Biology subjects

Wu, D.

Publications and source records attributed to Wu, D..

At least 109 records · Page 6Linked to original sources

bioRxiv2025

Generative modeling for RNA splicing predictions and design

Alternative splicing (AS) of pre-mRNA plays a crucial role in tissue-specific gene regulation, with disease implications due to splicing defects. Predicting and manipulating AS can therefore uncover new regulatory mechanisms and aid in therapeutics design. We introduce TrASPr+BOS, a generative AI model with Bayesian Optimization for predicting and designing RNA for tissue-specific splicing outcomes. TrASPr is a multi-transformer model that can handle different types of AS events and generalize to unseen cellular conditions. It then serves as an oracle, generating labeled data to train a Bayesian Optimization for Splicing (BOS) algorithm to design RNA for condition-specific splicing outcomes. We show TrASPr+BOS outperforms existing methods, enhancing tissue-specific AUPRC by up to 1.8 fold and capturing tissue-specific regulatory elements. We validate hundreds of predicted novel tissue-specific splicing variations and confirm new regulatory elements using dCas13. We envision TrASPr+BOS as a light yet accurate method researchers can probe or adopt for specific tasks.

bioinformatics↗

bioRxiv2025

Precision mRNA Nanomedicine for Targeted Vascular Therapies in ARDS and Atherosclerosis

Vascular diseases, including acute respiratory distress syndrome (ARDS) and atherosclerosis, are leading causes of global morbidity and mortality, underscoring the critical need for innovative therapies. While human genetics has uncovered specific molecular mechanisms in endothelial cells that drive acute and chronic vascular inflammation central to these conditions, effective in vivo strategies to spatiotemporally target these disease-causing pathways using therapeutic mRNAs remain limited. Here, we present a modular and tunable nanoparticle platform (PROGRAMMED nanoparticles) engineered to selectively deliver therapeutic mRNAs to target cells with distinct molecular signatures, such as inflamed endothelial cells expressing vascular cell adhesion molecule 1 (VCAM1). This approach restores key genetics-informed endothelial pathways implicated in disease, including the loss of Kruppel-like factor 2 (KLF2) in acute inflammation underlying ARDS and the suppression of phospholipid phosphatase 3 (PLPP3) in chronic inflammation driving atherosclerosis. In multiple preclinical models, these precision mRNA nanomedicine strategies demonstrated compelling therapeutic efficacy. VCAM1-targeting PROGRAMMED nanoparticles delivering KLF2 mRNA to inflamed lung microvascular endothelial cells significantly improved microvascular health and alleviated virus-induced ARDS. Similarly, PLPP3 mRNA delivery by VCAM1-targeting PROGRAMMED nanoparticles attenuated arterial inflammation, slowed atherosclerosis progression, and promoted regression of advanced plaques. This innovative platform offers a transformative approach for spatiotemporal mRNA therapies targeting inflamed endothelial cells, providing a minimally invasive and highly specific strategy to address acute and chronic vascular diseases. Moreover, the modularity of PROGRAMMED nanoparticles, particularly their ability to display diverse targeting motifs, holds substantial potential for addressing a broad spectrum of vascular complications and other diseases that require precise spatiotemporal correction of dysregulated genes.

bioengineering↗

bioRxiv2025

The RING Finger E3 Ligase RNF25 Protects DNA Replication Forks Independently of its Canonical Roles in Ubiquitin Signaling

The DNA damage response (DDR) mechanisms that allow cells to tolerate DNA replication stress are critically important for genome stability and cell viability. Using an unbiased genetic screen we identify a role for the RING finger E3 ubiquitin ligase RNF25 in promoting DNA replication stress tolerance. In response to DNA replication stress, RNF25-deficient cells generate aberrantly high levels of single-stranded DNA (ssDNA), accumulate in S-phase and show reduced mitotic entry. Using single-molecule DNA fiber analysis, we show that RNF25 protects reversed DNA replication forks generated by the fork remodeler HLTF from nucleolytic degradation by MRE11 and CtIP. Mechanistically, RNF25 interacts with the replication fork protection factor REV7 and recruits REV7 to nascent DNA after replication stress. The role of RNF25 in protecting replication forks is fully separable from its canonical functions in ubiquitin conjugation. This work reveals the RNF25-REV7 signaling axis as an important protective mechanism in cells experiencing replication stress.

