Biology subjects

Mills, A.

Publications and source records attributed to Mills, A..

14 recordsLinked to original sources

bioRxiv2026

Functional divergence of WWC family proteins in human endothelial cells

The Hippo signaling pathway is increasingly recognized as a key regulator of endothelial cell (EC) proliferation, migration and vascular development. However, the roles of its upstream scaffold proteins remain poorly understood. Although WWC family proteins are widely regarded as functionally redundant activators of LATS1/2 kinases, the human genome contains a third family member, WWC3, that is absent from mice, raising the possibility of species-specific regulation of endothelial Hippo signaling. Here, we assessed the roles of WWC2 and WWC3 in human ECs using siRNA-mediated knockdown. Surprisingly, we found that WWC3 is the predominant regulator of canonical Hippo signaling, with a substantially greater effect than WWC2 on LATS1/2 phosphorylation, YAP/TAZ localization and expression of Hippo target genes. Loss of WWC3 also altered endothelial morphology and induced a partial endothelial-to-mesenchymal transition-like (EndoMT-like) phenotype. By contrast, WWC2 had a lesser effect on canonical Hippo signaling, but it was required for normal VEGF signaling dynamics. Despite these distinct molecular functions, depletion of either WWC2 or WWC3 impaired EC proliferation, migration, and cord formation in vitro. Together, our findings demonstrate that WWC family proteins perform overlapping but distinct functions in human ECs, with WWC3 acting as the predominant canonical Hippo regulator, whereas WWC2 more efficiently modulates VEGF signaling. These results reveal unexpected functional specialization among WWC proteins and suggest that regulation of Hippo signaling in human ECs differs from that inferred from mouse studies.

cell biology↗

bioRxiv2026

OpenAntigens: a structure-aware database for antigen construct design across the human cell-surface and secreted proteome

Choosing which region of a protein to express remains poorly standardized in antibody discovery, recombinant reagent generation, structural biology and computational binder design. For human cell-surface and secreted proteins, this requires reconciling topology, processing, predicted and experimental structure, modifications, interaction partners, orthologs, paralogs and cross-reactivity risk before ordering DNA. OpenAntigens is a free, no-login database of construct-design reports for 5328 human secreted, GPI-anchored, single-pass and multipass proteins. It integrates UniProt topology, AlphaFold pLDDT and PAE, PDB precedent, InterPro and Pfam domains, mouse and cynomolgus orthologs, paralog and family context, Open Targets disease associations, partner and assembly context, and BLAST searches. It provides 55 305 construct suggestions spanning full design regions, PDB-backed boundaries, annotated domains, pLDDT/PAE-derived regions and membrane-expression options, plus 148 722 sequence-similarity hits to help choose constructs and assess cross-reactivity. For targets with compatible AlphaFold models, the interactive designer links sequence, structure, pLDDT and PAE, allowing users to revise boundaries and export species-equivalent sequences with real-time cysteine and modification warnings. OpenAntigens places reproducible construct suggestions, comparative context and browser editing in one workflow, reducing manual reconciliation across resources. OpenAntigens is available at openantigens.org.

bioinformatics↗

bioRxiv2026

Transient architecture of the embryonic pancreas determines endocrine mass

During organogenesis, epithelial tissues undergo extensive three-dimensional (3D) remodeling while simultaneously generating specialized cell types. Whether these transient architectural states actively instruct lineage allocation remains unclear. Here we identify a morphogenetic stage in which resolution of epithelial stratification is required for lineage allocation and establishment of endocrine cell mass. We show that loss of the Hippo pathway regulator Merlin disrupts lumen morphogenesis and prevents formation of the transient 3D epithelial architecture that characterizes normal pancreas development. Failure to establish this architectural state alters lineage allocation, impairing acinar differentiation, markedly reducing adult endocrine cell mass, and disrupting glucose homeostasis. Mosaic analyses reveal that these lineage defects arise non-cell autonomously, demonstrating that epithelial architecture itself instructs cell fate decisions. Mechanistically, Merlin coordinates PI3K-regulated polarized membrane trafficking required for apical membrane biogenesis and lumen formation. Together, these findings identify Merlin-dependent membrane trafficking as a mechanism coupling epithelial morphogenesis to lineage allocation and demonstrate that transient developmental architectures can determine the cellular composition of mature organs.

