viral immune responses in human tonsil histocultures

Dengue virus (DENV) is the most prevalent mosquito-borne viral disease with over ten million cases worldwide. There are four antigenically distinct, co-circulating DENV serotypes (DENV 1-4) capable of infecting humans. Given the lack of immunocompetent animal models and the limitations of known cell culture models, more physiologically relevant experimental models are needed to recapitulate DENV infection and host immune responses. Building on previous observations that DENV-2 and DENV-4 serotypes elicit distinct innate immune responses in monocyte-derived dendritic cells (moDC) in vitro, we utilized a human tonsil histoculture (HC) model to further investigate serotype-specific differences within the physiologically relevant human lymphoid environment. We show that human tonsil HCs preserved their tissue cytoarchitecture for up to 6 days in culture, including maintaining functional germinal centers and diverse immune populations within T and B cell compartments. Exposure of tonsil HCs to DENV-2 and DENV-4 showed that DENV-4 replication peaked earlier and induced enhanced innate immune activation compared to DENV-2, consistent with previous observations in human DCs. Moreover, by leveraging the structural and cellular complexity of the HC system, we further identified that DENV E protein co-localized with HLA-DR+ antigen-presenting cells, confirming that DENV-infected cells within tonsil HCs predominantly express antigen-presenting cell markers. Altogether, these findings demonstrate that different DENV serotypes can exhibit different viral replication dynamics and induce distinct immune responses within human lymphoid tissue. This establishes human tonsil HCs as human model system with intact cytoarchitecture that closely mirrors lymph node structure and function, providing a powerful platform to study antigen-driven and virus-specific human immune responses and ultimately to evaluate vaccine candidates and antiviral therapeutics.

microbiology↗

bioRxiv2025

Impaired Lung BCAA Metabolism Promotes Ferroptosis and Resultant Pulmonary Arterial Hypertension-Associated Hepatopathy

BackgroundDysregulated branched chain amino acid (BCAA) homeostasis occurs in pulmonary arterial hypertension (PAH) as BCAA metabolites accumulate and cause metabolic alterations in pulmonary artery smooth muscle cells (PASMC). In other cells, altered BCAA metabolism promotes ferroptosis, a PAH-inducing metabolic pathway. However, the interplay between BCAAs, lung ferroptosis, and PAH is unexplored, as is the impact of PAH severity on liver molecular regulation, a key unknown as recent clinical data highlight the importance of the lung-right heart-liver axis in PAH outcomes. MethodsHuman metabolomic and transcriptomic studies examined BCAA metabolism and ferroptosis pathways. The relationship between BCAAs and ferroptotic-phenotypes in PASMCs was evaluated. Multi-omics and physiological analyses evaluated how modulation of BCAA catabolism impacted preclinical PAH multi-organ physiology. Confocal microscopy and proteomic analyses assessed hepatic alterations in human PAH. ResultsMetabolomic analyses identified alterations in BCAA metabolites across multiple physiological gradients in patients with pulmonary vascular disease. RNA sequencing demonstrated deficits in the BCAA catabolic and ferroptosis pathways in PAH lungs and smooth muscle cells. In vitro, excess BCAAs induced mitochondrial fragmentation, reactive oxygen species generation, and lipid peroxidation in PASMC. Moreover, BCAAs promoted PASMC death, which ferrostatin-1, a ferroptosis antagonist, rescued. BT2, a small-molecule inducer of BCAA catabolism, reduced PAH severity, improved RV function, and enhanced maximal exercise capacity in monocrotaline rats. BT2 blunted pro-ferroptotic changes in lung metabolites and proteins, and combatted peri-vascular complement deposition. In the liver, BT2 blocked mechanical shear stress phenotypes including hepatocyte nuclear expansion and restructured mitochondrial protein regulation and the metabolomic signature. Additionally, a low BCAA diet modestly combatted preclinical PAH severity. Finally, human PAH livers exhibited increased hepatocyte nuclear size and derangements in liver metabolic regulation. ConclusionsImpaired BCAA metabolism promotes PAH via ferroptosis. PAH severity is associated with hepatic pathological shear stress phenotypes and metabolic alterations, which are combatted by a BCAA-targeted therapy. Graphical Abstract/Summary Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/672819v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@1649980org.highwire.dtl.DTLVardef@199c40aorg.highwire.dtl.DTLVardef@1586fdorg.highwire.dtl.DTLVardef@15166c1_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Crackling Cloud: an event-driven, cloud-based CRISPR-Cas9 guide RNA design tool