molecular biology↗

bioRxiv2025

Decoding the Molecular Language of Proteins with Evola

Proteins, natures intricate molecular machines, are the products of billions of years of evolution and play fundamental roles in sustaining life. Yet, deciphering their molecular language--understanding how sequences and structures encode biological functions--remains a cornerstone challenge. Here, we introduce Evolla, an interactive protein-language model designed to transcend static classification by interpreting protein function through natural language queries. Trained on 546 million protein-text pairs and refined via Direct Preference Optimization, Evolla couples high-dimensional molecular representations with generative semantic decoding. Benchmarking establishes Evollas superiority over general large language models in functional inference, demonstrates zero-shot performance parity with the state-of-the-art supervised model, and exposes remote functional relationships invisible to conventional alignment. We validate Evolla through two distinct applications: identifying candidate eukaryotic signature proteins in Asgard archaea, with functional Vps4 homologs validated via yeast complementation; and interactively discovering a novel deep-sea polyethylene terephthalate (PET) hydrolase, PsPETase, confirmed to degrade plastic films. These results position Evolla not merely as a predictor, but as a generative engine capable of complex hypothesis formulation, shifting the paradigm from static annotation to interactive, actionable discovery. The Evolla online service is available at http://www.chat-protein.com/.

bioinformatics↗

bioRxiv2025

A kinetic model for USP14 regulated substrate degradation in 26S proteasome

Despite high-resolution structural studies on the USP14-proteasome-substrate complexes, time-resolved cryo-electron microscopy (cryo-EM) results on USP14-regulated allostery of the 26S proteasome are still very limited and a quantitative understanding of substrate degradation dynamics remains elusive. In this study, we propose a mean field model of ordinary differential equations (ODEs) for USP14 regulated substrate degradation in 26S proteasome. The kinetic model incorporates recent cryo-EM findings on the allostery of 26S proteasome and generates results in good agreement with time-resolved experimental observations. The model elucidates that USP14 typically reduces the substrate degradation rate and reveals the functional dependence of this rate on the concentrations of substrate and adenosine triphosphate (ATP). The half-maximal effective concentration (EC50) of the substrate for different ATP concentrations is predicted. When multiple substrates are present, the model suggests that substrates that are easier to insert into the OB-ring and disengage from the proteasome, or less likely to undergo deubiquitination would be more favored to be degraded by the USP14-bound proteasome. Author SummaryThe proteasome is a crucial protein complex involved in the degradation of damaged or unnecessary proteins within cells, requiring ATP and ubiquitin for its functioning. It is regulated by cellular factors that transiently associate with it, often referred to as proteasome-associated proteins. USP14 is such a protein that activates its deubiquitination activity through reversible binding to the proteasome, thereby decreasing the substrate degradation activity of the proteasome. In this study, we developed a kinetic model to describe how USP14 regulates substrate degradation in proteasome based on recent experimental findings. The model yields result consistent with experimental observations, demonstrating that the mean-field description of mass action law for chemical reactions also applies to complex biomolecular machineries.

biophysics↗

bioRxiv2024

Chemically-inducible CRISPR/Cas9 circuits for ultra-high dynamic range gene perturbation

CRISPR/Cas9 technologies provide unique capabilities for modeling disease and understanding gene-to-phenotype connections. In cultured cells, chemical-mediated control of Cas9 activity can limit off-target effects and enable mechanistic study of essential genes. However, widely-used Tet-On systems often show "leaky" Cas9 expression, leading to unintended edits, as well as weak activity upon induction. Leakiness can be distinctly problematic in the context of Cas9 nuclease activity, which may result in cumulative DNA damage and degradation of the target cell genome over time. To overcome these deficiencies, we established transgenic platforms that minimize Cas9 functionality in the off-state along with maximized and uncompromised on-state gene editing efficiency. By combining conditional destabilization and inhibition of Cas9, we developed an all-in-one (one or multiple guide RNAs and Cas9) ultra-tight, Tet-inducible system with exceptional dynamic range (on vs. off-state) across various cell lines and targets. As an alternative to Tet-mediated induction, we created a branaplam-regulated splice switch module for low-baseline and robust Cas9 activity control. Lastly, for circumstances where DNA damage needs to be avoided, we constructed a dual-control, Tet-inducible CRISPRi module for tight and potent transcriptional silencing. This upgraded suite of inducible CRISPR systems has broad applications for numerous cell types and experimental conditions.

