Biology subjects

Wong, D.

Publications and source records attributed to Wong, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Once in a blue moon: an unexpected evolutionary route to blue floral pigmentation in orchids

Blue flowers are rare in the hyperdiverse orchid family, and the genetic mechanisms enabling their blue delphinidin-based pigmentation remain poorly understood. Within the Australian orchid subtribe Caladeniinae (~350 spp.), repeated transitions from maroon to blue flowers, followed by reversions to maroon, provide a unique opportunity to investigate blue flower evolution. Using phylotranscriptomics, anthocyanin profiling, molecular evolutionary analyses, and gene expression data from 51 species, we reconstructed the genetic changes associated with these transitions. Despite its near-universal role in delphinidin biosynthesis across flowering plants, unexpectedly, we found no evidence for a functional CYP75A-mediated flavonoid 3'5'-hydroxylase (F3'5'H) pathway. Instead, blue-flowered species possess a duplicated CYP75B lineage gene (CYP75B-D) with apparent 5'-hydroxylase activity, a derived D-type dihydroflavonol reductase (DFR), and a distinct anthocyanin biosynthetic pathway co-expression pattern. Phylogenetic analyses suggest that the D-type DFR pre-dated blue flower evolution, representing a permissive evolutionary shift later coupled with the evolution of CYP75B-D. Notably, blue flower evolution coincided with repeated pollinator transitions and major palaeoenvironmental change during the Middle Miocene. Together, these findings reveal an unexpected route to blue floral pigmentation and suggest that its evolution arose through the interaction of molecular innovation, regulatory evolution, and ecological opportunity.

evolutionary biology↗

bioRxiv2026

An early-diverging Caladeniinae orchid reveals diversification of terpene and apocarotenoid pathways underlying floral scent evolution

Orchids exhibit extraordinary floral diversity, yet the molecular mechanisms underlying floral scent evolution remain poorly understood. Guided by recent phylogenomic reconstruction of the Australian orchid subtribe Caladeniinae, we used Glossodia major, an earlier-diverging food-deceptive lineage, as an evolutionary anchor for investigating floral volatile diversification. Scent-producing petals showed coordinated expression of terpene synthase (TPS), carotenoid and carotenoid cleavage dioxygenase (CCD) pathways. Functional characterisation identified GmTPS-b3, GmTPS-b4 and GmTPS-a1 as contributors to floral monoterpene and sesquiterpene production, while GmCCD1 produced geranylacetone, the dominant floral volatile, from {zeta}-carotene. GmCCD4 and GmCCD7a showed additional, partially overlapping apocarotenoid-forming activities. Integration of tissue-specific expression, enzyme activity and subcellular localisation linked these pathways to the emitted floral bouquet. Functional comparisons with characterised Caladenia homologues revealed biochemical conservation and catalytic and compartmental specialisation, while expression profiling across 31 species identified pollination strategy-associated regulatory divergence in TPSa1 and TPSb3, consistent with corresponding shifts in terpenoid-rich floral scent. We propose that differential modification and deployment of conserved biosynthetic pathways contributed to the attenuation and re-emergence of terpenoid-rich floral scent during Caladeniinae diversification. More broadly, our findings provide candidate mechanistic links between molecular evolution, pollinator-mediated selection and floral diversification.

plant biology↗

bioRxiv2026

Single-Cell Profiling of Dynamic Epicardial Cell States During Myocardial Infarction

