👤 Dan B Faulkner

Malignant pleural mesothelioma (PM) is an aggressive cancer with limited treatment options. Although first-line nivolumab plus ipilimumab improves outcomes for some patients, a majority fail to respond. Mechanisms of immune resistance in PM remain poorly understood, underscoring the need for new clinically actionable drug targets to overcome immunotherapy resistance. We established an in silico pipeline to investigate the molecular basis of T-cell exclusion in bulk RNA-sequencing data from 448 patients across three immune checkpoint blockade (ICB)-naïve PM cohorts. We assessed genome-wide correlations between gene expression and a previously validated cytotoxic T-cell signature score. Candidate immune evasion genes were prioritized based on clinical relevance, drug availability, and experimental feasibility for follow-up translational research. The in silico pipeline produced a highly reproducible catalogue of genes whose expression inversely correlates with T-cell infiltration, including established immune evasion factors (e.g. SOX4, KDM5B, CMTM4) and five novel FDA-approved drug targets (SMO, GANAB, ERBB2, GABRA1, ODC1). Seven additional targets (ARNT, BMPR1B, GSK3B, ACVR1, BACE1, RPS6KB1, ULK1) with preclinical inhibitors were also identified. Notably, we identified a possible link between primary cilia, Hedgehog signaling and T-cell exclusion. We found that SMO expression correlated with poor clinical response to second-line nivolumab plus ipilimumab in PM, highlighting SMO as a promising therapeutic target and potential biomarker for treatment resistance. This comprehensive transcriptomic characterization of T-cell exclusion in PM reveals that targeting cilium-based Hedgehog signaling, in addition to multiple other actionable drug targets, could enhance the efficacy of ICB treatment in PM. Show less

Acute prolonged sitting increases blood pressure (BP) and arterial stiffness (AS). Both of these may be mitigated via light physical activity (LPA). Whether long COVID (LC), which partly manifests as vascular sequelae, predisposes a heightened sensitivity to sitting or diminished benefits from its interruption is unknown. The aims of this study were to identify whether individuals with LC: (i) exhibit a worse BP/AS response to uninterrupted sitting and (ii) a diminished mitigation of BP/AS response to sitting interrupted with LPA, compared to healthy controls. Thirty participants with LC and 15 controls completed 2 h of uninterrupted sitting and sitting interrupted with LPA. Central and peripheral systolic and diastolic BP and carotid-femoral pulse wave velocity (cfPWV) were determined pre and post sitting. Linear mixed-effects models demonstrated no three-way or two-way interactions for any variable. There was a significant main effect of time, with increases in central systolic (MD = 3.37 mmHg, SE = 0.93 mmHg, p < 0.001) and central diastolic (MD = 3.00 mmHg, SE = 0.58 mmHg, p < 0.001) BP. cfPWV was not altered in sitting in either group (MD = 0.13 m/s, SE = 0.09 m/s, p = 0.170). Uninterrupted sitting increases BP similarly, but AS is unchanged. Interrupting sitting with LPA did not mitigate sitting-induced increase in BP regardless of LC diagnosis. Show less

Understanding the factors which control endothelial cell (EC) function and angiogenesis is crucial for developing the horse as a disease model, but equine ECs remain poorly studied. In this study, we have optimised methods for the isolation and culture of equine aortic endothelial cells (EAoECs) and characterised their angiogenic functions in vitro. Mechanical dissociation, followed by magnetic purification using an anti-VE-cadherin antibody, resulted in EC-enriched cultures suitable for further study. Fibroblast growth factor 2 (FGF2) increased the EAoEC proliferation rate and stimulated scratch wound closure and tube formation by EAoECs on the extracellular matrix. Pharmacological inhibitors of FGF receptor 1 (FGFR1) (SU5402) or mitogen-activated protein kinase (MEK) (PD184352) blocked FGF2-induced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and functional responses, suggesting that these are dependent on FGFR1/MEK-ERK signalling. In marked contrast, vascular endothelial growth factor-A (VEGF-A) had no effect on EAoEC proliferation, migration, or tubulogenesis and did not promote ERK1/2 phosphorylation, indicating a lack of sensitivity to this classical pro-angiogenic growth factor. Gene expression analysis showed that unlike human ECs, FGFR1 is expressed by EAoECs at a much higher level than both VEGF receptor (VEGFR)1 and VEGFR2. These results suggest a predominant role for FGF2 versus VEGF-A in controlling the angiogenic functions of equine ECs. Collectively, our novel data provide a sound basis for studying angiogenic processes in horses and lay the foundations for comparative studies of EC biology in horses versus humans. Show less