Gene editing has been revolutionised by the CRISPR-Cas9 technology. The versatility and ease-of-use of the technology far exceeds its predecessors, however, the selection of a high-quality guide RNA (gRNA) is critical to directing it to a target site. Selecting gRNA calls upon high-performance algorithms that evaluate nuclease activity at on-target and off-target sites. While there are a suite of programs available, many struggle to analyse the largest genomes, or their predictive accuracy is low. We have previously published a program, named Crackling that is amongst the fastest and most accurate tools available, however, it requires an end-user to have access to a traditional high-performance computing environment. Here, we present an adaptation of Crackling, named Crackling Cloud, that takes advantage of modern serverless cloud technologies that are widely available to anyone, and do not consume resources and incur costs when sitting idle, but can scale to use large volumes of resources when analyses require that. Crackling Cloud is provided as a templated solution using technologies of Amazon Web Services, and is available for free on GitHub under the terms of the BSD 3-clause licence: https://github.com/bmds-lab/Crackling-AWS

bioinformatics↗

bioRxiv2024

Inferring gene regulatory networks by hypergraph variational autoencoder

In constructing Gene Regulatory Networks (GRNs), it is crucial to consider cellular heterogeneity and differential gene regulatory modules. However, traditional methods have predominantly focused on cellular heterogeneity, approaching the subject from a relatively narrow scope. We present HyperG-VAE, a Bayesian deep generative model that utilizes a hypergraph to model single-cell RNA sequencing (scRNA-seq) data. HyperG-VAE employs a cell encoder with a Structural Equation Model to address cellular heterogeneity and build GRNs, alongside a gene encoder using hypergraph self-attention to identify gene modules. Encoders are synergistically optimized by a decoder, enabling HyperG-VAE to excel in GRN inference, single-cell clustering, and data visualization, evidenced by benchmarks. Additionally, HyperG-VAE effectively reveals gene regulation patterns and shows robustness in varied downstream analyses, demonstrated using B cell development data in bone marrow. The interplay of encoders by the overlapping genes between predicted GRNs and gene modules is further validated by gene set enrichment analysis, underscoring that the gene encoder boosts the GRN inference. HyperG-VAE proves efficient in scRNA-seq data analysis and GRN inference.

bioinformatics↗

bioRxiv2023

ATP13A3 Variants Promote Pulmonary Arterial Hypertension by Disrupting Polyamine Transport

AimsPotential loss-of-function variants of ATP13A3, the gene encoding a P5B-type transport ATPase of undefined function, were recently identified in pulmonary arterial hypertension (PAH) patients. ATP13A3 is implicated in polyamine transport but its function has not been fully elucidated. Here, we sought to determine the biological function of ATP13A3 in vascular endothelial cells and how PAH-associated mutations may contribute to disease pathogenesis. We also generated mice harbouring an Atp13a3 variant analogous to a human disease-associated variant to establish whether these mice develop PAH. Methods and ResultsWe studied the impact of ATP13A3 deficiency and overexpression in endothelial cell (EC) models (human pulmonary ECs, blood outgrowth ECs (BOECs) and HMEC-1 cells), including a PAH patient-derived BOEC line harbouring an ATP13A3 variant (LK726X). ATP13A3 localised to the recycling endosomes of human ECs. Knockdown of ATP13A3 in ECs generally reduced the basal polyamine content, consistently reduced putrescine uptake, and altered the expression of enzymes involved in polyamine metabolism. Conversely, overexpression of wild-type ATP13A3 increased polyamine uptake, with an overall preference of putrescine > spermidine > spermine. Functionally, loss of ATP13A3 was associated with reduced EC proliferation, increased apoptosis in serum starvation and increased monolayer permeability to thrombin. Assessment of five PAH-associated missense ATP13A3 variants (L675V, M850I, V855M, R858H, L956P) confirmed loss-of-function phenotypes represented by impaired polyamine transport and dysregulated EC function. Furthermore, mice carrying a heterozygous germ-line Atp13a3 frameshift variant representing a human mutation spontaneously developed a PAH phenotype, with increased pulmonary pressures, right ventricular remodelling and muscularisation of pulmonary vessels. ConclusionWe identify ATP13A3 as a polyamine transporter, deficiency of which leads to EC dysfunction and predisposes to PAH. This suggests a need for targeted therapies to alleviate the imbalances in polyamine homeostasis and EC dysfunction in PAH. Translational perspectiveRare missense ATP13A3 disease-associated variants have been identified in patients with pulmonary arterial hypertension (PAH), though their pathogenicity has not been confirmed as the function of ATP13A3 is not known. We have identified ATP13A3 as a polyamine transporter, showing that ATP13A3 deficiency impaired polyamine homeostasis and uptake, and drove endothelial dysfunction. Conversely, overexpression increased polyamine uptake and rescued the proapoptotic phenotype of cells harbouring a disease-associate variant. Mice heterozygous for a disease-associated Atp13a3 mutation spontaneously develop PAH. These findings support the rationale for exploring dysregulated polyamine homeostasis in PAH and suggest a potential for therapeutic targeting of polyamine pathways in PAH.

cell biology↗

bioRxiv2023

Polymer biodegradation by Halanaerobium promotes reservoir souring during hydraulic fracturing.