developmental biology↗

bioRxiv2026

ARID5B mutations cause a neurodevelopmental syndrome with neuroinflammation episodes

Genetic disorders affecting the epigenetic machinery constitute a major group of neurodevelopmental conditions. Pathogenic variants in several ARID transcription factors--particularly ARID1A, ARID1B, and ARID2--cause Coffin-Siris syndromes, all characterized by intellectual disability (ID). These genes encode core subunits of the BRG1/BRM-associated factor (BAF) chromatin remodeling complex. In contrast, ARID family members that function in other regulatory complexes have remained largely unexplored in neurodevelopmental disease. Here, we identify 29 individuals carrying heterozygous ARID5B variants, of which 24 (83%) introduce premature termination codons in the exceptionally long final exon, one affects the exon 9 splice donor site, and four are missense variants in conserved domains within the N-terminal half of the protein. Using a CRISPR-Cas9 knock-in mouse model harboring the p.Q522Ter variant, together with in vitro assays, we investigated the functional consequences of C-terminal ARID5B truncations. All affected individuals presented with global developmental delay or ID--most commonly mild--and frequent speech and language impairment. Recurrent features included kidney malformations, behavioral difficulties, and recurrent infections of the respiratory and urinary tracts. Two individuals experienced central nervous system inflammation, and two infants presented with persistent pulmonary hypertension. Remarkably, 19 of 29 variants (66%) cluster within the first quarter of exon 10, are de novo, and escape nonsense-mediated mRNA decay (NMD), which we confirmed for two variants affecting seven individuals. Variants outside this region were inherited. Heterozygous mice exhibited developmental and behavioral abnormalities, while homozygous mutations was perinatally lethal. Truncations and a small deletion within a predicted nuclear localization signal (NLS) caused cytosolic mislocalization of ARID5B, whereas the isolated C-terminal half retained nuclear localization, suggesting an independent distal NLS. Collectively, these findings define ARID5B-related neurodevelopmental disorder as a distinct clinical entity and reveal how disruption of specific ARID5B domains impacts protein localization, mammalian development, immune and neurobehavioral function. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/698931v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@158e8d9org.highwire.dtl.DTLVardef@1f014dorg.highwire.dtl.DTLVardef@18e6e7forg.highwire.dtl.DTLVardef@1f886e9_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2025

Quadruple Parametric Optical Mapping to Measure Cardiac Metabolism-Excitation-Contraction Coupling

Cardiac excitation contraction coupling has been studied by recording transmembrane potential (Vm) and intracellular calcium (Ca2+), simultaneously. Recently, imaging metabolic substrates such as NADH in addition to Vm and Ca2+ has been reported to improve our understanding of cardiac physiology and the interaction between these different aspects of heart function, known as metabolism-excitation-contraction-coupling (MECC). In this study, we developed and validated the quadruple parametric optical mapping technique to simultaneously measure Vm, Ca2+ and two metabolic substrates, NADH and FAD. Langendorff perfused murine hearts of both sexes were subjected to either pharmacological interventions: 0.1 {micro}M nifedipine or 0.1 {micro}M flecainide, or pathological intervention: ischemia/reperfusion and the four facets of cardiac function were measured. Hearts were stained with RH237 and Rhod2-AM dyes to measure Vm and Ca2+, respectively. Two LED light sources (520 and 365 nm) were used to excite the dyes and induce NADH/FAD autofluorescence, respectively, and all four optical signals were simultaneously recorded and analyzed to measure12 parameters of cardiac function. Nifedipine significantly altered Ca2+ related parameters, including increased Ca2+ rise-time and Ca2+ decay, and prolonged Vm-Ca2+ delay. Flecainide significantly affected all three aspects of MECC with increased Vm rise-time and Ca2+ decay, while decreasing conduction velocity (CV) and the redox ratio. Ischemia also altered MECC with increased Vm rise-time and NADH intensity, decreased CV and redox ratio. Using quadruple parametric optical mapping, we demonstrate known cardiac drug response to nifedipine and flecainide and report novel aspects of cardiac function that are modulated by these drugs. These novel drug effects would not have been identified without such a multi-parametric approach to assessing cardiac physiology. Further, we were able to determine chronological changes in MECC during ischemia and reperfusion to better understand the interplay between electrical, mechanical, and metabolic function in the heart.

