OBJECTIVE:Zinc finger E-box-binding homeobox 1 (ZEB1) is a transcription factor primarily known for its regulatory roles in epithelial-to-mesenchymal transition (EMT) and cell fate determination. Recent studies suggest that endothelial ZEB1 signaling promotes blood vessel growth and reduces junctional integrity, although the underlying mechanisms remain unclear. Notably, the role of ZEB1 in the lymphatic vasculature is unknown, and the regulation of lymphatic integrity by VE-cadherin remains poorly defined. METHODS:Here, using an integrated proteomic and transcriptomic approach, we identify ZEB1-dependent signaling pathways associated with cell-cell junction reorganization in lymphatic endothelial cells (LECs). RESULTS:Loss of ZEB1 reduced VE-cadherin phosphorylation at pY731 and pY685 and was accompanied by decreased monolayer resistance and impedance, together with increased leukocyte transendothelial migration. ZEB1 knockdown also reduced YES tyrosine kinase expression and altered YAP1 expression and junctional localisation, changes that were associated with reduced VE-cadherin phosphorylation. Silencing YAP1 in HDLECs similarly reduced VE-cadherin phosphorylation and impaired barrier integrity, recapitulating aspects of the phenotype observed following ZEB1 knockdown. CONCLUSIONS:Collectively, these findings suggest that ZEB1 contributes to lymphatic endothelial barrier maintenance in association with altered YAP1 and YES signaling.

Choroidal neovascularisation (CNV) is a hallmark of wet/neovascular age-related macular degeneration (wAMD), characterized by aberrant blood vessel growth from the choroid into the retina. Both pathological angiogenesis and inflammation contribute to disease progression. Here we show that analsysis of single-cell RNA sequencing of experimental CNV lesions revealed upregulation of Zeb1 in angiogenic endothelial cells (ECs). We generated an endothelial-specific Zeb1 knockout (Zeb1iECKO) mouse model to assess its functional role. CNV was induced via laser photocoagulation, and vascular leakage and inflammation were evaluated using fluorescein angiography, immunohistochemistry, and transcriptomic analyses. Zeb1iECKO mice exhibited increased fluorescein leakage and enhanced vascular invasion during CNV, indicating destabilized neovascular structures. However, leukocyte infiltration within CNV lesions was not elevated. In vitro, ZEB1 knockdown in human ECs led to downregulation of inflammatory signalling pathways and reduced expression of adhesion molecules in response to TNF-α stimulation yet retained angiogenic capacity. ZEB1 coordinates angiogenic and inflammatory responses in CNV. Its loss enhances neovascularisation without promoting inflammation, suggesting a potential therapeutic target for modulating pathological angiogenesis in wAMD while minimizing inflammatory damage.

We describe a modular, diverse, and customizable supramolecular-materials platform that can deliver nucleic acids in vitro and in vivo. The chemistries deployed enable the generation of multiple supramolecular polycations, which can associate with RNA to form polyelectrolyte complexes, but which have the unique feature of reversible cross-links, via host-guest interactions of monomers that display aromatic amino acid termini with cucurbit[8]uril (CB[8]). Families of supramolecular polymers can be prepared by simple variation in monomer structure, enabling the tuning of properties. We demonstrate that these supramolecular polyelectrolyte complexes with RNA can be prepared easily via automatable procedures to generate nanoparticles that meet Critical Quality Attributes for manufactured RNA vaccines and therapeutics. We show that these materials can deliver RNA to a range of cell types, displaying reporter-protein expression at levels equivalent to, or greater than, commercial transfection reagents, with no acute adverse phenotypic effects. Finally, we demonstrate the success of our materials platform across a range of nucleic acid types, with expression of mRNA within tumors of an orthotopic Triple-Negative Breast Cancer mouse model, knockdown of a kinase implicated in cancer progression via siRNA, and effective protection against H1N1 influenza virus challenge in mice following injections of self-amplifying RNA.

Ovarian cancer is one of the most common gynaecological cancers affecting more than 300,000 women worldwide each year. S100 calcium-binding protein A6 (S100A6) is a member of the S100 family of calcium-binding proteins. Upon activation by calcium (Ca2+) signalling, S100A6 regulates numerous cellular processes including cell proliferation and metastasis. The role of S100A6 is well established across multiple tumour types where increased S100A6 expression contributes to tumourigenesis and worse patient outcome. However, the role of S100A6 in ovarian cancer is not well established. The impact of cytoplasmic and nuclear S100A6 expression on overall survival and clinicopathological criteria was investigated in 462 ovarian tumours by immunohistochemistry. Additionally, S100A6 expression in an ovarian cancer cohort from The Cancer Genome Atlas (TCGA) PanCancer dataset (n = 299) was evaluated and associations with overall survival and progression-free survival were identified. Immunohistochemical staining revealed that high cytoplasmic expression of S100A6 was significantly associated with better overall survival (p = 0.014). Additionally, high nuclear expression was significantly associated with better overall survival (p = 0.036). In contrast, analysis of mRNA S100A6 suggests no significant association of mRNA levels for overall survival (p = 0.903) and progression free survival (p = 0.278). Our data provides novel insights regarding the clinical implications of S100A6 expression in ovarian cancer, providing strong rationale for functional investigations of S100A6 in ovarian cancer.