Background: The epicardium is reactivated after myocardial infarction (MI); however, the gene expression profiles of post-MI adult epicardial subpopulations remain incompletely defined. Methods: Single-cell RNA sequencing was performed on lineage-traced Wt1+ epicardial cells from Wt1CreERT2/+; R26tdT/+; PdgfranGFP/+ adult mice after sham surgery or at 7 and 14 days after permanent artery ligation to induce MI. Immunostaining was performed on Wt1-lineage-traced cardiac tissue to validate spatial expression after ischemic injury. Results: Unbiased clustering identified nine transcriptionally distinct epicardial populations, encompassing mesothelial, fibroblast/mesenchymal, transitional, and proliferative phenotypes. Fibroblast-like epicardial cells (Wt1+/Pdgfra+) showed time-dependent expression profiles associated with upregulation of epithelial-to-mesenchymal transition (EMT) and extracellular matrix (ECM) gene programs. At 7 days post-MI, there was notable enrichment of genes related to chemokines and Wnt components. By 14 days post-MI, the expression profile shifted toward immune regulation. In contrast, a Wt1high/Msln+ population showed minimal upregulation of EMT gene programs but enhanced paracrine signaling related to wound healing and semaphorins, suggesting reactivation of reparative and angiogenic functions akin to those of the epicardium during embryonic development. Immunostaining and in situ hybridization fluorescence analyses validated laminar epicardial cell placement after MI, comprising a surface Msln+ sheet, an overlapping Wt1-lineage band, and a subadjacent PDGFR+ and Periostin+ compartment that expands 7-14 days after MI and regresses by day 28 post-ischemia. Conclusions: Our data define epicardial gene programs in which a signaling epithelial cell surface overlays an effector mesenchymal cell stroma to coordinate angiogenesis, leukocyte recruitment, and ECM remodeling. This study presents the first integrated single-cell atlas of epicardial-derived cells across multiple post-ischemic timepoints, offering new insights into their reparative potential and dynamic signaling diversity in the injured adult heart.

molecular biology↗

bioRxiv2026

Inter-haplotype inversions and repeat expansion in the sexually deceptive orchid Chiloglottis trapeziformis

Chiloglottis trapeziformis is a sexually deceptive Australian orchid that provides a valuable system for studying orchid genome evolution, structural variation, and the molecular basis of specialized pollination. However, high-quality nuclear genome resources remain scarce for most orchids, particularly Australias diverse terrestrial lineages. To address this gap, we integrated PacBio HiFi, Oxford Nanopore ultra-long reads, and Hi-C chromatin-contact data to generate the first chromosome-scale, haplotype-resolved nuclear genome assembly for an Australian terrestrial orchid, Chiloglottis trapeziformis. Hi-C guided scaffolding resolved two haplotypes into 20 chromosomes each, consistent with the reported karyotype and genome size (2n=40, haplotype sizes of 1.58 Gb and 1.91 Gb). Genome completeness was high for both haplotypes, recovering 95.1% and 95.5% complete BUSCO genes for haplotype 1 and haplotype 2, respectively. De novo repeat annotation revealed a repeat-rich genome (85.79- 88.25% repetitive sequence), dominated by LTR retrotransposons. Evidence-guided annotation identified 16,287 and 16,548 protein-coding genes in Haplotype 1 and Haplotype 2, respectively. Phylogenetically informed comparisons placed C. trapeziformis as sister to Anoectochilus roxburghii among sampled Orchidoideae and showed broad gene-order conservation. Comparing the two haplotypes for structural variation, we identified large inter-haplotype inversions containing functionally annotated genes with detectable RNA expression, with focal examples further supported by local Hi-C contact patterns and breakpoint-level inspection. Inversion-overlapping genes did not show elevated dS relative to collinear background. This assembly and annotation resource provides a foundation for population and conservation genomics, structural and comparative analyses, and genome-enabled hypothesis testing of molecular traits underlying sexual deception in orchids. Significance statementSexually deceptive orchids use remarkable chemical, visual, and tactile mimicry to attract specific pollinators, but the genome resources needed to understand how these complex traits evolved remain scarce. By generating a chromosome-scale, haplotype-resolved genome for Chiloglottis trapeziformis, we provide the first genomic framework for the large and unique Australasian tribe Diurideae. We show extensive structural variation between haplotypes in an otherwise highly collinear genome, with several large inversions potentially impacting expressed genes. The absence of elevated coding divergence in these regions highlights structural variation as a potentially underappreciated driver of orchid genome evolution. This resource fills a major gap for Australian orchids and provides a foundation for linking genome structure with orchid diversification, conservation, and the evolution of sexual deception.

genomics↗

bioRxiv2025

Whole-Proteome Screening and Multi-Modal Profiling of Antigen-Specific CD4+ T Cells at Single-Cell Resolution