Perfluorooctanoic acid (PFOA) is a typical C8 representative compound of perfluoroalkyl and polyfluoroalkyl substances (PFAS) widely used in industrial and domestic products. It is a persistent organic pollutant found in the environment as well as in the tissues of humans and wildlife. Despite emerging scientific and public interest, the precise mechanisms of PFOA toxicity remain unclear. In this study, male rats were exposed to 1.25, 5, and 20 mg PFOA/kg body weight/day for 14 days by gavage; food intake and bodyweight changes were recorded every day. After 14 days, blood was collected for sera biochemistry, livers were quickly stripped and weighed after execution. Part of the liver tissue was frozen by liquid nitrogen for iTRAQ-Based Quantitative Proteomics Analysis; and some was fixed in 4% paraformaldehyde (PFA) for histological section and hematoxylin-eosin (HE) staining. Urine samples were also collected and monitored by raising rats in metabolic cages. Real-time quantitative PCR and western blot was used to validate the proteomics assay after bioinformatics analysis. The results demonstrate that 20 mg/kg/d PFOA exposure cause body weight loss and significant liver swelling and reduced urea metabolism. The sera biochemistry assay shows that ALT, GGT, BILD and UREA levels have significant changes compared with normal control group and reference range of rat sera. The subsequent iTRAQ-based quantitative proteomics analysis of rat livers identified 3,327 non-redundant proteins of which 112 proteins were significantly upregulated and 80 proteins were downregulated. Gene ontology analysis revealed proteins are primarily involved in cellular, metabolic and single-organism processes. Among them, eight proteins (ACOX1, ACOX2, ACOX3, ACSL1, EHHADH, GOT2, MTOR and ACAA1) were related to oxidation of fatty acids and two proteins (ASS1 and CPS1) were found to be associated with urea cycle disorder. The downregulation of urea synthesis proteins ASS1 and CPS1 after exposure to PFOA was then confirmed through qPCR and western blot analysis. Together, these data demonstrate that PFOA exposure directly influences urea metabolism and provides insight into specific mechanisms of hepatotoxicity as a result of PFOA exposure. Show less

Adipocyte differentiation is probably controlled by transcriptional and post-transcriptional regulation. Longissimus lumborum from Angus steers (aged 155 d; seven animals per diet) fed high-starch or low-starch diets for 112 d (growing phase) followed by a common high-starch diet for an additional 112 d (finishing phase) was biopsied at 0, 56, 112 and 224 d for transcript profiling via quantitative PCR of twenty genes associated with adipogenesis and energy metabolism. At 56 d steers fed high starch had greater expression of PPARgamma as well as the lipogenic enzymes ATP citrate lyase (ACLY), glucose-6-phosphate dehydrogenase (G6PD), fatty acid synthase (FASN), fatty acid binding protein 4 (FABP4), stearoyl-CoA desaturase (SCD), glycerol-3-phosphate acyltransferase, mitochondrial (GPAM), and diacylglycerol O-acyltransferase homologue 2 (DGAT2), and the adipokine adiponectin (ADIPOQ). Expression of insulin-induced gene 1 (INSIG1) was also greater with high starch at 56 d. Steers fed low starch experienced a marked increase in FASN, FABP4, SCD, DGAT2 and thyroid hormone-responsive (SPOT14 homologue, rat) (THRSP) between 56 and 112 d of feeding. A greater expression of the transcription factors sterol regulatory element-binding transcription factor 1 (SREBF1) and MLX interacting protein-like (MLXIPL) was observed at 224 d in steers fed high starch, suggesting a nutritional imprinting effect. Carryover effects of low starch feeding were discerned by greater expression at 224 d of THRSP, FABP4, SCD and DGAT2. These steers also had greater PPARgamma at 224 d. Despite these responses, low starch led to greater expression at 224 d of nuclear receptor subfamily 2, group F, member 2 (NR2F2), a known repressor of rodent adipocyte differentiation through its negative effects on PPARgamma, ADIPOQ and FABP4. Results suggested that early exposure to high starch induced precocious intramuscular adipocyte proliferation and metabolic imprinting of lipogenic transcription regulators. Low starch might have blunted the PPARgamma-driven adipogenic response through up-regulation of NR2F2 but the endogenous ligand for this nuclear receptor remains unknown. Show less