molecular biology↗

bioRxiv2024

Vascular endothelial growth factor-D improves lung vascular integrity during acute lung injury

Disorders in pulmonary vascular integrity are a prominent feature in many lung diseases. Paracrine signaling is highly enriched in the lung and plays a crucial role in regulating vascular homeostasis. However, the specific local cell-cell crosstalk signals that maintain pulmonary microvascular stability in adult animals and humans remain largely unexplored. In this study, we employed single-cell RNA-sequencing (scRNAseq)-based computational pipelines to systematically profile ligand-receptor (L/R) interactions within the lung microvascular niche and identified vascular endothelial growth factor-D (VEGF-D) as a key local factor with previously unrecognized barrier-protective properties in models of acute lung injury. Our scRNAseq data revealed that, under physiological conditions, soluble L/R interactions between mesenchymal cells, in particular alveolar fibroblast, and microvascular endothelial cells are predominantly associated with pathways involved in maintaining vascular integrity as compared to all other cells. Upon treatment with top identified ligands, we found that VEGF-D significantly enhanced endothelial barrier function and conferred protection against inflammatory challenges induced by tumor necrosis factor- (TNF-), interleukin-1{beta} (IL-1{beta}), and thrombin. This barrier-protective effect of VEGF-D was significantly attenuated by inhibition of VEGFR2, either through siRNA knockdown or pharmacological blockade using specific VEGFR2 inhibitors. Intravenous administration of recombinant VEGF-D in lipopolysaccharides (LPS)-induced acute lung injury models significantly reduced vascular permeability (7339 {+/-} 2510 (LPS) v.s. 5350 {+/-} 1821 (LPS + VEGF-D), p < 0.05), immune cell infiltration (0.791 {+/-} 0.199 x 106 WBC/mL (LPS) v.s. 0.540 {+/-} 0.190 x 106 WBC/mL (LPS + VEGF-D), p < 0.01), and the expression of pro-inflammatory markers TNF- and IL-6 in the lung tissue. This effect was abolished in VEGFR2iECKO mice, confirming that VEGF-D mediates its effects via VEGFR2-dependent signaling. This study demonstrates an unexpected protective role for VEGF-D in promoting lung endothelial barrier integrity and suggests that paracrine signaling from the alveolar fibroblast niche contributes critically to lung capillary homeostasis.

molecular biology↗

bioRxiv2024

Telomere-to-Telomere Genome Assembly Uncovers Wolbachia-Driven Sex-Specific Demography and Challenges Fisher's Principle in a Sawfly

Wolbachia, a widespread endosymbiotic bacterium, can reshape the evolutionary fates of its insect hosts by distorting reproduction and altering population dynamics. Despite extensive laboratory research, its long-term effects on host evolution in nature remain poorly understood, particularly regarding genetic mechanisms underlying changes in sex determination and reproduction. Here, we report the first telomere-to-telomere (T2T) genome assembly of the sawfly Analcellicampa danfengensis and the complete genome of it symbiotic Wolbachia. Comparative population genomics of six closely related Analcellicampa species revealed that Wolbachia-infected populations experience marked changes in sex-specific demography. While uninfected species maintain balanced genetic features between males and females, infected species show a persistent reduction in male effective population size alongside a stable or even growing female population, ultimately driving males toward extinction. Genomic scans identified positively selected genes associated with reproductive functions, sensory perception, neural development, and longevity, suggesting that Wolbachia manipulates critical host biological pathways to promote its transmission. These findings provide direct genomic evidence that Wolbachia acts as a powerful evolutionary force, reshaping host genomes in a way that disrupts Fishers principle, ultimately driving female-biased demography and the extinction of males at evolutionary timescales. This work provides deeper insights into host- endosymbiont coevolution and has important implications for evolutionary theory and pest management strategies.