Systematic whole-proteome screening and comprehensive profiling of antigen-specific CD4+ T cells are crucial for advancing our understanding of CD4+ T cell immunity, yet such efforts remain technically challenging. Here, we present a high-throughput platform that employs large-scale class II single-chain trimer libraries to detect antigen-specific CD4+ T cells, while simultaneously profiling their antigen specificity, TCR/{beta} sequences, MHC restriction, whole transcriptomes, and patient/timepoint origins at single-cell resolution. We benchmarked SCTs against conventional pMHCs and validated the SCT library-based approach in direct ex vivo identification of antigen-specific CD4+ T cells in healthy donors. We then applied the platform to screen the entire SARS-CoV-2 receptor-binding domain in a longitudinal patient cohort, identifying 2,188 antigen-specific CD4+ T cells and revealing key features that define antigen immunogenicity. Extending to cancer, we performed whole-proteome screening of HPV-16 E6/E7 for TCR repertoire profiling in a precancerous cohort, uncovering functional heterogeneity of HPV-specific TCRs. By integrating high-throughput antigen screening with high-dimensional, multi-modal cellular characterization, our approach offers an unprecedented window into CD4+ T cell immunity across diverse disease contexts and empowers the development of new therapies.

bioengineering↗

bioRxiv2025

Integrated Techniques for Extracellular Particle Separation and Single-Particle Multiparametric Characterization to Track Cancer Biomarkers from Tissue to Biofluids

Gastric cancer (GC) remains a formidable global health challenge, with late-stage diagnosis and high recurrence rates resulting in poor patient outcomes. This study explores the potential of advanced technologies, namely Bessel Beam Excitation Separation Technology (BEST) and multiparametric biochip assay (MBA), to track single extracellular vesicle and particle (EVP) cargo from organs of pathology into biofluids such as plasma and saliva. Using GC as a study model, we conducted high throughput, multiparametric analyses of EVPs derived from plasma, saliva, and tissue samples. Our findings demonstrate the feasibility of these techniques in isolating and characterizing EVPs, revealing consistent EVP morphology and size across biofluids. Furthermore, differential expression patterns of the developed and validated salivary GC biomarkers, miR-140-5p and miR-301a-3p, were observed in GC patient biofluids, supporting the diagnostic relevance of EVP cargo. Notably, saliva emerged as the most promising biofluid for GC diagnosis, achieving superior Receiver Operating Characteristic (ROC) curve values compared to plasma and tissue. This study highlights the role of BEST and MBA in advancing single-EVP analysis and elucidating EVP trafficking, paving the way for future diagnostic applications of EVP cargo.

cancer biology↗

bioRxiv2024

Microenvironment T-Type calcium channels regulate neuronal and glial processes to promote glioblastoma growth

BackgroundGlioblastoma (GBM) is the most common primary malignant brain tumor. The aim of this study was to elucidate the role of microenvironment and intrinsic T-type calcium channels (Cav3) in regulating tumor growth and progression. MethodsWe grafted syngeneic GBM cells into Cav3.2 knockout mice to assess the role of microenvironment T-Type calcium channels on GBM tumor growth. We performed single-cell RNA-seq (scRNA-seq) of tumors from WT and Cav3.2 KO mice to elucidate the regulation of tumors by the microenvironment. We used neurons from WT and Cav3.2 KO mice in co-culture with GBM stem cells (GSC) to assess the effects of Cav3.2 on neuron/GSC synaptic connections and tumor cell growth. ResultsCav3.2 KO in the microenvironment led to significant reduction of GBM growth and prolongation of animal survival. scRNA-seq showed that microenvironment Cav3.2 regulates neuronal and glial biological processes. Microenvironment Cav3.2 downregulated numerous genes associated with regulating the OPC cell state in GBM tumors such as SOX10 and Olig2. Neuronal Cav3.2 promoted neuron/GSC synaptic connections and GSC growth. Treatment of GSCs with the Cav3 blocker mibefradil downregulated genes associated with neuronal processes. The Cav3 blocker drug mibefradil synergized with temozolomide (TMZ) and radiation to reduce in vivo tumor growth and prolong animal survival. ConclusionsTogether these data reveal a role for microenvironment Cav3 in promoting GBM tumor progression through regulating neuronal and glial processes particularly associated with the OPC-cell state. Targeting both intrinsic and microenvironment Cav3 with the inhibitor mibefradil significantly enhanced the anti-GBM effects of TMZ and radiation. Key PointsO_LIMicroenvironment Cav3.2 promotes GBM progression C_LIO_LIMicroenvironment Cav3.2 promotes neuronal and glial processes C_LIO_LIPharmacological targeting of intrinsic and microenvironment Cav3 synergizes with TMZ/radiation C_LI Importance of the StudyIn this study, we demonstrate for the first time that microenvironment Cav3.2 contributes to GBM progression and growth by regulating neuronal and glial processes. Our findings highlight the importance of T-type calcium channels in the microenvironment as well as the tumor and provides preclinically relevant data for the use of mibefradil to inhibit GBM growth in combination with standard of care therapies.