bioengineering↗

bioRxiv2025

Nicotine Influence on Vascular and Neurocognitive Function with In-utero Electronic Cigarette Exposure

Emerging studies find arteriolar dysfunction in offspring with in-utero electronic cigarette (Ecig) exposure, but the long-term effect on offsprings cerebrovascular vascular and neurocognitive health is poorly understood. Ecigs provides a unique opportunity to directly evaluate the contributions of inhaled nicotine from the vehicle e-liquid - which was not possible with traditional cigarettes. Moreover, many Ecigs have variable power settings, which can alter the toxicity of the aerosol cloud produced. We hypothesize maternal vaping at different wattages will have variable effects on cerebrovascular function in the offspring, and that these effects would be independent of nicotine. We used time-mated female Sprague-Dawley rats with Ecig exposure from gestation day (GD)2-21. We studied male and female offspring for vascular and neurocognitive function at 1-, 3-, 6- and 12-months of age. We found that, both sexes, offspring with in-utero exposure (at 5w and 30w Ecig conditions) exhibited impaired middle cerebral artery (MCA) reactivity. While the magnitude of impairment was greater at higher that lower watts, Ecig at 5-watts still exhibited significant impairments in MCA function (suggesting the harm threshold for blood vessels is very low). Vascular dysfunction was evident with or without nicotine in the e-liquid, but nicotine exposure resulted in short-term memory deficits, evidence of neuronal damage, and increased astrocyte interaction with endothelial cells in 6- and 12-month-old offspring. We also observed altered expression of clock genes and antioxidant signaling pathways, along with a decrease in sirtuin-1 expression, decreased ratio of beta-amyloid A 42/40 protein expression, and increased in NOX1, which are consistent with redox imbalance, neuroinflammation, and advancing cellular senescence. These preclinical data provide evidence suggesting that in utero exposure to Ecigs from maternal vaping can be expected to adversely affect the brain health of offspring in their adult life and that neurocognitive outcomes are worsened with exposure to nicotine. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=174 SRC="FIGDIR/small/638202v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1e7a7a1org.highwire.dtl.DTLVardef@1ae5bceorg.highwire.dtl.DTLVardef@d323eorg.highwire.dtl.DTLVardef@5db4af_HPS_FORMAT_FIGEXP M_FIG C_FIG Created in BioRender

developmental biology↗

bioRxiv2024

Lats1/2 are essential for developmental vascular remodeling and biomechanical adaptation to shear stress