evolutionary biology↗

bioRxiv2024

Degradation of intrinsically disordered proteins using proteolysis targeting nanobody conjugate

Intrinsically disordered proteins (IDPs) play crucial roles in diverse cellular processes and are implicated in numerous diseases. However, due to the inherently unstructured nature, it is challenging to modulate their function using small molecule degraders or inhibitors. To address this, we introduced proteolysis targeting nanobody conjugate (PROTNC) composed of a nanobody that binds to an IDP, an E3 ligase ligand, and a cyclic cell-penetrating peptide for efficient intracellular delivery. We first showed a general-purpose PROTNC system capable to degrade different endogenous IDPs based on a co-condensate formation mechanism. We also introduced a straightforward strategy to degrade individual IDPs using specific PROTNCs equipped with a nanobody recognizing that particular IDP. Notably, we demonstrated the degradation of TPX2 which is an undruggable oncogenic IDP responsible for microtubule nucleation. This targeted degradation effectively inhibited cancer cell proliferation by disrupting bipolar mitotic spindle assembly in metaphase and directed cells to apoptotic pathway.

cell biology↗

bioRxiv2024

De novo non-compartmentalized microorganism for directed evolution of nanobodies

The creation of de novo microorganisms is a fascinating task, and microorganisms offer a wide range of applications for humanity. Herein, we designed a de novo microorganism, termed Syn-phage, as an artificial unencapsulated mimic of naturally occurring phage capable of evolving nanobodies with improved properties. Syn-phage composes unified genomic and phenotypic materials with phenotypic proteins encoded from its genetic information. Since Syn-phage can undergo amplification in E. coli. host, we established a protocol for the evolution of nanobodies with improved avidity or affinity, allowing super-resolution live-cell immunoimaging of endogenous proteins, a challenge posed in immunolabeling with antibodies. Utilizing a Syn-phage evolved nanobody, we visualized the microtubule nucleation factor hTPX2, recorded the dynamics of it during mitotic progression, and imaged it at a resolution beyond the diffraction limit of light via an AI-powered imaging pipeline. Hence, this study demonstrated the possibility to design de novo microorganisms for capitative applications.

biochemistry↗

bioRxiv2024

A survey of hypothalamic phenotypes identifies molecular and behavioral consequences of MYT1L haploinsufficiency in male and female mice.

The transcription factor MYT1L supports proper neuronal differentiation and maturation during brain development. MYT1L haploinsufficiency results in a neurodevelopmental disorder characterized by intellectual disability, developmental delay, autism, behavioral disruptions, aggression, obesity and epilepsy. While MYT1L is expressed throughout the brain, how it supports proper neuronal function in distinct regions has not been assessed. Some features of MYT1L Neurodevelopmental Syndrome suggest disruption of hypothalamic function, such as obesity and endocrine issues, and previous research showed changes in hypothalamic neuropeptide expression following knockdown in zebrafish. Here, we leveraged our heterozygous Myt1l mutant, previously shown to recapitulate aspects of the human syndrome such as hyperactivity, social challenges, and obesity, to examine the impact of MYT1L loss on hypothalamic function. Examining the molecular profile of the MYT1L haploinsufficient hypothalamus revealed a similar scale of disruption to previously studied brain regions, yet with region-specific roles for MYT1L, including regulation of neuropeptide systems. Alterations in oxytocin and arginine vasopressin cell numbers were also found. Behaviors studied included maternal care, social group hierarchies, and aggression, all of which were unchanged. Feeding and metabolic markers were also largely unchanged in MYT1L haploinsufficient mice, yet an interaction was observed between diet and MYT1L genotype on weight gain. Our findings here suggest that gross endocrine function was not altered by MYT1L haploinsufficiency, and that key sex-specific behaviors related to proper hypothalamic function remain intact. Further study is needed to understand the functional impact of the altered hypothalamic molecular profile and changes in neuropeptide cell numbers that result from MYT1L haploinsufficiency.