cancer biology↗

bioRxiv2024

Utility of Cellular Measurements of Non-Specific Endocytosis to Assess the Target-Independent Clearance of Monoclonal Antibodies

Past studies have demonstrated higher clearance for monoclonal antibodies possessing increased rates of non-specific endocytosis. However, this metric is oftentimes evaluated indirectly using biophysical techniques or cell surface binding studies that may not provide insight into the specific rates of cellular turnover. Furthermore, few examples evaluating non-specific endocytosis have been reported for a therapeutic antibody that reached clinical assessment. In the current report, we evaluated a therapeutic human immunoglobulin G2 monoclonal antibody targeted against the interleukin-4 receptor alpha chain (IL-4R) that exhibited elevated target independent clearance in previous Phase 1 and 2 studies. We confirmed high non-specific clearance of the anti-IL-4R antibody as compared to a reference antibody during pharmacokinetic assessments in wild type mice where target-mediated disposition was absent. We then developed a cell-based method capable of measuring cellular protein endocytosis and demonstrated the anti-IL-4R antibody exhibited marked non-specific uptake relative to the reference compound. Antibody homology modeling identified the anti-IL-4R antibody possessed positive charge patches whose removal via targeted mutations substantially reduced its non-specific endocytosis. We then expanded the scope of the study by evaluating a panel of consisting of both preclinical and clinical monoclonal antibodies and demonstrate those with the highest rates of non-specific uptake in vitro exhibit elevated target independent clearance, low subcutaneous bioavailability, or both. Our results support the observation that high non-specific endocytosis is a negative attribute in monoclonal antibody development and demonstrate the utility of a generic cell-based screen as a quantitative tool to measure non-specific endocytosis of protein therapeutics at the single-cell level. Highlights- Developed a novel, reproducible cellular assay to directly quantify non-specific endocytosis of therapeutic proteins. - A previous clinical candidate monoclonal antibody with rapid target-independent clearance in mice and humans possessed extensive non-specific endocytosis that was due to exposed positive charge features. - Demonstration of distinct rates of endocytosis into mammalian cells for disparate monoclonal antibodies, even those with common specificity for targets or isoelectric points. - Cell-based assay to quantify the potential impact of non-specific endocytosis on target-independent clearance and/or subcutaneous bioavailability of monoclonal antibodies.

biochemistry↗

bioRxiv2023

Long non-coding RNA TUG1 is down-regulated in Friedreich's ataxia.