Blood vessels in mammalian embryos develop from initial aggregates of endothelial cell (EC) progenitors, which coordinate the opening and stabilization of central vascular lumens, all while under progressively increasing flow and pressure from blood circulation. Mechanical cues exerted by shear stress from the blood flow remodel an initial vascular plexus into a ramifying array of large and small vessels. As plasma starts to fill vascular lumens, these forces trigger changes in EC gene expression and dynamic alterations in cell shape and cell adhesion, as cuboidal angioblasts elongate and flatten into ECs. Little is known about how embryonic ECs sense and transduce hemodynamic signals as vessels form in vivo. Here, we report a critical requirement for the Lats1 and Lats2 Hippo pathway kinases during this process. We show that when Lats1/2 are genetically deleted in ECs, embryos develop severe defects in blood vessel formation, which lead to embryonic lethality by E11.5. We find that initial vessel patterning and circulation initiate properly, however remodeling of the initial vascular plexus fails due to lumen collapse and altered blood flow. When Lats1/2 are knocked down using siRNA approaches in cultured ECs, cells fail to elongate and polarize, similar to ECs in the mutant embryos. In addition, VE-cadherin (VEcad) based junctions fail to mature under shear stress. These data show that Lats1/2 deficient cells no longer respond to laminar shear stress, both in vivo and in vitro. This work identifies the Hippo pathway kinases Lats1 and Lats2 as critical transducers of biomechanical cues during the early steps of blood vessel remodeling. This study will provide new targets for treatment of vascular diseases and new directions for efforts to generate vascularized tissues for replacement therapies. HighlightsO_LILats1 and Lats2 mRNA and protein are expressed in murine embryonic endothelial cells (ECs). C_LIO_LIDeletion of Lats1/2 in embryonic endothelium results in severe vascular defects and embryonic lethality. C_LIO_LILoss of Lats1/2 leads to failure of both vascular remodeling and EC elongation upon exposure to flow, in vivo and in vitro. C_LIO_LILats1/2 are required for cell-cell VE-cadherin adhesion maturation under flow. C_LIO_LILoss of Lats1/2 results in cytoskeletal disorganization in response to shear stress. C_LI

developmental biology↗

bioRxiv2024

MAPK14/p38α Shapes the Molecular Landscape of Endometrial Cancer and promotes Tumorigenic Characteristics

The molecular underpinnings of High Grade Endometrial Carcinoma (HGEC) metastatic growth and survival are poorly understood. Here we show that ascites-derived and primary tumor HGEC cell lines in 3D spheroid culture faithfully recapitulate key features of malignant peritoneal effusion and exhibit fundamentally distinct transcriptomic, proteomic and metabolomic landscapes when compared with conventional 2D monolayers. Using genetic screening platform we identify MAPK14 (which encodes the protein kinase p38) as a specific requirement for HGEC in spheroid culture. MAPK14/p38 has broad roles in programing the phosphoproteome, transcriptome and metabolome of HGEC spheroids, yet has negligible impact on monolayer cultures. MAPK14 promotes tumorigenicity in vivo and is specifically required to sustain a sub-population of spheroid cells that is enriched in cancer stemness markers. Therefore, spheroid growth of HGEC activates unique biological programs, including p38 signaling, that cannot be captured using 2D culture models and are highly relevant to malignant disease pathology.

cancer biology↗

bioRxiv2023

An AAV capsid reprogrammed to bind human Transferrin Receptor mediates brain-wide gene delivery

Developing vehicles that efficiently deliver genes throughout the human central nervous system (CNS) will broaden the range of treatable genetic diseases. We engineered an AAV capsid, BI-hTFR1, that binds human Transferrin Receptor (TfR1), a protein expressed on the blood-brain barrier (BBB). BI-hTFR1 was actively transported across a human brain endothelial cell layer and, relative to AAV9, provided 40-50 times greater reporter expression in the CNS of human TFRC knock-in mice. The enhanced tropism was CNS-specific and absent in wild type mice. When used to deliver GBA1, mutations of which cause Gaucher disease and are linked to Parkinsons disease, BI-hTFR1 substantially increased brain and cerebrospinal fluid glucocerebrosidase activity compared to AAV9. These findings establish BI-hTFR1 as a promising vector for human CNS gene therapy.

neuroscience↗

bioRxiv2023

Genomic profiling and pre-clinical modelling of breast cancer leptomeningeal metastasis (BCLM) reveals acquisition of a lobular-like phenotype