Hydraulically fractured shale reservoirs have facilitated studies of unexplored niches in the continental deep biosphere. Members of the genus Halanaerobium are ubiquitous in these environments. Polymers like guar gum used as gelling agents in hydraulic fracturing fluids are known to be fermentable substrates, but metabolic pathways encoding these processes have not been characterized. To explore this, produced water samples from the Permian Basin were incubated at 30{degrees}C to simulate above-ground storage conditions, and at 60{degrees}C to simulate subsurface reservoir conditions. Guar metabolism coincided with Halanaerobium enrichment only at 30{degrees}C, revealing genes for polymer biodegradation through the mixed-acid fermentation pathway in different metagenome-assembled genomes (MAGs). Whereas thiosulfate reduction to sulfide is often invoked to explain the dominance of Halanaerobium in these settings, Halanaerobium genomes did not uncover genes for this metabolism. Sulfide production was observed in 60{degrees}C incubations, with corresponding enrichment of Desulfohalobium and Desulfovibrionaceae that possess complete pathways for coupling mannose and acetate oxidation to sulfate reduction. These findings outline how production of fermentation intermediates (mannose, acetate) by Halanaerobium in topside settings can result in reservoir souring when these metabolites are introduced into the subsurface through produced water re-use. ImportanceHydraulically fractured shale oil reservoirs are ideal for studying extremophiles such as thermohalophiles. During hydraulic fracturing, reservoir production water is stored in surface ponds prior to re-use. Microorganisms in these systems therefore need to withstand various environmental changes such as the swing between warm downhole oil reservoir temperatures and cooler surface conditions. This study follows this water cycle during fracking and the associated microbial metabolic potential. Of particular interest are members of the genus Halanaerobium, that have been reported to reduce thiosulfate contributing to souring of oil reservoirs. Here, we show that some Halanaerobium strains were unable to grow under oil reservoir temperatures and do not possess genes for thiosulfate reduction. Rather, it is likely that these organisms metabolize complex organics in fracking fluids at lower temperatures, thereby generating substrates that support reservoir souring by thermophilic sulfate-reducing bacteria at higher temperatures.

microbiology↗

bioRxiv2023

ApoA-I Protects Pancreatic β-cells from Cholesterol-induced Mitochondrial Damage and Restores their Ability to Secrete Insulin

Objective: High cholesterol levels in pancreatic {beta}-cells cause oxidative stress and decrease insulin secretion. {beta}-cells can internalize apolipoprotein (apo) A-I, which increases insulin secretion. This study asks whether internalization of apoA-I improves {beta}-cell insulin secretion by reducing oxidative stress. Approach: Ins-1E cells were cholesterol-loaded by incubation with cholesterol-methyl-{beta}-cyclodextrin. Insulin secretion in the presence of 2.8 or 25 mM glucose was quantified by radioimmunoassay. Internalization of fluorescently labelled apoA-I by {beta}-cells was monitored by flow cytometry. The effects of apoA-I internalization on {beta}-cell gene expression was evaluated by RNA sequencing. ApoA-I binding partners on the {beta}-cell surface were identified by mass spectrometry. Mitochondrial oxidative stress was quantified in {beta}-cells and isolated islets with MitoSOX and confocal microscopy. Results: An F1-ATPase {beta}-subunit on the {beta}-cell surface was identified as the main apoA-I binding partner. {beta}-cell internalization of apoA-I was time-, concentration-, temperature-, cholesterol- and F1-ATPase {beta}-subunit-dependent. {beta}-cells with internalized apoA-I (apoA-I+ cells) had higher cholesterol and cell surface F1-ATPase {beta}-subunit levels than {beta}-cells without internalized apoA-I (apoA-I- cells). The internalized apoA-I co-localized with mitochondria and was associated with reduced oxidative stress and increased insulin secretion. The ATPase inhibitory factor 1, IF1, attenuated apoA-I internalization and increased oxidative stress in Ins-1E {beta}-cells and isolated mouse islets. Differentially expressed genes in apoA-I+ and apoA-I- Ins-1E cells were related to protein synthesis, the unfolded protein response, insulin secretion and mitochondrial function. Conclusions: These results establish that {beta}-cells are functionally heterogeneous and apoA-I restores insulin secretion in {beta}-cells with elevated cholesterol levels by improving mitochondrial redox balance.

cell biology↗

bioRxiv2022

Effect of insulin insufficiency on ultrastructure and function in skeletal muscle