Friedreichs Ataxia (FRDA) is a neurodegenerative disorder caused by reduced frataxin (FXN) levels. It leads to motor and sensory impairments and has a median life expectancy of around 35 years. As the most common inherited form of ataxia with no cure, FRDA lacks reliable, non-invasive biomarkers, prolonging and inflating the cost of clinical trials. This study identifies long non-coding RNA Tug1 as a potential blood-based FRDA biomarker. In a previous study using a frataxin knockdown mouse model (FRDAkd), we observed several hallmark FRDA symptoms and abnormalities in various tissues. Building on this, we hypothesized that a dual-source approach--comparing the data from peripheral blood samples from FRDA patients with tissue samples from affected areas in FRDAkd mice, tissues usually unattainable from patients--would effectively identify robust biomarkers. A comprehensive reanalysis was conducted on gene expression data from 183 age- and sex-matched peripheral blood samples of FRDA patients, carriers, and controls, as well as 192 tissue datasets from FRDAkd mice. Blood and tissue samples underwent RNA isolation and qRT-PCR, and frataxin knockdown was confirmed through ELISA. Tug1 RNA interaction was explored via RNA pull-down assays. Validation was performed in serum and blood samples on an independent set of 45 healthy controls, 45 FRDA patients; 66 heterozygous carriers, and 72 FRDA patients. Tug1 and Slc40a1 emerged as potential blood-based biomarkers, confirmed in the FRDAkd mouse model (One-way ANOVA, p [≤] 0.05). Tug1 was consistently downregulated after Fxn knockdown and correlated strongly with Fxn levels (R2 = 0.71 during depletion, R2 = 0.74 during rescue). Slc40a1 showed a similar but tissue-specific pattern. Further validation of Tug1s downstream targets strengthened its biomarker candidacy. In additional human samples, TUG1 levels were significantly downregulated in both whole blood and serum of FRDA patients compared to controls (Wilcoxon signed-rank test, p < 0.05). Regression analyses revealed a negative correlation between TUG1 levels and disease onset (p < 0.0037), and positive correlations with disease duration and Functional Disability Stage score (p < 0.04). This suggests that elevated TUG1 levels correlate with earlier onset and more severe cases. In summary, this study highlights Tug1 as a crucial blood-based biomarker for FRDA. Tug1s consistent expression variance across human and mouse tissues is closely associated to disease severity and key FRDA pathways. It also correlates strongly with Fxn levels, making it a promising early, non-invasive marker. TUG1 offers potential for FRDA monitoring and therapeutic development, warranting further clinical research.

neuroscience↗

bioRxiv2023

A mutant fitness compendium in Bifidobacteria reveals molecular determinants of colonization and host-microbe interactions

Bifidobacteria commonly represent a dominant constituent of human gut microbiomes during infancy, influencing nutrition, immune development, and resistance to infection. Despite interest as a probiotic therapy, predicting the nutritional requirements and health-promoting effects of Bifidobacteria is challenging due to major knowledge gaps. To overcome these deficiencies, we used large-scale genetics to create a compendium of mutant fitness in Bifidobacterium breve (Bb). We generated a high density, randomly barcoded transposon insertion pool in Bb, and used this pool to determine Bb fitness requirements during colonization of germ-free mice and chickens with multiple diets and in response to hundreds of in vitro perturbations. To enable mechanistic investigation, we constructed an ordered collection of insertion strains covering 1462 genes. We leveraged these tools to improve models of metabolic pathways, reveal unexpected host- and diet-specific requirements for colonization, and connect the production of immunomodulatory molecules to growth benefits. These resources will greatly reduce the barrier to future investigations of this important beneficial microbe.

microbiology↗

bioRxiv2023

Profiling novel lateral gene transfer events in the human microbiome

Lateral gene transfer (LGT) is an important mechanism for genome diversification in microbial populations, including the human microbiome. While prior work has surveyed LGT events in human-associated microbial isolate genomes, the scope and dynamics of novel LGT events arising in personal microbiomes are not well understood, as there are no widely adopted computational methods to detect, quantify, and characterize LGT from complex microbial communities. We addressed this by developing, benchmarking, and experimentally validating a computational method (WAAFLE) to profile novel LGT events from assembled metagenomes. Applying WAAFLE to >2K human metagenomes from diverse body sites, we identified >100K putative high-confidence but previously uncharacterized LGT events ([~]2 per assembled microbial genome-equivalent). These events were enriched for mobile elements (as expected), as well as restriction-modification and transport functions typically associated with the destruction of foreign DNA. LGT frequency was quantifiably influenced by biogeography, the phylogenetic similarity of the involved taxa, and the ecological abundance of the donor taxon. These forces manifest as LGT networks in which hub species abundant in a community type donate unequally with their close phylogenetic neighbors. Our findings suggest that LGT may be a more ubiquitous process in the human microbiome than previously described. The open-source WAAFLE implementation, documentation, and data from this work are available at http://huttenhower.sph.harvard.edu/waafle.