Breast cancer leptomeningeal metastasis (BCLM), where tumour cells grow along the lining of the brain and spinal cord, is a devastating development for patients. Investigating this metastatic site is hampered by difficulty in accessing tumour material. Here, we utilise cerebrospinal fluid (CSF) cell-free DNA (cfDNA) and CSF disseminated tumour cells (DTCs) to explore the clonal evolution of BCLM and heterogeneity between leptomeningeal and extracranial metastatic sites. Somatic alterations with potential therapeutic actionability were detected in 81% (17/21) of BCLM cases, with 19% detectable in CSF cfDNA only. BCLM was enriched in genomic aberrations in adherens junction and cytoskeletal genes, revealing a lobular-like breast cancer phenotype. CSF DTCs were cultured in 3D to establish BCLM patient-derived organoids, and used for the successful generation of BCLM in vivo models. These data reveal that BCLM possess a unique genomic aberration profile and highlights potential cellular dependencies in this hard-to-treat form of metastatic disease.

cancer biology↗

bioRxiv2021

The structure and flexibility analysis of the Arabidopsis Synaptotagmin 1 reveal the basis of its regulation at membrane contact sites

Cell function requires the maintenance of membrane lipid homeostasis as changes in cellular environment unbalance this equilibrium. The non-vesicular lipid transfer at endoplasmic reticulum (ER) and plasma membrane (PM) contact sites (CS) is central to restore it. Extended synaptotagmins (E-Syts) are ER proteins that play a central role in this process as they act as molecular tethers with PM and as lipid transfer proteins between these organelles. E-Syts are constitutively anchored to the ER through an N-terminal hydrophobic segment and bind to the PM via C-terminal C2 domains. In plants, synaptotagmins (SYTs) are orthologous of E-Syts and regulate the ER-PM communication by the activity of their two C2 domains in response to abiotic stresses. We have combined macromolecular crystallography, small-angle X-ray scattering, structural bioinformatics and biochemical data to analyze the regulation of plant synaptotagmin 1 (SYT1). Our data show that the binding of SYT1 to the PM is regulated by the interaction of the first C2 domain through a Ca2+-dependent lipid binding site and by a site for phosphorylated forms of phosphatidylinositol in such a way that two different molecular signals are integrated in response to stress. In addition, our data show that SYT1 is highly flexible by virtue of up to three hinge points, including one that connects the two C2 domains. This feature provides conformational freedom to SYT1 to define a large and complementary interaction surface with the PM. This structural plasticity, in turn, may facilitate lipid extraction, protein loading and subsequent transfer between PM and ER. Data DepositionThe atomic coordinates and structure factors have been deposited in the Protein Data Bank, https://www.pdb.org/ [PDB ID codes Ca2+ and Cd2+ complexes of SYT1C2A (7AS6 and 7ATP, respectively)]. The final SAXS models were deposited and are available at SASBDB https://www.sasbdb.org/ [ID codes SASDKG6 for the SMP2C2A construct SASDKJ9 for the C2AB construct and SASDKK9 in presence of Ca2+]

plant biology↗

bioRxiv2020

Synthesis of diagnostic quality cancer pathology images

Deep learning-based computer vision methods have recently made remarkable breakthroughs in the analysis and classification of cancer pathology images. However, there has been relatively little investigation of the utility of deep neural networks to synthesize medical images. In this study, we evaluated the efficacy of generative adversarial networks (GANs) to synthesize high resolution pathology images of ten histological types of cancer, including five cancer types from The Cancer Genome Atlas (TCGA) and the five major histological subtypes of ovarian carcinoma. The quality of these images was assessed using a comprehensive survey of board-certified pathologists (n = 9) and pathology trainees (n = 6). Our results show that the real and synthetic images are classified by histotype with comparable accuracies, and the synthetic images are visually indistinguishable from real images. Furthermore, we trained deep convolutional neural networks (CNNs) to diagnose the different cancer types and determined that the synthetic images perform as well as additional real images when used to supplement a small training set. These findings have important applications in proficiency testing of medical practitioners and quality assurance in clinical laboratories. Furthermore, training of computer-aided diagnostic systems can benefit from synthetic images where labeled datasets are limited (e.g., rare cancers). We have created a publicly available website where clinicians and researchers can attempt questions from the image survey at http://gan.aimlab.ca/.

bioinformatics↗

bioRxiv2019

Platinum chemotherapy induces lymphangiogenesis to prime tissues for tumor metastasis