animal behavior and cognition↗

bioRxiv2024

IMMClock reveals immune aging and T cell function at single-cell resolution

The aging of the immune system substantially impacts individual immune responses, yet accurately quantifying immune age remains a complex challenge. Here we developed IMMClock, a novel immune aging clock that uses gene expression data to predict the biological age of individual CD8 T cells, CD4 T cells, and NK cells. The accuracy of IMMClock is first validated across multiple independent datasets, demonstrating its robustness. Second, utilizing the IMMClock, we find that intrinsic cellular aging processes are more strongly altered during immune aging than differentiation processes. Thirdly, our analysis confirms the strong associations between immune aging and established processes such as cellular senescence, exhaustion, and telomere length at the single cell level. Furthermore, immune aging is accelerated under several disease conditions such as type 2 diabetes, heart disease, and cancer. Finally, we apply IMMClock to analyze a perturb-seq gene activation screen of T cell functionality. We find that the post-perturbation immune age of individual T cells is strongly correlated with their pre-perturbation immune age. Furthermore, the immune age at resting state of individual T cells is strongly predictive of their post-stimulation activation state. Overall, IMMClock advances our understanding of immune aging by providing precise, single-cell level age estimations. Its future applications hold promise for identifying interventions that concomitantly rejuvenate and activate T cells, potentially enhancing efforts to counteract age-related immune decline.

bioinformatics↗

bioRxiv2024

Real-time volumetric imaging of cells and molecules in deep tissues with Takoyaki ultrasound

Acoustic contrast agents and reporter genes play a critical role in allowing ultrasound to visualize blood flow, map molecules and track cellular function in opaque living organisms. However, existing ultrasound methods to image acoustic contrast agents predominantly focus on 2D planar imaging, while the biological phenomena of interest unfurl in three dimensions. Here, we introduce a method for efficient, dynamic imaging of contrast agents and reporter genes in 3D using multiplexed matrix array transducers. Our "Takoyaki" pulse sequence uses the simultaneous scanning of multiple focal points to excite contrast agents with sufficient acoustic pressure for nonlinear imaging while efficiently covering 3D space. Through in vitro experiments, we first show that the Takoyaki sequence produces highly sensitive volume images of gas vesicle contrast agents and compare its performance with alternative imaging schemes. We then establish its utility in cellular imaging in vivo by visualizing acoustic reporter gene-expressing tumors in a mouse model of glioblastoma. Finally, we demonstrate real-time volumetric imaging by tracking the dynamics of fluid motion in brain ventricles after intraventricular contrast injection. Takoyaki imaging enables a more comprehensive understanding of biological processes by providing spatiotemporal information in 3D within the constraints of accessible multiplexed matrix array systems.

bioengineering↗

bioRxiv2024

Immunosuppression Drugs Exhibit Differential Effects on Endothelial Cell Function

Immunosuppressive medications are widely used to treat patients with neoplasms, autoimmune conditions, and solid organ transplants. Prior studies indicate that immunosuppression drugs can cause adverse vascular remodeling. Given the systemic effects of the drugs, elucidating cell-type specific drug-effects has been challenging. We utilized induced pluripotent stem-cell derived endothelial cells to investigate the role of widely used immunosuppression drugs on endothelial function. We found that among immunosuppression agents, sirolimus reduced basic endothelial cell functions including cell migration, proliferation, acetylated LDL uptake, and angiogenesis properties; while tacrolimus only reduced nitric oxide release. This model allows for investigation of differential effect of immunosuppression drugs on endothelial function that can elucidate mechanisms contributing to adverse vascular profiles observed clinically.

cell biology↗

bioRxiv2024

Cross-Species Analysis Reveals No Universal Programmed Aging Mechanism: Insights from Single-Cell Transcriptomics in Zebrafish, Fruit Fly, and Nematode

The question of whether aging follows a universal programmed process has been a topic of debate for a long time. Previous arguments, either supporting or refuting programmed aging, were mainly based on different evolutionary biology theories. In this study, we analysed single-cell RNA sequencing data from zebrafish, fruit fly, and nematode at various stages of development to explore gene co-expression modules across these species. We successfully identified a co-expression module related to ribosomal protein genes that is shared across the early development stages in multiple tissues of all three species. However, we did not find any cross-species shared gene co-expression modules related to aging. Further analysis of gene regulatory networks (GRNs) demonstrated that although certain aging-related genes are conserved, their regulatory mechanisms vary significantly between species. These findings suggest that aging is not governed by a conserved universal program but rather by species-specific adaptations to damage and environmental conditions.