bioinformatics↗

bioRxiv2022

PI 3-kinase isoform p110alpha controls smooth muscle cell functionality and protects against aortic aneurysm formation

BackgroundCatalytic class IA PI 3-kinase isoform p110 is a crucial regulator of cellular proliferation and survival in numerous cell types. While p110 is critically involved in pathogenic vascular remodeling, its physiological role for vascular integrity under stress conditions has not been studied. We report a protective function of smooth muscle p110 against abdominal aortic aneurysm (AAA) formation. Methods & ResultsIn mice lacking p110 in smooth muscle cells (sm-p110-/-), perfusion of the infrarenal aorta with porcine pancreatic elastase (PPE) yielded substantially enhanced AAA formation compared to wild type controls. This disease phenotype is partly attributable to a subtle preexisting vascular phenotype under basal conditions, as sm-p110-/- mice displayed a smaller media area, deranged aortic wall structure (detached smooth muscle cells, increased apoptotic cell death), and a diminished functional responsiveness of aortic rings to vasodilators. Furthermore, p110 is also implicated in regenerative processes during AAA development: Whereas wild type mice showed increased media hypertrophy, neointima formation and proliferation upon PPE intervention, these vascular remodeling processes were diminished in sm-p110-/- mice. Concomitantly, increased numbers of elastic fiber breaks and ECM degradation were detected in sm-p110-/- aorta. Mechanistically, we found that lack of p110 expression impaired smooth muscle cell proliferation, expression of contractile marker genes and production of elastin fibers. This phenotype largely depended on reduced phosphorylation and inactivation of FOXO1, as specific FOXO1 inhibition fully rescued proliferation of p110-/- smooth muscle cells, and knockdown of FOXO1 increased expression of calponin and elastin. ConclusionsSmooth muscle p110 protects against AAA disease by maintaining aortic wall homoeostasis and promoting SMC proliferation to compensate for cell loss during AAA development. Our findings have potential implications for current approaches aimed at p110 inhibition for cancer therapy and suggest new pharmacological strategies to activate p110 signaling in AAA disease.

cell biology↗

bioRxiv2022

Deep Representation Learning Determines Drug Mechanism of Action from Cell Painting Images

Fluorescent-based microscopy screens carry a broad range of phenotypic information about how compounds affect cellular biology. From changes in cellular morphology observed in these screens, one key area of medicinal interest is determining a compounds mechanism of action. However, much of this phenotypic information is subtle and difficult to quantify. Hence, creating quantitative embeddings that can measure cellular response to compound perturbation has been a key area of research. Here we present a deep learning enabled encoder called MOAProfiler that captures phenotypic features for determining mechanism of action from Cell Painting images. We compared our method with both a traditional computer vision means of feature encoding via CellProfiler and a deep learning encoder called DeepProfiler. The results, on two independent and biologically different datasets, indicated that MOAProfiler encoded MOA-specific features that allowed for more accurate clustering and classification of compounds over hundreds of different MOAs.

bioinformatics↗

bioRxiv2022

Integrative single-cell meta-analysis reveals disease-relevant vascular cell states and markers in human atherosclerosis

Coronary artery disease (CAD) and atherosclerosis are characterized by plaque formation in the arteries wall. CAD progression involves complex interactions and phenotypic plasticity within and between distinct vascular and immune cell lineages. Single-cell RNA-seq (scRNA-seq) studies have highlighted lineage-specific transcriptomic signatures, but the reported cell phenotypes in humans remain controversial. Here, we meta-analyzed four scRNA-seq datasets, creating the first map of human cell diversity in atherosclerosis. We generated an atlas of 118,578 high-quality cells, characterized cell-type diversity and provided insights into smooth muscle cell (SMC) phenotypic modulation, transcription factor activity and cell-cell communication. We integrated genome-wide association study (GWAS) data and uncovered a critical role for modulated SMC phenotypes in CAD and coronary calcification. Finally, we identified candidate markers of fibromyocyte and fibrochondrogenic human SMCs (LTBP1 and CRTAC1) that may serve as proxies of atherosclerosis progression. Altogether, we created a unified cellular map of atherosclerosis informing cell state-specific mechanistic and translational studies of cardiovascular diseases.