Chemotherapy has been used to inhibit cancer growth for decades, but emerging evidence shows it can affect the tumor stroma unintentionally promoting cancer malignancy. After treatment of primary tumors, remaining drugs drain via lymphatics. Though all drugs interact with the lymphatics, we know little of their impact on them. Here, we show a previously unknown effect of platinums, a widely used class of chemotherapeutics, to directly induce systemic lymphangiogenesis and activation. These changes are dose-dependent, long-lasting, and occur in healthy and cancerous tissue in multiple mouse models of breast cancer. We saw similar effects in human ovarian and breast cancer patients whose treatment regimens included platinums. Carboplatin treatment of healthy mice prior to mammary tumor inoculation increases cancer metastasis as compared to no pre-treatment. These platinum-induced phenomena could be blocked by VEGFR3 inhibition. These findings have implications for cancer patients receiving platinums and may support the inclusion of anti-VEGFR3 therapy into treatment regimens or differential design of treatment regimens to alter these potential effects. SummaryPlatinum chemotherapy induces VEGFR3-dependent lymphangiogenesis, priming tissues for metastasis of breast cancer. Inhibition of VEGFR3 via antibody blockade can reverse these effects.

cancer biology↗

bioRxiv2018

Wolf-Hirschhorn Syndrome-associated genes are enriched in motile neural crest and affect craniofacial development in Xenopus laevis

Wolf-Hirschhorn Syndrome (WHS) is a human developmental disorder arising from a hemizygous perturbation, typically a microdeletion, on the short arm of chromosome four. In addition to pronounced intellectual disability, seizures, and delayed growth, WHS presents with a characteristic facial dysmorphism and varying prevalence of microcephaly, micrognathia, cartilage malformation in the ear and nose, and facial asymmetries. These affected craniofacial tissues all derive from a shared embryonic precursor, the cranial neural crest, inviting the hypothesis that one or more WHS-affected genes may be critical regulators of neural crest development or migration. To explore this, we characterized expression of multiple genes within or immediately proximal to defined WHS critical regions, across the span of craniofacial development in the vertebrate model system Xenopus laevis. This subset of genes, WHSC1, WHSC2, LETM1, and TACC3, are diverse in their currently-elucidated cellular functions; yet we find that their expression demonstrates shared tissue-specific enrichment within the anterior neural tube, pharyngeal arches, and later craniofacial structures. We examine the ramifications of this by characterizing craniofacial development and neural crest migration following individual gene depletion. We observe that several WHS-associated genes significantly impact facial patterning, cartilage formation, pharyngeal arch migration, and neural crest motility, and can separately contribute to forebrain scaling. Thus, we have determined that numerous genes within and surrounding the defined WHS critical regions potently impact craniofacial patterning, suggesting their role in WHS presentation may stem from essential functions during neural crest-derived tissue formation.\n\nAuthor SummaryWolf-Hirschhorn Syndrome (WHS), a developmental disorder caused by small deletions on chromosome four, manifests with pronounced and characteristic facial malformation. While genetic profiling and case studies provide insights into how broader regions of the genome affect the syndromes severity, we lack a key component of understanding its pathology; a basic knowledge of how individual WHS-affected genes function during development. Importantly, many tissues affected by WHS derive from shared embryonic origin, the cranial neural crest. This led us to hypothesize that genes deleted in WHS may hold especially critical roles in this tissue. To this end, we investigated the roles of four WHS-associated genes during neural crest cell migration and facial patterning. We show that during normal development, expression of these genes is enriched in migratory neural crest and craniofacial structures. Subsequently, we examine their functional roles during facial patterning, cartilage formation, and forebrain development, and find that their depletion recapitulates features of WHS craniofacial malformation. Additionally, two of these genes directly affect neural crest cell migration rate. We report that depletion of WHS-associated genes is a potent effector of neural crest-derived tissues, and suggest that this explains why WHS clinical presentation shares so many characteristics with classic neurochristopathies.

developmental biology↗