evolutionary biology↗

bioRxiv2024

LP-Micro Offers Interpretable Disease Outcome Prediction by Leveraging Microbial Biomarkers and Their Time-Varying Effects

Information generated from longitudinally-sampled microbial data has the potential to illuminate important aspects of development and progression for many human conditions and diseases. Identifying microbial biomarkers and their time-varying effects can not only advance our understanding of pathogenetic mechanisms, but also facilitate early diagnosis and guide optimal timing of interventions. However, longitudinal predictive modeling of highly noisy and dynamic microbial data (e.g., metagenomics) poses analytical challenges. To overcome these challenges, we introduce a robust and interpretable machine-learning-based longitudinal microbiome analysis framework, LP-Micro, that encompasses: (i) longitudinal microbial feature screening via a polynomial group lasso, (ii) disease outcome prediction implemented via machine learning methods (e.g., XGBoost, deep neural networks), and (iii) interpretable association testing between time points, microbial features, and disease outcomes via permutation feature importance. We demonstrate in simulations that LP-Micro can not only identify incident disease-related microbiome taxa but also offers improved prediction accuracy compared to existing approaches. Applications of LP-Micro in two longitudinal microbiome studies with clinical outcomes of childhood dental disease and weight loss following bariatric surgery yield consistently high prediction accuracy. The identified critical early predictive time points are informative and aligned with clinical expectations.

bioinformatics↗

bioRxiv2024

Inhibition of Androgen Receptor Exposes Replication Stress Vulnerability in Prostate Cancer

Standard initial systemic treatment for patients with metastatic prostate cancer includes agents that target androgen receptor (AR) signaling. Despite an initial positive response to these AR pathway inhibitors (ARPIs), acquired resistance remains a significant challenge. We show that treatment of AR-positive prostate cancer cells with the frontline ARPI enzalutamide induces DNA replication stress. Such stress is exacerbated by suppression of translesion DNA synthesis (TLS), leading to aberrant accumulation of single-stranded DNA (ssDNA) gaps and persistent DNA damage biomarkers. We further demonstrate that the TLS inhibitor, JH-RE-06, markedly sensitizes AR-positive prostate cancer cells, but not AR-negative benign cells, to enzalutamide in vitro. Combination therapy with enzalutamide and JH-RE-06 significantly suppresses cancer growth in a syngeneic murine tumor model over vehicle control or individual treatment groups. These findings suggest that AR inhibition broadly triggers DNA replication stress in hormone-sensitive prostate cancer, thereby exposing a unique vulnerability that can be exploited by a TLS-disrupting adjuvant for targeted therapy.

cancer biology↗

bioRxiv2024

Neural correlates differ between crystallized and fluid intelligence in adolescents

Fluid and crystallized intelligence are acknowledged as distinct facets of cognitive ability during brain development, but the specific neural substrates and molecular mechanisms underlying them remain unclear. This study used a sample comprising 7471 young adolescents (mean age 9.87 {+/-} 0.62 years) from the ABCD cohort to elucidate the differential neural correlates of fluid and crystallized intelligence. Our findings indicated that micro-level brain MRI phenotypes such as water diffusivity were closely associated with fluid intelligence, whereas macro-level brain MRI phenotypes such as gray matter cortical thickness were indicative of crystallized intelligence. We further investigated the molecular mechanisms underlying fluid and crystallized intelligence by correlating the characteristic MRI markers with spatial transcriptome profiles and PET imaging. Results showed that fluid intelligence had significant associations with serotonin and glutamate system, while crystallized intelligence was related to serotonin, dopamine and acetylcholine system. Furthermore, we examined the impacts of lifestyle factors on these two forms of intelligence and how the molecular pathways mediated these impacts. Our investigation suggested that physical activities, screen use and sleep duration influenced fluid intelligence mainly through mGlu5 receptors and crystallized intelligence through 5HT1a and D2 receptors. In conclusion, these findings illustrated a distinct neural basis between fluid and crystallized intelligence from the perspectives of neuroimaging, neurotransmitters, and lifestyles in young adolescents.

neuroscience↗