genomics↗

bioRxiv2022

FHL5 controls vascular disease-associated gene programs in smooth muscle cells

BackgroundGenome-wide association studies (GWAS) have identified hundreds of loci associated with common vascular diseases such as coronary artery disease (CAD), myocardial infarction (MI), and hypertension. However, the lack of mechanistic insights for a majority of these loci limits translation of these findings into the clinic. Among these loci with unknown functions is UFL1-FHL5 (chr6q16.1), a locus that reached genome-wide significance in a recent CAD/MI GWAS meta-analysis. In addition to CAD/MI, UFL1-FHL5 is also implicated to coronary calcium, intracranial aneurysm, and migraine risk, consistent with the widespread pleiotropy observed among other GWAS loci. MethodsWe apply a multimodal approach leveraging statistical fine-mapping, epigenomic profiling, and imaging of human coronary artery tissues to implicate Four-and-a-half LIM domain 5 (FHL5) as the top candidate causal gene. We unravel the molecular mechanisms of the cross-phenotype genetic associations through in vitro functional analyses and epigenomic profiling experiments. ResultsWe prioritized FHL5 as the top candidate causal gene at the UFL1-FHL5 locus through eQTL colocalization methods. FHL5 gene expression was enriched in the SMC and pericyte population in human artery tissues with coexpression network analyses supporting a functional role in regulating SMC contraction. Unexpectedly, under procalcifying conditions, FHL5 overexpression promoted vascular calcification and dysregulated processes related to extracellular matrix organization and calcium handling. Lastly, by mapping FHL5 binding sites and inferring FHL5 target gene function using artery tissue gene regulatory network analyses, we highlight regulatory interactions between FHL5 and downstream CAD/MI loci, such as FOXL1 and FN1 that have roles in vascular remodeling. ConclusionTaken together, these studies provide mechanistic insights into the pleiotropic genetic associations of UFL1-FHL5. We show that FHL5 mediates vascular disease risk through transcriptional regulation of downstream vascular remodeling loci. These trans-acting mechanisms may account for a portion of the heritable risk for complex vascular diseases.

genomics↗

bioRxiv2022

Genetic regulation of human aortic smooth muscle cell gene expression and splicing predict causal coronary artery disease genes

Coronary artery disease (CAD) is the leading cause of death worldwide. Recent meta-analyses of genome-wide association studies (GWAS) have identified over 175 loci associated with CAD. The majority of these loci are in non-coding regions and are predicted to regulate gene expression. Given that vascular smooth muscle cells (SMCs) play critical roles in the development and progression of CAD, we hypothesized that a subset of the CAD GWAS risk loci are associated with the regulation of transcription in distinct SMC phenotypes. Here, we measured gene expression in SMCs isolated from the ascending aortas of 151 ethnically diverse heart transplant donors in quiescent or proliferative conditions and calculated the association of their expression and splicing with [~]6.3 million imputed single nucleotide polymorphism (SNP) markers across the genome. We identified 4,910 expression and 4,412 splice quantitative trait loci (sQTL) that represent regions of the genome associated with transcript abundance and splicing. 3,660 of the eQTLs had not been observed in the publicly available Genotype-Tissue Expression dataset. Further, 29 and 880 of the eQTLs were SMC- and sex-specific, respectively. To identify the effector transcript(s) regulated by CAD GWAS loci, we used four distinct colocalization approaches and identified 84 eQTL and 164 sQTLs that colocalized with CAD loci, highlighting the importance of genetic regulation of mRNA splicing as a molecular mechanism for CAD genetic risk. Notably, 20% and 35% of the eQTLs were unique to quiescent or proliferative SMCs, respectively. Two CAD loci colocalized with a SMC sex-specific eQTL (AL160313.1 and TERF2IP) and another locus colocalized with SMC-specific eQTL (ALKBH8). Also, 27% and 37% of the sQTLs were unique to quiescent or proliferative SMCs, respectively. The most significantly associated CAD locus, 9p21, was an sQTL for the long non-coding RNA CDKN2B-AS1, also known as ANRIL, in proliferative SMCs. Collectively, these results provide evidence for the molecular mechanisms of genetic susceptibility to CAD in distinct SMC phenotypes.