BackgroundDecreased insulin availability and high blood glucose levels, the hallmark features of poorly controlled diabetes, drive disease progression and are associated with decreased skeletal muscle mass. We have shown that mice with {beta}-cell dysfunction and normal insulin sensitivity have decreased skeletal muscle mass. This project asks how insulin deficiency impacts on the structure and function of the remaining skeletal muscle in these animals. MethodsSkeletal muscle function was determined by measuring exercise capacity and specific muscle strength prior to and after insulin supplementation for 28 days in 12-week-old mice with conditional {beta}-cell deletion of the ATP binding cassette transporters ABCA1 and ABCG1 ({beta}-DKO mice). Abca1 and Abcg1 floxed (fl/fl) mice were used as controls. RNAseq was used to quantify changes in transcripts in soleus and extensor digitorum longus muscles. Skeletal muscle and mitochondrial morphology were assessed by transmission electron microscopy. Myofibrillar Ca2+ sensitivity and maximum isometric single muscle fibre force were assessed using MyoRobot biomechatronics technology. ResultsRNA transcripts were significantly altered in {beta}-DKO mice compared to fl/fl controls (32 in extensor digitorum longus and 412 in soleus). Exercise capacity and muscle strength were significantly decreased in {beta}-DKO mice compared to fl/fl controls (p = 0.012), and a loss of structural integrity was also observed in skeletal muscle from the {beta}-DKO mice. Supplementation of {beta}-DKO mice with insulin restored muscle integrity, strength and expression of 13 and 16 of the dysregulated transcripts in and extensor digitorum longus and soleus muscles, respectively. ConclusionsInsulin insufficiency due to {beta}-cell dysfunction perturbs the structure and function of skeletal muscle. These adverse effects are rectified by insulin supplementation.

physiology↗

bioRxiv2022

PTPN1 deficiency modulates BMPR2 signaling and induces endothelial dysfunction in Pulmonary Arterial Hypertension

Bone morphogenic protein receptor 2 (BMPR2) expression and signaling are impaired in pulmonary arterial hypertension (PAH). How BMPR2 signaling is decreased in PAH is poorly understood. Protein tyrosine phosphatases (PTPs) play important roles in vascular remodeling in PAH. To identify whether PTPs modify BMPR2 signaling we used a siRNA-mediated high throughput screening of 22,124 murine genes in mouse myoblastoma reporter cells using ID1 expression as read-out for BMPR2 signaling. We further experimentally validated the top hit, PTPN1 (PTP1B), in human healthy pulmonary arterial endothelial cells (PAECs) either silenced by siRNA or exposed to hypoxia and confirmed its relevance to PAH by measuring PTPN1 levels in blood and PAECs collected from PAH patients. We identified PTPN1 as a novel regulator of BMPR2 signaling in PAECs, which is downregulated in the blood of PAH patients and documented that downregulation of PTPN1 is linked to endothelial dysfunction in PAECs. These findings point to a potential involvement for PTPN1 in PAH and will aid in our understanding of the molecular mechanisms involved in the disease.

cell biology↗

bioRxiv2019

Genomic insights into the Archaea inhabiting an Australian radioactive legacy site

During the 1960s, small quantities of radioactive materials were co-disposed with chemical waste at the Little Forest Legacy Site (LFLS, Sydney, Australia). The microbial function and population dynamics in a waste trench during a rainfall event have been previously investigated using shotgun metagenomics. This revealed a broad abundance of candidate and potentially undescribed taxa in this iron-rich, radionuclide-contaminated environment. Here, applying genome-based metagenomic methods, we recovered 37 refined archaeal MAGs ([≥]50% completeness, [≤]10% redundancy) from 10 different major lineages. They were, mainly, included in four DPANN lineages without standing nomenclature (LFWA-I to IV) and Methanoperedenaceae (ANME-2D). While most of the new DPANN lineages show reduced genomes with limited central metabolism typical of other DPANN, the orders under the LFWA-III lineage, Ca. Gugararchaeales and Ca. Anstonellales, may constitute distinct orders with a more comprehensive central metabolism and anabolic capabilities within the Micrarchaeota phylum. The new Methanoperedens spp. MAGs, together with previously published data, suggests metal ions as the ancestral electron acceptors during the anaerobic oxidation of methane while the respiration of nitrate/nitrite via molybdopterin oxidoreductases would have been a secondary acquisition. The presence of genes for the biosynthesis of polyhydroxyalkanoates in most Methanoperedens also appears to be a widespread characteristic of the genus for carbon accumulation. We formally propose 22 new candidate taxa based on data analysed in this manuscript, as well as four missing formal taxa definitions and two new candidate species based on extant data. We present evidence of four new DPANN lineages and six non-conspecific Methanoperedens, while exploring their uniqueness, potential role in elemental cycling, and evolutionary history.

microbiology↗