Endothelial cells (EC) play a critical role in vascular homeostasis, and their function is influenced by oxygen tension. This study investigates long-term effects of EC culture under physiological oxygen tension on their basal and nitric oxide (NO)-modulated K⁺ channel activities. Electrophysiological experiments demonstrated for the first time that human umbilical vein EC (HUVEC) exhibit larger basal K⁺ outward and smaller inward currents under normoxic (5kPa) compared to hyperoxic (18kPa) O2 levels. Outward currents were only potentiated under hyperoxic conditions by NO. Human cerebral microvascular EC (hCMEC/D3) showed larger outward currents under normoxia which were further potentiated by NO. Protein expression of Kir6.1, KCa3.1, KCa1.1 and KCa2.3 channels was unaffected by ambient O2, suggesting that observed changes in K+ currents in both EC types were due to ion channel modulation. In HUVEC, changes in half-activation voltage and hyperpolarized membrane potentials were detected only under hyperoxic conditions following NO exposure, with both cell types exhibiting altered current activation kinetics of outward and inward currents between culture conditions. Together, we report novel insights into the modulation of K+ channels in EC, with implications for regulation of vascular tone and the design and use of experimental models in vitro for high throughput drug discovery and clinical translation.

Human endothelial cells (EC) play a critical role in vascular homeostasis and their function is influenced by oxygen tension. This study investigates for the first time the effects of long-term adaptation (5 days) of two major EC types to physiological oxygen tension (5kPa) on basal and nitric oxide (NO)-modulated K+ channel activities. Whole-cell patch clamp experiments demonstrate that human umbilical vein EC (HUVEC) exhibit larger basal K+ outward and smaller inward currents under 5kPa O2 compared to standard hyperoxic (18kPa) culture conditions. Outward currents were potentiated by NO only under hyperoxia. Human cerebral microvascular EC (hCMEC/D3) showed larger outward currents under 5kPa O2 which were further potentiated by NO. Pharmacological isolation of different K+ currents using tetraethylammonium, TRAM-34 and apamin revealed differential effects in EC adapted to 5kPa or 18kPa O2. Under 5kPa O2, both cell types show greater contributions of TEA-sensitive currents and in addition hCMEC/D3 cells exhibit higher proportions of TRAM-34 and apamin-sensitive currents under 5kPa O2. In HUVEC, changes in half-activation voltage and hyperpolarized membrane potentials were detected only under hyperoxic conditions following NO exposure, with both cell types exhibiting altered current activation kinetics of outward and inward currents. Notably, expression of KCa3.1, KCa1.1, KCa2.3 and Kir6.1 channels was unaffected by O2, suggesting that changes in whole-cell currents in both EC types were due to channel modulation. Thus, our findings reveal that physiological O2 tension shapes the electrophysiological phenotype of human EC by modulating K+ channel function and NO responsiveness. The novel insights into the modulation of EC K+ channels by O2 has implications for the regulation of vascular tone and design and use of experimental models in vitro for high throughput drug discovery and clinical translation.

Advances in artificial intelligence (AI) show significant promise in multiscale modeling and biomedical informatics, particularly in the analysis of phonon microscopy (high-frequency ultrasound) data for cancer detection. This study addresses critical issues in data engineering for time-resolved phonon microscopy of biomedical samples by tackling the ‘batch effect,’ which arises from unavoidable technical variations between experiments, creating confounding variables that AI models may inadvertently learn. We present a multi-task conditional neural network framework that simultaneously achieves inter-batch calibration by removing confounding variables and accurate cell classification from time-resolved phonon-derived signals. We validate our approach by training and validating on different experimental batches, achieving a balanced precision of 89.22% and an average cross-validated precision of 89.07% for classifying background, healthy and cancerous regions. Furthermore, our model enables reconstruction of denoised images, which enable the physical interpretation of salient features indicative of disease states, such as sound velocity, sound attenuation, and cell adhesion to substrates. This work demonstrates the potential of AI methodologies in improving health outcomes and advancing cancer-informatics platforms.