genetics↗

bioRxiv2021

The grape MYB24 mediates the coordination of light-induced terpene and flavonol accumulation in response to berry anthocyanin sunscreen depletion

The presence of naturally-occurring color mutants in plants has permitted the identification of many regulatory genes implicated in the synthesis of discrete metabolic compounds, mostly anthocyanins and carotenoids. Conversely, transcription factors that coordinate more than one specialized metabolic pathway seem challenging to screen from a forward genetics perspective. We explored the relationship between different branches of the phenylpropanoid and isoprenoid pathways while examining an infrequent berry skin color variegation in grapevine. Red and white berry skin sections were compared at the genetic, transcriptomic and metabolomic levels showing that, as in most cultivated white grape varieties, the uncolored skin section convened the non-functional alleles of the anthocyanin regulators MYBA1 and MYBA2, explaining the lack of pigments. In contrast, light-responsive flavonols and monoterpenes increased in anthocyanin-depleted areas. We disclosed an enrichment of the flavonol, terpene and carotenoid pathways among up-regulated genes from white-skin sections, accompanied by increased expressions of flavonol regulators and the still uncharacterized MYB24 gene. We used DAP-seq to examine the in vitro binding of affinity-purified MYB24 protein to genomic DNA and demonstrated its binding in the promoter regions of terpene (22) and carotenoid (6) genes, in addition to more than 30 photosynthesis and light-response genes, including the flavonol-regulator HY5 homologue (HYH). We confirmed the activation of TPS35 and HYH promoter:luciferase reporters in the presence of MYB24 and the grape bHLH MYC2, all of which correlate in their higher expression in white skin variegated sections. The integration of several datasets allowed to define a list of high confidence targets, suggesting MYB24 as a modulator of light responses including the synthesis of flavonoids (flavonols) and isoprenoids (terpenes, and putatively carotenoids). The correspondence between MYB24 and monoterpenes in all conditions surveyed implies that this regulatory network is broadly triggered towards berry ripening, and that the absence of anthocyanin sunscreens accelerates its activation most likely in a dose-dependent manner due to increased radiation exposure.

plant biology↗

bioRxiv2021

Effects of memantine and high dose vitamin D on gait in the APP/PS1 mouse model of Alzheimer's disease following vitamin D deprivation

BackgroundAltered gait is a frequent feature of Alzheimers disease (AD), as is vitamin D deficiency. Treatment with memantine and vitamin D can protect cortical axons from exposure to amyloid-{beta} and glutamate toxicity, suggesting this combination may mitigate altered gait in AD. ObjectiveInvestigate the effects of vitamin D deprivation and subsequent treatment with memantine and vitamin D enrichment on gait performance in APPswe/PS1dE9 mice. MethodsMale APPswe/PS1dE9 mice were split into four groups (n=14 each) at 2.5 months of age. A control group was fed a standard diet throughout while the other three groups started a vitamin D-deficient diet at month 6. The VitD- group remained on this deficient diet for the rest of the study. At month 9, the remaining two groups began treatment with either memantine alone or memantine combined with 10 IU/g of vitamin D. Gait performance was assessed at months 6, 9, 12, and 15. ResultsVitamin D deprivation led to a 13% increase in hind stride width by month 15 (p<0.001). Examination of the treatment groups at month 15 revealed that mice treated with memantine alone still showed an increase in hind stride width compared to controls (p<0.01), while mice treated with memantine and vitamin D did not (p=0.21). ConclusionVitamin D deprivation led to impaired postural control in the APPswe/PS1dE9 model. Treatment with memantine and vitamin D, but not memantine alone, prevented this impairment. Future work should explore the potential for treatments incorporating vitamin D supplementation to improve gait in people with AD.

animal behavior and cognition↗