Hypoxia is a feature of high grade serous ovarian cancer (HGSOC) microenvironment and contributes to platinum and PARP inhibitor resistance. Hypoxia induces activator protein-1 (AP-1) transcription factor (TF) activity leading to sustained proliferation, invasion, metastasis and angiogenesis. The role of AP-1 in DNA damage signaling and repair (DDR) and platinum/PARP resistance is unclear in HGSOC. PARP sensitive and resistant HGSOC cells (PEO1, PEO1R) were monitored for the activity of 48 different TFs using a luciferase-based reporter assay. AP-1 subunits including c-JUN, JUND, JUNB, cFOS and FOSL2 were profiled for protein expression under normoxia and hypoxia (1% O2). PEO1 and PEO1R tumor xenografts were immunohistochemically evaluated for JUNB, FOSL2, MRE11, CA-9 and CD-31 expression. CRISPR knock outs (KO) of JUNB and FOSL2 were generated and investigated for DNA repair gene expression (by DNA repair profiler PCR arrays), whole genome RNA sequencing, proliferation, invasion, cisplatin sensitivity (clonogenic and 3D-spheroids). Functional assays included DNA double strand break (DSB) accumulation, cell cycle progression, apoptosis, immunofluorescence, protein stability assay, co-immunoprecipitation and chromatin immunoprecipitation. The clinicopathological significance of FOSL2, JUNB and MRE11 expression was investigated in 331 clinical epithelial ovarian cancers. In platinum/PARP resistant PEO1R cells, upregulation of AP-1 transcription activity was evident compared to PEO1. Overexpression of JUNB and FOSL2 proteins was observed in normoxia and hypoxia (1% O2). PEO1 and PEO1R tumor xenografts showed high levels of JUNB and FOSL2 in tumor hypoxic areas. Compared to controls, JUNB and FOLS2_KO cells were less proliferative and have increased sensitivity to cisplatin/PARPi (olaparib), which was associated with increased DSBs, G2/M cell cycle arrest and increased apoptosis. DNA repair profiling revealed down regulation of several DNA repair genes in KO cells including MRE11, a key DDR factor. JUNB/FOSL2 proteins physically interacted with MRE11 and promoted its stability. RNA sequencing revealed enrichment of pathways such as platinum response, oxidative phosphorylation, translation and others in KO cells compared to control. In clinical cohorts, high FOSL2, high JUNB and high MRE11 expression was significantly associated with shorter progression-free survival (PFS) and worse overall survival (OS). Our data provides evidence that JUNB and FOSL2 may operate at the hypoxia-DDR interface in HGSOC. JUNB and FOSL2 not only have predictive and prognostic significance but could also be attractive anti-cancer targets including in platinum/PARP resistant HGSOC. Shatha Alqahtani, Rinad Mahmoud, Mashael Algethami, Asmaa Ibrahim, Ahmed Shoqafi, Jennie N. Jeyapalan, Sophie Kellaway, Nigel P. Mongan, Emad A. Rakha, Alan McIntyre, Srinivasan Madhusudan. Unravelling the role of AP-1 transcription factor in DNA damage signaling and response (DDR), platinum and PARP inhibitor resistance in ovarian cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2911.

Table S4: Well counts for single cell colonies two months after cisplatin treatment.

Figure S12: Survival of polyploid cells is reduced by the addition of NOTCH inhibitor during cisplatin treatment.

Figure S9: Transcription factors regulating downregulated genes in HCC1806 cells surviving 10 DPT as quantified using RNAseq and CHEA3 analysis.

Figure S4: Distribution fitting and cell population identification for cell line HCC1806.

Figure S8: Pathways downregulated in HCC1806 cells surviving 10 DPT, as quantified with RNAseq and Reactome analysis.

Figure S7: Drug-resilient 786-0 cells exhibit 1-2 whole genome duplications with high fidelity

Figure S11: Survival of polyploid cells is reduced by the addition of NOTCH inhibitor during cisplatin treatment.

Oxidation–reduction post-translational modifications (redox-PTMs) are chemical alterations to amino acids of proteins. Redox-PTMs participate in the regulation of protein conformation, localization and function, acting as signalling effectors that impact many essential biochemical processes in the cells. Crucially, the dysregulation of redox-PTMs of proteins has been implicated in the pathophysiology of numerous human diseases, including neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. This review aims to highlight the current gaps in knowledge in the field of redox-PTMs biology and to explore new methodological advances in proteomics and computational modelling that will pave the way for a better understanding of the role and therapeutic potential of redox-PTMs of proteins in neurodegenerative diseases. Here, we summarize the main types of redox-PTMs of proteins while providing examples of their occurrence in neurodegenerative diseases and an overview of the state-of-the-art methods used for their detection. We explore the potential of novel computational modelling approaches as essential tools to obtain insights into the precise role of redox-PTMs in regulating protein structure and function. We also discuss the complex crosstalk between various PTMs that occur in living cells. Finally, we argue that redox-PTMs of proteins could be used in the future as diagnosis and prognosis biomarkers for neurodegenerative diseases.

Figure S1: Dose response curves for cell lines after 72h cisplatin treatment

Drug-resilient cells exhibit one to two whole-genome duplications with high fidelity. A, Copy numbers in untreated and treated surviving HCC1806 cells 5 DPT, as visualized with AneuFinder (reads per 10 Mb over total amount of reads) from scWGS each row representing a single nucleus. B, Ratio of DNA content within each cell in untreated and surviving HCC1806 cells 5 DPT. The heat maps show the normalized read depth (reads per 10 Mb bins over total amount of reads in the cell) of scWGS, where blue areas show a lower number of reads, and red areas show a higher number of reads. The blocks R1, R2, and R3 in the left represent replicates 1, 2, and 3, respectively. C, Copy number of chromosome X in untreated (CTL), surviving HCC1806 cells at 5 DPT and their progeny, as visualized with chromosomal FISH of cells in interphase.

Table S1: LD50 values for cell lines after 72h cisplatin treatment.

Figure S13: Effects of T2445 treatment on cellular proliferation at 10nM.